From c858819e5bb4644637fc1790307b2eb72d38391b Mon Sep 17 00:00:00 2001 From: jykr Date: Mon, 25 Mar 2024 14:39:04 -0400 Subject: [PATCH 001/107] Change the default behavior of bean-qc to not remove the replicates even when it has bad replicates --- README.md | 105 +++++++++++------ bean/framework/Edit.py | 6 +- ...Resso2Align.cpython-38-x86_64-linux-gnu.so | Bin 269256 -> 0 bytes bean/model/run.py | 2 +- bean/qc/utils.py | 106 ++++++++++-------- bin/bean-qc | 4 +- notebooks/sample_quality_report.ipynb | 42 +++---- tests/test_res/.gitkeep | 0 8 files changed, 164 insertions(+), 101 deletions(-) delete mode 100755 bean/mapping/CRISPResso2Align.cpython-38-x86_64-linux-gnu.so create mode 100644 tests/test_res/.gitkeep diff --git a/README.md b/README.md index eead4ed..01047e1 100644 --- a/README.md +++ b/README.md @@ -206,9 +206,18 @@ Above command produces `prefix_editing_preference.[html,ipynb]` as editing prefe ## `bean-qc`: QC of reporter screen data ```bash bean-qc \ - my_sorting_screen.h5ad `# Input ReporterScreen .h5ad file path` \ + my_sorting_screen.h5ad `# Input ReporterScreen .h5ad file path` \ -o my_sorting_screen_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r qc_report_my_sorting_screen `# Prefix for QC report` + -r qc_report_my_sorting_screen `# Prefix for QC report` \ + +# Inspect the output qc_report_my_sorting_screen.html to tweak QC threshold + +bean-qc \ + my_sorting_screen.h5ad \ + -o my_sorting_screen_masked.h5ad \ + -r qc_report_my_sorting_screen \ + #[--count-correlation-thres 0.7 ...]\ + -b ``` `bean-qc` supports following quality control and masks samples with low quality. Specifically: @@ -229,36 +238,68 @@ Above command produces #### Additional Parameters -* `--tiling` (default: `None`): If set as `True` or `False`, it sets the screen object to be tiling (`True`) or variant (`False`)-targeting screen when calculating editing rate. -* `--replicate-label` (default: `"rep"`): Label of column in `bdata.samples` that describes replicate ID. -* `--condition-label` (default: `"condition"`)": Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time, etc.). -* `--sample-covariates` (default: `None`): Comma-separated list of column names in `bdata.samples` that describes non-selective experimental condition (drug treatment, etc.). The values in the `bdata.samples` should NOT contain `.`. -* `--no-editing` (default: `False`): Ignore QC about editing. Can be used for QC of other editing modalities. - -Editing rate quantification -* `--ctrl-cond` (default: `"bulk"`): Value in of column in `ReporterScreen.samples[condition_label]` where guide-level editing rate to be calculated - - Editing rate is calculated with following parameters in variant screens: - * `--target-pos-col` (default: `"target_pos"`): Target position column in `bdata.guides` specifying target edit position in reporter. - - For tiling screens: - * `--rel-pos-is-reporter` (default: `False`): Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If `False`, those are relative to spacer position. - * `--edit-start-pos` (default: `2`): Edit start position to quantify editing rate on, 0-based inclusive. - * `--edit-end-pos` (default: `7`): Edit end position to quantify editing rate on, 0-based exclusive. - -LFC of positive controls -* `--posctrl-col` (default: `group`): Column name in .h5ad.guides DataFrame that specifies guide category. -* `--posctrl-val` (default: `PosCtrl`): Value in .h5ad.guides[`posctrl_col`] that specifies guide will be used as the positive control in calculating log fold change. -* `--lfc-conds` (default: `"top,bot"`): Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated between, delimited by comma - - -Sample filtering thresholds -* `--count-correlation-thres` (default: `0.7`): Threshold of guide count correlation to mask out. -* `--edit-rate-thres` (default: `0.1`): Mean editing rate threshold per sample to mask out. -* `--lfc-thres` (default: `0.1`): Positive guides' correlation threshold to filter out. - -Other -* `--recalculate-edits` (default: `False`): Even when `ReporterScreen.layers['edit_count']` exists, recalculate the edit counts from `ReporterScreen.uns['allele_count']`." +##### Optional arguments: +* `-o OUT_SCREEN_PATH`, `--out-screen-path OUT_SCREEN_PATH` + Path where quality-filtered ReporterScreen object to be written to +* `-r OUT_REPORT_PREFIX`, `--out-report-prefix OUT_REPORT_PREFIX` + Output prefix of qc report (prefix.html, prefix.ipynb) + +##### QC thresholds: +* `--count-correlation-thres COUNT_CORRELATION_THRES` + Correlation threshold to mask out. +* `--edit-rate-thres EDIT_RATE_THRES` + Mean editing rate threshold per sample to mask out. +* `--lfc-thres LFC_THRES` + Positive guides' correlation threshold to filter out. + +##### Run options: +* `-b`, `--remove-bad-replicates` + Remove replicates with at least two of its samples meet the QC threshold (bean-run does not support having only one sorting bin sample for a replicate). +* `-i`, `--ignore-missing-samples` + If the flag is not provided, if the ReporterScreen object does not contain all condiitons for + each replicate, make fake empty samples. If the flag is provided, don't add dummy samples. +* `--no-editing` Ignore QC about editing. Can be used for QC of other editing modalities. +* `--dont-recalculate-edits` + When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from + ReporterScreen.uns['allele_count']. + +##### Input `.h5ad` formatting: +Note that these arguements will change the way the QC metrics are calculated for guides, samples, or replicates. +* `--tiling TILING` Specify that the guide library is tiling library without 'n guides per target' design +* `--replicate-label REPLICATE_LABEL` + Label of column in `bdata.samples` that describes replicate ID. +* `--sample-covariates SAMPLE_COVARIATES` + Comma-separated list of column names in `bdata.samples` that describes non-selective + experimental condition. (drug treatment, etc.) +* `--condition-label CONDITION_LABEL` + Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time, + etc.) +###### Editing rate calculation + * `--ctrl-cond CTRL_COND` + Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate + to be calculated + * `--rel-pos-is-reporter` + Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If + `False`, those are relative to spacer position. + Editing rate is calculated with following parameters in + * Variant screens: + * `--target-pos-col TARGET_POS_COL` + Target position column in `bdata.guides` specifying target edit position in reporter + * tiling screens: + * `--edit-start-pos EDIT_START_POS` + Edit start position to quantify editing rate on, 0-based inclusive. + * `--edit-end-pos EDIT_END_POS` + Edit end position to quantify editing rate on, 0-based exclusive. +###### LFC of positive controls + * `--posctrl-col POSCTRL_COL` + Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all + gRNAs, feed empty string ''. + * `--posctrl-val POSCTRL_VAL` + Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the + positive control in calculating log fold change. + * `--lfc-conds LFC_CONDS` + Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated + between, delimited by comma

diff --git a/bean/framework/Edit.py b/bean/framework/Edit.py index 0a51fb5..5abba2e 100644 --- a/bean/framework/Edit.py +++ b/bean/framework/Edit.py @@ -175,7 +175,11 @@ def get_range(self): min(edit.pos for edit in self.edits), max(edit.pos for edit in self.edits), ) - + + def set_uid(self, uid): + self.edits = {edit.set_uid(uid) for edit in self.edits} + return self + def get_uid(self): uid = None if ( diff --git a/bean/mapping/CRISPResso2Align.cpython-38-x86_64-linux-gnu.so b/bean/mapping/CRISPResso2Align.cpython-38-x86_64-linux-gnu.so deleted file mode 100755 index 0ad2bee3060070f20255b0a6a734a27cb415a432..0000000000000000000000000000000000000000 GIT binary patch literal 0 HcmV?d00001 literal 269256 zcmeFadt6l2`aeFPbSBeg@-#VFohHLVyMaYPN)3v-F|6^HbOl8WO@VMG(Y!!FF-;Vw zD|I?2y3^$pb=aq22B{=F8OO@~eLw42YglZV-{*XPuit;a&-OL$ zXT9I=^{lm?b=iCN?7dk(HD_9UT%1j9Lu?n>`cpQWR}zM6sFDCnv)S?gXxs5Z+s{NL zQNx+TrrO(_oTf4o$n+YQOva}fpAbIJ;&UfHs@q`Nmg93NJ|pp2hR?b9s4Ybr 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If the flag is provided, don't add dummy samples.", action="store_true", ) - parser.add_argument( - "-r", - "--out-report-prefix", - help="Output prefix of qc report (prefix.html, prefix.ipynb)", - type=str, + run_parser.add_argument( + "--no-editing", + help="Ignore QC about editing. Can be used for QC of other editing modalities.", + action="store_true", ) - parser.add_argument( + run_parser.add_argument( + "--dont-recalculate-edits", + help="When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from ReporterScreen.uns['allele_count'].", + action="store_true", + ) + + input_parser.add_argument( "--tiling", dest="tiling", type=lambda x: bool(distutils.util.strtobool(x)), help="Specify that the guide library is tiling library without 'n guides per target' design", ) - parser.add_argument( + input_parser.add_argument( "--replicate-label", help="Label of column in `bdata.samples` that describes replicate ID.", type=str, default="rep", ) - parser.add_argument( + input_parser.add_argument( "--sample-covariates", help="Comma-separated list of column names in `bdata.samples` that describes non-selective experimental condition. (drug treatment, etc.)", type=str, default=None, ) - parser.add_argument( + input_parser.add_argument( "--condition-label", help="Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time, etc.)", type=str, default="condition", ) - parser.add_argument( - "--no-editing", - help="Ignore QC about editing. Can be used for QC of other editing modalities.", - action="store_true", - ) - parser.add_argument( + input_parser.add_argument( "--target-pos-col", help="Target position column in `bdata.guides` specifying target edit position in reporter", type=str, default="target_pos", ) - parser.add_argument( + input_parser.add_argument( "--rel-pos-is-reporter", help="Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If `False`, those are relative to spacer position.", action="store_true", default=False, ) - parser.add_argument( + input_parser.add_argument( "--edit-start-pos", help="Edit start position to quantify editing rate on, 0-based inclusive.", default=2, ) - parser.add_argument( + input_parser.add_argument( "--edit-end-pos", help="Edit end position to quantify editing rate on, 0-based exclusive.", default=7, ) - parser.add_argument( - "--count-correlation-thres", - help="Correlation threshold to mask out.", - type=float, - default=0.7, - ) - parser.add_argument( - "--edit-rate-thres", - help="Mean editing rate threshold per sample to mask out.", - type=float, - default=0.1, - ) - parser.add_argument( + + input_parser.add_argument( "--posctrl-col", help="Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all gRNAs, feed empty string ''.", type=str, default="target_group", ) - parser.add_argument( + input_parser.add_argument( "--posctrl-val", help="Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the positive control in calculating log fold change.", type=str, default="PosCtrl", ) - parser.add_argument( - "--lfc-thres", - help="Positive guides' correlation threshold to filter out.", - type=float, - default=-0.1, - ) - parser.add_argument( + + input_parser.add_argument( "--lfc-conds", help="Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated between, delimited by comma", type=str, default="top,bot", ) - parser.add_argument( + input_parser.add_argument( "--ctrl-cond", help="Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate to be calculated", type=str, default="bulk", ) - parser.add_argument( - "--recalculate-edits", - help="Even when ReporterScreen.layers['edit_count'] exists, recalculate the edit counts from ReporterScreen.uns['allele_count'].", - action="store_true", - ) + + args = parser.parse_args() if args.out_screen_path is None: args.out_screen_path = f"{args.bdata_path.rsplit('.h5ad', 1)[0]}.filtered.h5ad" diff --git a/bin/bean-qc b/bin/bean-qc index 1f79ab9..970cb19 100644 --- a/bin/bean-qc +++ b/bin/bean-qc @@ -33,9 +33,9 @@ def main(): comp_cond2=args.lfc_cond2, ctrl_cond=args.ctrl_cond, exp_id=args.out_report_prefix, - recalculate_edits=args.recalculate_edits, + recalculate_edits=~args.dont_recalculate_edits, base_edit_data=args.base_edit_data, - + remove_bad_replicates=args.remove_bad_replicates, ), kernel_name="bean_python3", ) diff --git a/notebooks/sample_quality_report.ipynb b/notebooks/sample_quality_report.ipynb index 65a0d92..ff3a213 100644 --- a/notebooks/sample_quality_report.ipynb +++ b/notebooks/sample_quality_report.ipynb @@ -47,7 +47,7 @@ "edit_quantification_start_pos = 2\n", "edit_quantification_end_pos = 7\n", "target_pos_col = \"target_pos\"\n", - "corr_X_thres = 0.7\n", + "count_correlation_thres = 0.7\n", "edit_rate_thres = 0.1\n", "posctrl_col = \"group\"\n", "posctrl_val = \"PosCtrl\"\n", @@ -57,9 +57,10 @@ "comp_cond1 = \"top\"\n", "comp_cond2 = \"bot\"\n", "ctrl_cond = \"bulk\"\n", - "recalculate_edits = False\n", + "recalculate_edits = True\n", "tiling = None\n", - "base_edit_data = True" + "base_edit_data = True\n", + "remove_bad_replciates = False" ] }, { @@ -396,7 +397,7 @@ "source": [ "bdata.samples[\"mask\"] = 1\n", "bdata.samples.loc[\n", - " bdata.samples.median_corr_X.isnull() | (bdata.samples.median_corr_X < corr_X_thres),\n", + " bdata.samples.median_corr_X.isnull() | (bdata.samples.median_corr_X < count_correlation_thres),\n", " \"mask\",\n", "] = 0\n", "if \"mean_editing_rate\" in bdata.samples.columns.tolist():\n", @@ -421,23 +422,24 @@ "outputs": [], "source": [ "# leave replicate with more than 1 sorting bin data\n", - "rep_n_samples = bdata_filtered.samples.groupby(replicate_label)[\"mask\"].sum()\n", - "print(rep_n_samples)\n", - "rep_has_too_small_sample = rep_n_samples.loc[rep_n_samples < 2].index.tolist()\n", - "print(\n", - " f\"Excluding reps {rep_has_too_small_sample} that has less than 2 samples per replicate.\"\n", - ")\n", - "if isinstance(replicate_label, str):\n", - " samples_include = ~bdata_filtered.samples[replicate_label].isin(\n", - " rep_has_too_small_sample\n", + "if remove_bad_replciates:\n", + " rep_n_samples = bdata_filtered.samples.groupby(replicate_label)[\"mask\"].sum()\n", + " print(rep_n_samples)\n", + " rep_has_too_small_sample = rep_n_samples.loc[rep_n_samples < 2].index.tolist()\n", + " print(\n", + " f\"Excluding reps {rep_has_too_small_sample} that has less than 2 samples per replicate.\"\n", " )\n", - "else:\n", - " bdata_filtered.samples[\"_rc\"] = bdata_filtered.samples[\n", - " replicate_label\n", - " ].values.tolist()\n", - " samples_include = ~bdata_filtered.samples[\"_rc\"].isin(rep_has_too_small_sample)\n", - " bdata_filtered.samples.pop(\"_rc\")\n", - "bdata_filtered = bdata_filtered[:, samples_include]" + " if isinstance(replicate_label, str):\n", + " samples_include = ~bdata_filtered.samples[replicate_label].isin(\n", + " rep_has_too_small_sample\n", + " )\n", + " else:\n", + " bdata_filtered.samples[\"_rc\"] = bdata_filtered.samples[\n", + " replicate_label\n", + " ].values.tolist()\n", + " samples_include = ~bdata_filtered.samples[\"_rc\"].isin(rep_has_too_small_sample)\n", + " bdata_filtered.samples.pop(\"_rc\")\n", + " bdata_filtered = bdata_filtered[:, samples_include]" ] }, { diff --git a/tests/test_res/.gitkeep b/tests/test_res/.gitkeep new file mode 100644 index 0000000..e69de29 From bfdf121ccb4e07fa5723936a4980bdc0ceaad890 Mon Sep 17 00:00:00 2001 From: jykr Date: Mon, 25 Mar 2024 15:22:03 -0400 Subject: [PATCH 002/107] allow & test feeding no --allele-df-key for tiling run --- bean/model/run.py | 2 +- bean/preprocessing/data_class.py | 1 - tests/test_run.py | 4 ++-- 3 files changed, 3 insertions(+), 4 deletions(-) diff --git a/bean/model/run.py b/bean/model/run.py index 9615d8a..e09dcd5 100644 --- a/bean/model/run.py +++ b/bean/model/run.py @@ -222,7 +222,7 @@ def parse_args(): parser.add_argument( "--allele-df-key", type=str, - default="allele_counts", + default=None, help="screen.uns[allele_df_key] will be used as the allele count.", ) parser.add_argument( diff --git a/bean/preprocessing/data_class.py b/bean/preprocessing/data_class.py index 4bfc862..ed4a2c3 100644 --- a/bean/preprocessing/data_class.py +++ b/bean/preprocessing/data_class.py @@ -567,7 +567,6 @@ def _post_init( else: allele_counts_selected = self.screen.uns[allele_df_key] if control_guide_tag is not None: - def _set_uid_to_row(row): if control_guide_tag in row.guide: row[allele_col].set_uid(row.guide) diff --git a/tests/test_run.py b/tests/test_run.py index 1455707..3c877b1 100644 --- a/tests/test_run.py +++ b/tests/test_run.py @@ -120,8 +120,8 @@ def test_run_tiling_with_wo_negctrl_uniform(): @pytest.mark.order(24) -def test_run_tiling_negctrl(): - cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad --scale-by-acc --acc-bw-path tests/data/accessibility_signal.bw -o tests/test_res/tiling/ --allele-df-key allele_counts_spacer_0_19_A.G_translated_prop0.1_0.3 --fit-negctrl --negctrl-col strand --negctrl-col-value neg --control-guide-tag neg --repguide-mask None --n-iter 10" +def test_run_tiling_negctrl_allelekey(): + cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad --scale-by-acc --acc-bw-path tests/data/accessibility_signal.bw -o tests/test_res/tiling/ --fit-negctrl --negctrl-col strand --negctrl-col-value neg --control-guide-tag neg --repguide-mask None --n-iter 10" try: subprocess.check_output( cmd, From cdfad7ac712f26d1ccb15b7a9d2bf49b75d3a7c2 Mon Sep 17 00:00:00 2001 From: jykr Date: Mon, 25 Mar 2024 15:35:00 -0400 Subject: [PATCH 003/107] udpate readme to include read structure schematic --- README.md | 7 ++++++- imgs/read_struct.png | Bin 0 -> 35866 bytes 2 files changed, 6 insertions(+), 1 deletion(-) create mode 100755 imgs/read_struct.png diff --git a/README.md b/README.md index 01047e1..82ab214 100644 --- a/README.md +++ b/README.md @@ -78,9 +78,14 @@ bean-count-samples \ -o . `# output directory` \ -r `# read edit/allele information from reporter` \ -t 12 `# number of threads` \ - --name my_sorting_screen `# name of this sample run` + --name my_sorting_screen `# name of this sample run` \ + --guide-start-seq ATGCTTGCC `# Change this according to your read design ``` +You may need to adjust the command arguments according to your read structure. + Read structuren +See full detail in [Parameters](#parameters). + ### Input file format #### 1. gRNA_library.csv File should contain following columns. diff --git a/imgs/read_struct.png b/imgs/read_struct.png new file mode 100755 index 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...n_annotated.translation_ref_mismatches.csv | 1 + 4 files changed, 74 insertions(+) create mode 100644 tests/data/var_mini_counts.csv create mode 100644 tests/data/var_mini_guides.csv create mode 100644 tests/data/var_mini_samples.csv create mode 100644 tests/data/var_mini_screen_annotated.translation_ref_mismatches.csv diff --git a/tests/data/var_mini_counts.csv b/tests/data/var_mini_counts.csv new file mode 100644 index 0000000..745ef5d --- /dev/null +++ b/tests/data/var_mini_counts.csv @@ -0,0 +1,31 @@ +name,rep5_top,rep5_high,rep5_bulk,rep5_low,rep5_bot,rep6_top,rep6_high,rep6_bulk,rep6_low,rep6_bot +CONTROL_8_g1,368.0,245.0,302.0,798.0,84.0,342.0,1088.0,910.0,670.0,462.0 +CONTROL_8_g2,63.0,54.0,201.0,418.0,20.0,31.0,314.0,100.0,46.0,51.0 +CONTROL_8_g3,253.0,61.0,238.0,221.0,8.0,460.0,1295.0,786.0,1188.0,1019.0 +CONTROL_8_g4,236.0,133.0,324.0,425.0,35.0,338.0,953.0,429.0,864.0,406.0 +CONTROL_8_g5,69.0,55.0,56.0,267.0,16.0,129.0,777.0,140.0,253.0,204.0 +CONTROL_9_g1,259.0,177.0,170.0,514.0,38.0,84.0,234.0,148.0,103.0,79.0 +CONTROL_9_g2,335.0,164.0,225.0,516.0,37.0,654.0,2132.0,1079.0,1188.0,657.0 +CONTROL_9_g3,14.0,57.0,48.0,111.0,20.0,218.0,265.0,228.0,195.0,152.0 +CONTROL_9_g4,302.0,129.0,158.0,423.0,27.0,517.0,1258.0,362.0,935.0,605.0 +CONTROL_9_g5,124.0,67.0,156.0,320.0,21.0,152.0,605.0,321.0,428.0,226.0 +ACAT2_SA_44_g1,558.0,344.0,374.0,1366.0,105.0,512.0,1091.0,570.0,774.0,489.0 +ACAT2_SA_44_g2,1151.0,304.0,867.0,1224.0,144.0,953.0,2993.0,2069.0,1454.0,1038.0 +ACAT2_SA_44_g3,516.0,211.0,535.0,663.0,80.0,976.0,2389.0,1485.0,2520.0,1160.0 +ACAT2_SA_44_g4,432.0,90.0,352.0,459.0,69.0,392.0,1629.0,850.0,1203.0,658.0 +ACAT2_SA_44_g5,602.0,277.0,570.0,637.0,42.0,1024.0,2968.0,1547.0,1020.0,999.0 +ACAT2_SA_45_g1,353.0,146.0,262.0,508.0,54.0,345.0,1355.0,582.0,566.0,399.0 +ACAT2_SA_45_g2,389.0,139.0,280.0,369.0,66.0,446.0,848.0,458.0,584.0,498.0 +ACAT2_SA_45_g3,618.0,240.0,510.0,992.0,60.0,608.0,2514.0,920.0,882.0,773.0 +ACAT2_SA_45_g4,775.0,253.0,559.0,889.0,93.0,860.0,1810.0,991.0,978.0,673.0 +ACAT2_SA_45_g5,1046.0,471.0,830.0,1472.0,164.0,995.0,1966.0,1123.0,1394.0,1192.0 +ACAT2_SA_46_g1,735.0,266.0,496.0,975.0,89.0,2261.0,5981.0,4085.0,5552.0,2804.0 +ACAT2_SA_46_g2,825.0,201.0,444.0,835.0,67.0,394.0,1098.0,731.0,1105.0,723.0 +ACAT2_SA_46_g3,591.0,284.0,414.0,809.0,61.0,241.0,532.0,389.0,425.0,297.0 +ACAT2_SA_46_g4,294.0,147.0,237.0,407.0,50.0,325.0,893.0,642.0,992.0,392.0 +ACAT2_SA_46_g5,342.0,173.0,159.0,438.0,39.0,173.0,425.0,209.0,209.0,27.0 +ACAT2_SA_47_g1,446.0,149.0,90.0,484.0,34.0,164.0,412.0,176.0,227.0,150.0 +ACAT2_SA_47_g2,508.0,274.0,626.0,762.0,106.0,757.0,1840.0,844.0,987.0,903.0 +ACAT2_SA_47_g3,262.0,177.0,203.0,406.0,50.0,325.0,504.0,328.0,189.0,204.0 +ACAT2_SA_47_g4,186.0,115.0,205.0,317.0,13.0,95.0,176.0,161.0,210.0,93.0 +ACAT2_SA_47_g5,401.0,238.0,381.0,599.0,83.0,339.0,985.0,580.0,533.0,376.0 diff --git a/tests/data/var_mini_guides.csv b/tests/data/var_mini_guides.csv new file mode 100644 index 0000000..c0ab3d8 --- /dev/null +++ b/tests/data/var_mini_guides.csv @@ -0,0 +1,31 @@ +name,Unnamed: 0,Target gene/variant,Target descriptor,Arbitrary number,gRNA position category,Target base position in gRNA,Target base position in reporter,BE,target_group,sequence,Reporter,barcode,5-nt PAM,offset,target,target_pos,Group2,masked_sequence,masked_barcode,chrom,genomic_pos +CONTROL_8_g1,90,CONTROL,,8,g1,4,10,ABE,NegCtrl,AAAATTATCGGAAACGGTAG,GCTCGAAAAATTATCGGAAACGGTAGAATCTT,GAAC,AATCT,-10,CONTROL_8,9,NegCtrl,GGGGTTGTCGGGGGCGGTGG,GGGC,, +CONTROL_8_g2,91,CONTROL,,8,g2,5,11,ABE,NegCtrl,AAAAATTATCGGAAACGGTA,GGCTCGAAAAATTATCGGAAACGGTAGAATCT,CGTG,GAATC,-11,CONTROL_8,10,NegCtrl,GGGGGTTGTCGGGGGCGGTG,CGTG,, +CONTROL_8_g3,92,CONTROL,,8,g3,5,12,ABE,NegCtrl,AAAAATTATCGGAAACGGT,TGGCTCGAAAAATTATCGGAAACGGTAGAATC,ATCA,AGAAT,-12,CONTROL_8,11,NegCtrl,GGGGGTTGTCGGGGGCGGT,GTCG,, +CONTROL_8_g4,93,CONTROL,,8,g4,7,13,ABE,NegCtrl,CGAAAAATTATCGGAAACGG,ATGGCTCGAAAAATTATCGGAAACGGTAGAAT,CAAG,TAGAA,-13,CONTROL_8,12,NegCtrl,CGGGGGGTTGTCGGGGGCGG,CGGG,, +CONTROL_8_g5,94,CONTROL,,8,g5,8,14,ABE,NegCtrl,TCGAAAAATTATCGGAAACG,TATGGCTCGAAAAATTATCGGAAACGGTAGAA,TTCA,GTAGA,-14,CONTROL_8,13,NegCtrl,TCGGGGGGTTGTCGGGGGCG,TTCG,, +CONTROL_9_g1,95,CONTROL,,9,g1,4,10,ABE,NegCtrl,AGCATTCTCACCAAGACCGA,TAAGATAGCATTCTCACCAAGACCGAATGCCC,TCCC,ATGCC,-10,CONTROL_9,9,NegCtrl,GGCGTTCTCGCCGGGGCCGG,TCCC,, +CONTROL_9_g2,96,CONTROL,,9,g2,5,11,ABE,NegCtrl,TAGCATTCTCACCAAGACCG,CTAAGATAGCATTCTCACCAAGACCGAATGCC,GGCC,AATGC,-11,CONTROL_9,10,NegCtrl,TGGCGTTCTCGCCGGGGCCG,GGCC,, +CONTROL_9_g3,97,CONTROL,,9,g3,6,12,ABE,NegCtrl,ATAGCATTCTCACCAAGACC,GCTAAGATAGCATTCTCACCAAGACCGAATGC,GGAC,GAATG,-12,CONTROL_9,11,NegCtrl,GTGGCGTTCTCGCCGGGGCC,GGGC,, +CONTROL_9_g4,98,CONTROL,,9,g4,6,13,ABE,NegCtrl,ATAGCATTCTCACCAAGAC,CGCTAAGATAGCATTCTCACCAAGACCGAATG,AGCG,CGAAT,-13,CONTROL_9,12,NegCtrl,GTGGCGTTCTCGCCGGGGC,GGCG,, +CONTROL_9_g5,99,CONTROL,,9,g5,8,14,ABE,NegCtrl,AGATAGCATTCTCACCAAGA,GCGCTAAGATAGCATTCTCACCAAGACCGAAT,CACG,CCGAA,-14,CONTROL_9,13,NegCtrl,GGGTGGCGTTCTCGCCGGGG,CGCG,, +ACAT2_SA_44_g1,100,ACAT2,SA,44,g1,4,10,ABE,PosCtrl,TGTAGGTTCCTTCAATGGTG,TTCTATTGTAGGTTCCTTCAATGGTGCCTTAG,GGTT,CCTTA,-10,ACAT2_SA_44,9,PosCtrl_inc,TGTGGGTTCCTTCGGTGGTG,GGTT,chr6,160183949.0 +ACAT2_SA_44_g2,101,ACAT2,SA,44,g2,5,11,ABE,PosCtrl,TTGTAGGTTCCTTCAATGGT,TTTCTATTGTAGGTTCCTTCAATGGTGCCTTA,CATA,GCCTT,-11,ACAT2_SA_44,10,PosCtrl_inc,TTGTGGGTTCCTTCGGTGGT,CGTG,chr6,160183949.0 +ACAT2_SA_44_g3,102,ACAT2,SA,44,g3,6,12,ABE,PosCtrl,ATTGTAGGTTCCTTCAATGG,CTTTCTATTGTAGGTTCCTTCAATGGTGCCTT,GACG,TGCCT,-12,ACAT2_SA_44,11,PosCtrl_inc,GTTGTGGGTTCCTTCGGTGG,GGCG,chr6,160183949.0 +ACAT2_SA_44_g4,103,ACAT2,SA,44,g4,7,13,ABE,PosCtrl,TATTGTAGGTTCCTTCAATG,CCTTTCTATTGTAGGTTCCTTCAATGGTGCCT,AGTG,GTGCC,-13,ACAT2_SA_44,12,PosCtrl_inc,TGTTGTGGGTTCCTTCGGTG,GGTG,chr6,160183949.0 +ACAT2_SA_44_g5,104,ACAT2,SA,44,g5,8,14,ABE,PosCtrl,CTATTGTAGGTTCCTTCAAT,GCCTTTCTATTGTAGGTTCCTTCAATGGTGCC,AAGC,GGTGC,-14,ACAT2_SA_44,13,PosCtrl_inc,CTGTTGTGGGTTCCTTCGGT,GGGC,chr6,160183949.0 +ACAT2_SA_45_g1,105,ACAT2,SA,45,g1,4,10,ABE,PosCtrl,TCTAGGCTGTGGGCAGAATC,CCTCTTTCTAGGCTGTGGGCAGAATCCTGTTA,GATT,CTGTT,-10,ACAT2_SA_45,9,PosCtrl_inc,TCTGGGCTGTGGGCGGGGTC,GGTT,chr6,160188035.0 +ACAT2_SA_45_g2,106,ACAT2,SA,45,g2,5,11,ABE,PosCtrl,TTCTAGGCTGTGGGCAGAAT,TCCTCTTTCTAGGCTGTGGGCAGAATCCTGTT,TCAC,CCTGT,-11,ACAT2_SA_45,10,PosCtrl_inc,TTCTGGGCTGTGGGCGGGGT,TCGC,chr6,160188035.0 +ACAT2_SA_45_g3,107,ACAT2,SA,45,g3,6,12,ABE,PosCtrl,TTTCTAGGCTGTGGGCAGAA,TTCCTCTTTCTAGGCTGTGGGCAGAATCCTGT,TGTC,TCCTG,-12,ACAT2_SA_45,11,PosCtrl_inc,TTTCTGGGCTGTGGGCGGGG,TGTC,chr6,160188035.0 +ACAT2_SA_45_g4,108,ACAT2,SA,45,g4,7,13,ABE,PosCtrl,CTTTCTAGGCTGTGGGCAGA,GTTCCTCTTTCTAGGCTGTGGGCAGAATCCTG,TTCT,ATCCT,-13,ACAT2_SA_45,12,PosCtrl_inc,CTTTCTGGGCTGTGGGCGGG,TTCT,chr6,160188035.0 +ACAT2_SA_45_g5,109,ACAT2,SA,45,g5,8,14,ABE,PosCtrl,TCTTTCTAGGCTGTGGGCAG,TGTTCCTCTTTCTAGGCTGTGGGCAGAATCCT,TAGG,AATCC,-14,ACAT2_SA_45,13,PosCtrl_inc,TCTTTCTGGGCTGTGGGCGG,TGGG,chr6,160188035.0 +ACAT2_SA_46_g1,110,ACAT2,SA,46,g1,4,10,ABE,PosCtrl,TCTAGGCTCCTCACTTGGCT,CTGACTTCTAGGCTCCTCACTTGGCTTACTTG,CGAC,TACTT,-10,ACAT2_SA_46,9,PosCtrl_inc,TCTGGGCTCCTCGCTTGGCT,CGGC,chr6,160189541.0 +ACAT2_SA_46_g2,111,ACAT2,SA,46,g2,5,11,ABE,PosCtrl,TTCTAGGCTCCTCACTTGGC,TCTGACTTCTAGGCTCCTCACTTGGCTTACTT,AGAC,TTACT,-11,ACAT2_SA_46,10,PosCtrl_inc,TTCTGGGCTCCTCGCTTGGC,GGGC,chr6,160189541.0 +ACAT2_SA_46_g3,112,ACAT2,SA,46,g3,6,12,ABE,PosCtrl,CTTCTAGGCTCCTCACTTGG,TTCTGACTTCTAGGCTCCTCACTTGGCTTACT,GAGA,CTTAC,-12,ACAT2_SA_46,11,PosCtrl_inc,CTTCTGGGCTCCTCGCTTGG,GGGG,chr6,160189541.0 +ACAT2_SA_46_g4,113,ACAT2,SA,46,g4,7,13,ABE,PosCtrl,ACTTCTAGGCTCCTCACTTG,TTTCTGACTTCTAGGCTCCTCACTTGGCTTAC,GCGT,GCTTA,-13,ACAT2_SA_46,12,PosCtrl_inc,GCTTCTGGGCTCCTCGCTTG,GCGT,chr6,160189541.0 +ACAT2_SA_46_g5,114,ACAT2,SA,46,g5,7,14,ABE,PosCtrl,ACTTCTAGGCTCCTCACTT,ATTTCTGACTTCTAGGCTCCTCACTTGGCTTA,GATG,GGCTT,-14,ACAT2_SA_46,13,PosCtrl_inc,GCTTCTGGGCTCCTCGCTT,GGTG,chr6,160189541.0 +ACAT2_SA_47_g1,115,ACAT2,SA,47,g1,4,10,ABE,PosCtrl,CCCAGCTGAAAATGTAGCCA,TCCTTTCCCAGCTGAAAATGTAGCCAAAAAAT,CATC,AAAAA,-10,ACAT2_SA_47,9,PosCtrl_inc,CCCGGCTGGGGGTGTGGCCG,CGTC,chr6,160196200.0 +ACAT2_SA_47_g2,116,ACAT2,SA,47,g2,5,11,ABE,PosCtrl,TCCCAGCTGAAAATGTAGCC,CTCCTTTCCCAGCTGAAAATGTAGCCAAAAAA,CTTC,AAAAA,-11,ACAT2_SA_47,10,PosCtrl_inc,TCCCGGCTGGGGGTGTGGCC,CTTC,chr6,160196200.0 +ACAT2_SA_47_g3,117,ACAT2,SA,47,g3,6,12,ABE,PosCtrl,TTCCCAGCTGAAAATGTAGC,TCTCCTTTCCCAGCTGAAAATGTAGCCAAAAA,ACCG,CAAAA,-12,ACAT2_SA_47,11,PosCtrl_inc,TTCCCGGCTGGGGGTGTGGC,GCCG,chr6,160196200.0 +ACAT2_SA_47_g4,118,ACAT2,SA,47,g4,7,13,ABE,PosCtrl,TTTCCCAGCTGAAAATGTAG,TTCTCCTTTCCCAGCTGAAAATGTAGCCAAAA,GGTG,CCAAA,-13,ACAT2_SA_47,12,PosCtrl_inc,TTTCCCGGCTGGGGGTGTGG,GGTG,chr6,160196200.0 +ACAT2_SA_47_g5,119,ACAT2,SA,47,g5,8,14,ABE,PosCtrl,CTTTCCCAGCTGAAAATGTA,TTTCTCCTTTCCCAGCTGAAAATGTAGCCAAA,TTTC,GCCAA,-14,ACAT2_SA_47,13,PosCtrl_inc,CTTTCCCGGCTGGGGGTGTG,TTTC,chr6,160196200.0 diff --git a/tests/data/var_mini_samples.csv b/tests/data/var_mini_samples.csv new file mode 100644 index 0000000..2ac8712 --- /dev/null +++ b/tests/data/var_mini_samples.csv @@ -0,0 +1,11 @@ +,condition,rep,lower_quantile,upper_quantile +rep5_top,top,rep5,0.8,1.0 +rep5_high,high,rep5,0.6,0.8 +rep5_bulk,bulk,rep5,0.0,1.0 +rep5_low,low,rep5,0.2,0.4 +rep5_bot,bot,rep5,0.0,0.2 +rep6_top,top,rep6,0.8,1.0 +rep6_high,high,rep6,0.6,0.8 +rep6_bulk,bulk,rep6,0.0,1.0 +rep6_low,low,rep6,0.2,0.4 +rep6_bot,bot,rep6,0.0,0.2 diff --git a/tests/data/var_mini_screen_annotated.translation_ref_mismatches.csv b/tests/data/var_mini_screen_annotated.translation_ref_mismatches.csv new file mode 100644 index 0000000..0681467 --- /dev/null +++ b/tests/data/var_mini_screen_annotated.translation_ref_mismatches.csv @@ -0,0 +1 @@ +,gene,rel_pos,ref,edit From fb5aaf6536a47f9d9e0e27819cae856f4578d664 Mon Sep 17 00:00:00 2001 From: jykr Date: Mon, 25 Mar 2024 16:13:05 -0400 Subject: [PATCH 005/107] add testing for bean-create-samples --- tests/test_create.py | 13 +++++++++++++ 1 file changed, 13 insertions(+) create mode 100755 tests/test_create.py diff --git a/tests/test_create.py b/tests/test_create.py new file mode 100755 index 0000000..8adc87c --- /dev/null +++ b/tests/test_create.py @@ -0,0 +1,13 @@ +import pytest +import subprocess + +def test_create_screen(): + cmd = "bean-create-screen tests/data/var_mini_guides.csv tests/data/var_mini_samples.csv tests/data/var_mini_counts.csv" + try: + subprocess.check_output( + cmd, + shell=True, + universal_newlines=True, + ) + except subprocess.CalledProcessError as exc: + raise exc From 94954c6b5e6f432b9853642cc8d994cf5ddc14b0 Mon Sep 17 00:00:00 2001 From: jykr Date: Tue, 26 Mar 2024 12:38:17 -0400 Subject: [PATCH 006/107] Allow R2 adapter --- README.md | 10 +++++----- bean/mapping/GuideEditCounter.py | 32 ++++++++++++++++++++----------- bean/mapping/utils.py | 6 ++++++ bin/bean-count-samples | 24 +++++++++++++++++++---- imgs/read_struct.png | Bin 35866 -> 0 bytes imgs/sequence_struct.png | Bin 0 -> 189401 bytes tests/test_count.py | 16 ++++++++++++++-- 7 files changed, 66 insertions(+), 22 deletions(-) delete mode 100755 imgs/read_struct.png create mode 100644 imgs/sequence_struct.png diff --git a/README.md b/README.md index 82ab214..974e0ab 100644 --- a/README.md +++ b/README.md @@ -79,11 +79,10 @@ bean-count-samples \ -r `# read edit/allele information from reporter` \ -t 12 `# number of threads` \ --name my_sorting_screen `# name of this sample run` \ - --guide-start-seq ATGCTTGCC `# Change this according to your read design ``` -You may need to adjust the command arguments according to your read structure. - Read structuren +By default, `bean-count[-samples]` assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure. + Read structuren See full detail in [Parameters](#parameters). ### Input file format @@ -149,8 +148,9 @@ Output formats Read structure -* `--guide-start-seq`: Guide starts after this sequence in R1 (default: '') -* `--guide-end-seq`: Guide ends after this sequence in R1 (default: '') +* `--guide-start-seq` (default: ''): Guide starts after this sequence in R1 +* `--guide-end-seq` (default: ''): Guide ends after this sequence in R1 +* `--barcode-start-seq` (default: ''): Barcode + reporter starts after this sequence in R2, denoted as the sense direction (the same sequence direction as R1). * `--guide-start-seqs-file` (default: `None`): CSV file path with per-sample `guide_start_seq` to be used, if provided. Formatted as `sample_id, guide_start_seq` * `--guide-end-seqs-file` (default: `None`): CSV file path with per-sample `guide_end_seq` to be used, if provided. Formatted as `sample_id,guide_end_seq` * `-l`, `--reporter-length` (default: `32`): Length of the reporter sequence. diff --git a/bean/mapping/GuideEditCounter.py b/bean/mapping/GuideEditCounter.py index 55161d3..4ffd66a 100644 --- a/bean/mapping/GuideEditCounter.py +++ b/bean/mapping/GuideEditCounter.py @@ -124,6 +124,7 @@ def __init__(self, **kwargs): self.guide_start_seq = kwargs["guide_start_seq"].upper() self.guide_end_seq = kwargs["guide_end_seq"].upper() + self.barcode_start_seq = kwargs["barcode_start_seq"].upper() if not self.guide_start_seq == "": info( f"{self.name}: Using guide_start_seq={self.guide_start_seq} for {self.output_dir}" @@ -438,6 +439,7 @@ def _count_reporter_edits( matched_guide_idx: int, R1_seq: str, R2_record: SeqIO.SeqRecord, + R2_start: int = 0, single_base_qual_cutoff: str = 30, guide_allele: Allele = None, ): @@ -453,7 +455,7 @@ def _count_reporter_edits( guide_allele: Allele from baseedit in gRNA spacer sequence when paired with guide allele. """ ref_reporter_seq = self.screen.guides.reporter.iloc[matched_guide_idx] - read_reporter_seq, read_reporter_qual = self.get_reporter_seq_qual(R2_record) + read_reporter_seq, read_reporter_qual = self.get_reporter_seq_qual(R2_record, R2_start) guide_strand, offset = self._get_strand_offset_from_guide_index( matched_guide_idx @@ -514,10 +516,9 @@ def _get_guide_counts_bcmatch_semimatch( R1_seq = str(r1.seq) R2_seq = str(r2.seq) - bc_match, semimatch = self._match_read_to_sgRNA_bcmatch_semimatch( + bc_match, semimatch, R2_start = self._match_read_to_sgRNA_bcmatch_semimatch( R1_seq, R2_seq ) - if len(bc_match) == 0: if len(semimatch) == 0: # no guide match if self.keep_intermediate: @@ -555,10 +556,10 @@ def _get_guide_counts_bcmatch_semimatch( # TODO: what if reporter seq doesn't match barcode & guide? if self.count_guide_reporter_alleles: self._count_reporter_edits( - matched_guide_idx, R1_seq, r2, guide_allele=guide_allele + matched_guide_idx, R1_seq, r2, R2_start = R2_start, guide_allele=guide_allele ) else: - self._count_reporter_edits(matched_guide_idx, R1_seq, r2) + self._count_reporter_edits(matched_guide_idx, R1_seq, r2, R2_start = R2_start, ) tqdm_reads.postfix = f"n_read={self.bcmatch}" tqdm_reads.update() @@ -643,19 +644,28 @@ def get_reporter_seq(self, R1_seq, R2_seq): R2_seq[self.guide_bc_len : (self.guide_bc_len + self.reporter_length)] ) - def get_reporter_seq_qual(self, R2_record: SeqIO.SeqRecord): + def get_reporter_seq_qual(self, R2_record: SeqIO.SeqRecord, R2_start = 0): seq = R2_record[ - self.guide_bc_len : (self.guide_bc_len + self.reporter_length) + (R2_start + self.guide_bc_len) : (R2_start + self.guide_bc_len + self.reporter_length) ].reverse_complement() return (str(seq.seq), seq.letter_annotations["phred_quality"]) def get_barcode(self, R1_seq, R2_seq): - """This can be edited by user based on the read construct.""" - return revcomp(R2_seq[: self.guide_bc_len]) + if self.barcode_start_seq != "": + barcode_start_idx = revcomp(R2_seq).replace( + self.base_edited_from, self.base_edited_to + ).find( + self.barcode_start_seq.replace(self.base_edited_from, self.base_edited_to) + ) + if barcode_start_idx == -1: + return -1, "" + else: + barcode_start_idx = 0 + return barcode_start_idx, revcomp(R2_seq[barcode_start_idx: self.guide_bc_len]) def _match_read_to_sgRNA_bcmatch_semimatch(self, R1_seq: str, R2_seq: str): # This should be adjusted for each experimental recipes. - guide_barcode = self.get_barcode(R1_seq, R2_seq) + bc_start_idx, guide_barcode = self.get_barcode(R1_seq, R2_seq) bc_match_idx = np.array([]) semimatch_idx = np.array([]) for guide_length in self.guide_lengths: @@ -677,7 +687,7 @@ def _match_read_to_sgRNA_bcmatch_semimatch(self, R1_seq: str, R2_seq: str): ) semimatch_idx = np.append(semimatch_idx, _seq_match) - return (bc_match_idx.astype(int), semimatch_idx.astype(int)) + return bc_match_idx.astype(int), semimatch_idx.astype(int), bc_start_idx def _get_guide_position_seq_of_read(self, seq): guide_start_idx = self._get_guide_start_idx(seq) diff --git a/bean/mapping/utils.py b/bean/mapping/utils.py index bc50f7c..34054ce 100644 --- a/bean/mapping/utils.py +++ b/bean/mapping/utils.py @@ -74,6 +74,12 @@ def _get_input_parser(): help="Guide starts after this sequence in R1", default="", ) + parser.add_argument( + "--barcode-start-seq", + type=str, + help="Barcode + reporter starts after this sequence in R2, denoted as the sense direction (the same sequence direction as R1).", + default="", + ) parser.add_argument( "-r", "--count-reporter", help="Count reporter edits.", action="store_true" ) diff --git a/bin/bean-count-samples b/bin/bean-count-samples index 7f54c78..06c3cdb 100644 --- a/bin/bean-count-samples +++ b/bin/bean-count-samples @@ -59,6 +59,13 @@ def get_input_parser() -> argparse.Namespace: + "Formatted as `sample_id,guide_end_seq`", default=None, ) + parser.add_argument( + "--barcode-start-seqs-file", + type=str, + help="CSV file path with per-sample `barcode_start_seq` to be used." + + "Formatted as `sample_id,guide_end_seq`", + default=None, + ) parser.add_argument( "--rerun", help="Recount each sample", action="store_true", default=False @@ -91,6 +98,11 @@ def count_sample(R1: str, R2: str, sample_id: str, args: argparse.Namespace): and args_dict["guide_end_seqs_tbl"] is not None ): args_dict["guide_end_seq"] = args_dict["guide_end_seqs_tbl"][sample_id] + if ( + "barcode_start_seqs_tbl" in args_dict + and args_dict["barcode_start_seqs_tbl"] is not None + ): + args_dict["barcode_start_seq"] = str(args_dict["barcode_start_seqs_tbl"][sample_id]) counter = bean.mp.GuideEditCounter(**args_dict) if os.path.exists(f"{counter.output_dir}.h5ad") and not args_dict["rerun"]: screen = bean.read_h5ad(f"{counter.output_dir}.h5ad") @@ -146,7 +158,7 @@ def check_arguments(args: argparse.Namespace) -> argparse.Namespace: for read_length in first_read_lengths: _check_read_length(args, read_length, warn) - def _check_return_guide_seqs_tbl(guide_seqs_file, sample_tbl): + def _check_return_guide_seqs_tbl(guide_seqs_file, sample_tbl, label): """Check if the provided `guide_[start,end]_seqs_file` contains information about all samples in `sample_tbl`.""" guide_seqs_tbl = pd.read_csv(guide_seqs_file, header=None, dtype=str).fillna("") if len(guide_seqs_tbl.columns) == 1: @@ -155,18 +167,22 @@ def check_arguments(args: argparse.Namespace) -> argparse.Namespace: sample_has_seq = sample_tbl.iloc[:, 2].isin(guide_seqs_tbl[0]) if not sample_has_seq.all(): raise InputFileError( - f"Sample ID(s) {sample_tbl.iloc[:,2][~sample_has_seq]} not in guides_seqs_file {guide_seqs_tbl}" + f"Sample ID(s) {sample_tbl.iloc[:,2][~sample_has_seq]} not in {label}_seqs_file {guide_seqs_tbl}" ) guide_seqs_tbl.columns = ["sample", "seq"] return guide_seqs_tbl.set_index("sample")["seq"] if args.guide_start_seqs_file is not None: args.guide_start_seqs_tbl = _check_return_guide_seqs_tbl( - args.guide_start_seqs_file, sample_tbl + args.guide_start_seqs_file, sample_tbl, "guide_start" ) if args.guide_end_seqs_file is not None: args.guide_end_seqs_tbl = _check_return_guide_seqs_tbl( - args.guide_end_seqs_file, sample_tbl + args.guide_end_seqs_file, sample_tbl, "guide_end" + ) + if args.barcode_start_seqs_file is not None: + args.barcode_start_seqs_tbl = _check_return_guide_seqs_tbl( + args.barcode_start_seqs_file, sample_tbl, "barcode_start" ) return args diff --git a/imgs/read_struct.png b/imgs/read_struct.png deleted file mode 100755 index d9e6627903c1d0f02c5076fec11dad8c8527ed7c..0000000000000000000000000000000000000000 GIT binary patch literal 0 HcmV?d00001 literal 35866 zcmYgY1zeQr*BwL=MZyAUlo1dKr8}fMrArt}x;vBwRE8Fi8d|!$5rmIUw1AjxRVL=T3 zxMeG;=>UN+NMHSn;o|Nn2Z2yQq(p^PT@%))6f8;A5zOu@BcbzW48AHOzm1gb1k%v;4o8r*3p`{qHr1BM}t+-%owI zVepm)qF{0S^;x&+9IuqMJ6L{Pul~GYd`-w}%#zS1MBch`VrP>T(sbbuBRk+kEZq>M zswD_Tt{DCAPQLEoMi*-IQKK6~Vs~w_FwQ742cKE=o@cNgq_Y0v>&097h(y!ZKE;0D 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tests/test_res/ -r --barcode-start-seq=GGAA" + try: + subprocess.check_output( + cmd, + shell=True, + universal_newlines=True, + ) + except subprocess.CalledProcessError as exc: + raise exc -@pytest.mark.order(5) +@pytest.mark.order(6) def test_count_samples_tiling(): cmd = "bean-count-samples --input tests/data/sample_list_tiling.csv -b A -f tests/data/test_guide_info_tiling_chrom.csv -o tests/test_res/ -r" try: @@ -54,7 +66,7 @@ def test_count_samples_tiling(): raise exc -@pytest.mark.order(6) +@pytest.mark.order(7) def test_count_chroms(): cmd = "bean-count --R1 tests/data/test_tiling_R1.fastq --R2 tests/data/test_tiling_R2.fastq -b A -f tests/data/test_guide_info_tiling_chrom.csv -o tests/test_res/ -r" try: From 5217e84534216aab35dbcfd7bb62b400027b165d Mon Sep 17 00:00:00 2001 From: jykr Date: Tue, 26 Mar 2024 13:04:27 -0400 Subject: [PATCH 007/107] Debug barcode start site --- README.md | 3 ++- bean/mapping/GuideEditCounter.py | 13 ++++++------- tests/test_count.py | 12 ++++++++++++ 3 files changed, 20 insertions(+), 8 deletions(-) diff --git a/README.md b/README.md index 974e0ab..b78a3f2 100644 --- a/README.md +++ b/README.md @@ -90,7 +90,7 @@ See full detail in [Parameters](#parameters). File should contain following columns. * `name`: gRNA ID column * `sequence`: gRNA sequence -* `barcode`: R2 barcode to help match reporter to gRNA +* `barcode`: R2 barcode to help match reporter to gRNA, written in the sense direction (as in R1) * In order to use accessibility in the [variant effect quantification](#bean-run-quantify-variant-effects), provide accessibility information in one of two options. (For non-targeting guides, provide NA values (empty cell).) * Option 1: `chrom` & `genomic_pos`: Chromosome (ex. `chr19`) and genomic position of guide sequence. You will have to provide the path to the bigwig file with matching reference version in `bean-run`. * Option 2: `accessibility_signal`: ATAC-seq signal value of the target loci of each guide. @@ -153,6 +153,7 @@ Read structure * `--barcode-start-seq` (default: ''): Barcode + reporter starts after this sequence in R2, denoted as the sense direction (the same sequence direction as R1). * `--guide-start-seqs-file` (default: `None`): CSV file path with per-sample `guide_start_seq` to be used, if provided. Formatted as `sample_id, guide_start_seq` * `--guide-end-seqs-file` (default: `None`): CSV file path with per-sample `guide_end_seq` to be used, if provided. Formatted as `sample_id,guide_end_seq` +* `--guide-start-seqs-file` (default: `None`): CSV file path with per-sample `barcode_start_seq` to be used, if provided. Formatted as `sample_id, guide_start_seq` * `-l`, `--reporter-length` (default: `32`): Length of the reporter sequence. * `--gstart-reporter` (default: `6`): Start position of the guide sequence in the reporter * `--guide-bc` (default: `True`): Construct has guide barcode diff --git a/bean/mapping/GuideEditCounter.py b/bean/mapping/GuideEditCounter.py index 4ffd66a..f1d60e8 100644 --- a/bean/mapping/GuideEditCounter.py +++ b/bean/mapping/GuideEditCounter.py @@ -47,7 +47,7 @@ class NoReadsAfterQualityFiltering(Exception): strand_str_to_int = {"neg": -1, "pos": 1, "-": -1, "+": 1} - +base_revcomp = {"A":"T", "T":"A", "G":"C", "C":"G"} def _get_stranded_guide_offset(strand: int, start_pos: int, guide_len: int) -> int: if strand == -1: @@ -652,16 +652,15 @@ def get_reporter_seq_qual(self, R2_record: SeqIO.SeqRecord, R2_start = 0): def get_barcode(self, R1_seq, R2_seq): if self.barcode_start_seq != "": - barcode_start_idx = revcomp(R2_seq).replace( - self.base_edited_from, self.base_edited_to - ).find( - self.barcode_start_seq.replace(self.base_edited_from, self.base_edited_to) - ) + barcode_start_idx = R2_seq.replace( + base_revcomp[self.base_edited_from], base_revcomp[self.base_edited_to] + ).find(revcomp(self.barcode_start_seq).replace(base_revcomp[self.base_edited_from], base_revcomp[self.base_edited_to])) if barcode_start_idx == -1: return -1, "" + barcode_start_idx += len(self.barcode_start_seq) else: barcode_start_idx = 0 - return barcode_start_idx, revcomp(R2_seq[barcode_start_idx: self.guide_bc_len]) + return barcode_start_idx, revcomp(R2_seq[barcode_start_idx: (barcode_start_idx + self.guide_bc_len)]) def _match_read_to_sgRNA_bcmatch_semimatch(self, R1_seq: str, R2_seq: str): # This should be adjusted for each experimental recipes. diff --git a/tests/test_count.py b/tests/test_count.py index bf7e685..fd9500a 100644 --- a/tests/test_count.py +++ b/tests/test_count.py @@ -1,5 +1,8 @@ import pytest import subprocess +from bean.mapping import GuideEditCounter +from bean.mapping.utils import _get_input_parser +from bean.mapping._supporting_fn import revcomp @pytest.mark.order(2) @@ -52,6 +55,15 @@ def test_count_samples_bcstart(): except subprocess.CalledProcessError as exc: raise exc +def test_barcode_start_idx(): + args = vars(_get_input_parser().parse_args(["-b", "A", "-f", "tests/data/test_guide_info.csv",])) + args["barcode_start_seq"] = "gtttgaattcgctagctaggtcttg" + args["R1"] = "tests/data/test_R1.fastq" + args["R2"] = "tests/data/test_R2.fastq" + gc = GuideEditCounter(**args) + sidx, bc = gc.get_barcode("", revcomp("agtggcaccgagtcggtgcttttttTAACAGTGTAATCTGGCGAGCCACTGTTCTTTGTACCAGAAgtttgaattcgctagctaggtcttgctgg".upper())) + assert sidx == 29 + assert bc == "AGAA" @pytest.mark.order(6) def test_count_samples_tiling(): From 3f85e25d14766c0e610e4db1eb1dbcb3bdc20024 Mon Sep 17 00:00:00 2001 From: jykr Date: Wed, 27 Mar 2024 11:36:33 -0400 Subject: [PATCH 008/107] debug negative strand gene translation --- bean/annotate/translate_allele.py | 81 ++++++++++++++++++++++--------- bean/annotate/utils.py | 10 ++-- bean/framework/AminoAcidEdit.py | 3 ++ bean/mapping/GuideEditCounter.py | 2 +- bin/bean-filter | 2 + tests/test_filter.py | 11 +++++ 6 files changed, 83 insertions(+), 26 deletions(-) diff --git a/bean/annotate/translate_allele.py b/bean/annotate/translate_allele.py index a8fe178..927e395 100644 --- a/bean/annotate/translate_allele.py +++ b/bean/annotate/translate_allele.py @@ -169,24 +169,24 @@ def get_cds_seq_pos_from_fasta(fasta_file_name: str) -> Tuple[List[str], List[in return (exon_chrom, translated_seq, genomic_pos, strand) -def _translate_single_codon(nt_seq_string: str, aa_pos: int) -> str: +def _translate_single_codon( + codon: List[str], +) -> str: # nt_seq_string: str, aa_pos: int) -> str: """Translate `aa_pos`-th codon of `nt_seq_string`.""" - codon = "".join(nt_seq_string[aa_pos * 3 : (aa_pos * 3 + 3)]) if len(codon) != 3: print("reached the end of CDS, frameshift.") return "/" try: + codon = "".join(codon) return codon_map[codon] except KeyError: if codon[-1] == "N" and codon[0] in BASE_SET and codon[1] in BASE_SET: aa_set = {codon_map[codon[:2] + N] for N in BASE_SET} if len(aa_set) == 1: return next(iter(aa_set)) - print(f"warning: no matching aa with codon {codon}") + raise ValueError(f"warning: no matching aa with codon {codon}") else: - print(f"Cannot translate codon due to ambiguity: {codon}") - - return "_" + raise ValueError(f"Cannot translate codon due to ambiguity: {codon}") class CDS: @@ -196,14 +196,17 @@ class CDS: def __init__(self): self.edited_aa_pos = set() self.edits_noncoding = set() + self.edited_nt: List[str] = [] + self.nt: List[str] = [] + self.strand: int = 1 @classmethod def from_fasta(cls, fasta_file_name, gene_name, suppressMessage=True): cds = cls() - if fasta_file_name is None: - if not suppressMessage: - print("No fasta file provided as reference: using LDLR") - fasta_file_name = os.path.dirname(be.__file__) + "/annotate/ldlr_exons.fa" + # if fasta_file_name is not None: + # if not suppressMessage: + # raise ValueError("No fasta file provided as reference: using LDLR") + # fasta_file_name = os.path.dirname(be.__file__) + "/annotate/ldlr_exons.fa" if gene_name not in cls.gene_info_dict: chrom, translated_seq, genomic_pos, strand = get_cds_seq_pos_from_fasta( fasta_file_name @@ -242,28 +245,36 @@ def from_gene_name(cls, gene_name, ref_version: str = "GRCh38"): cds.chrom = cls.gene_info_dict[gene_name]["chrom"] cds.translated_seq = deepcopy(cls.gene_info_dict[gene_name]["translated_seq"]) cds.genomic_pos = cls.gene_info_dict[gene_name]["genomic_pos"] - cds.nt = cds.gene_info_dict[gene_name]["translated_seq"] + cds.nt = cds.gene_info_dict[gene_name]["translated_seq"] # in sense strand + # print(cds.gene_name + ":" + "".join(cds.nt)) + # if cds.strand == -1: + # cds.nt = revcomp(cds.nt) cds.pos = np.array(cds.genomic_pos) cds.edited_nt = deepcopy(cds.nt) return cds - def translate(self): - if self.strand == -1: - self.edited_nt = revcomp(self.edited_nt) - self.aa = _translate(self.edited_nt, codon_map) + # def translate(self): + # if self.strand == -1: + # self.edited_nt = revcomp(self.edited_nt) + # self.aa = _translate(self.edited_nt, codon_map) def _get_relative_nt_pos(self, absolute_pos): + """0-based relative position""" nt_relative_pos = np.where(self.pos == absolute_pos)[0] assert len(nt_relative_pos) <= 1, nt_relative_pos return nt_relative_pos.astype(int).item() if nt_relative_pos else -1 def _edit_pos_to_aa_pos(self, edit_pos): + """0-based nt position. Adds in sense direction, needs to be reversed for antisense gene""" nt_relative_pos = self._get_relative_nt_pos(edit_pos) if nt_relative_pos != -1: self.edited_aa_pos.add(nt_relative_pos // 3) + return nt_relative_pos def edit_single(self, edit_str): + """Add a mutation induced by a single `edit_str` to CDS. + For the negative CDS, nt and edited_nt are antisense.""" edit = Edit.from_str(edit_str) rel_pos = self._edit_pos_to_aa_pos(edit.pos) if edit.strand == "-": @@ -285,13 +296,16 @@ def edit_single(self, edit_str): ) ) raise RefBaseMismatchException( - f"{self.gene_name + ';' if hasattr(self, 'gene_name') else ''}ref:{self.nt[rel_pos]} at pos {rel_pos}, got edit {edit}" + f"{self.gene_name + ';' if hasattr(self, 'gene_name') else ''}ref:{self.nt[rel_pos]} at pos {rel_pos}, got edit {edit}. Gene sequence: {''.join(self.nt)}" ) else: self.edited_nt[rel_pos] = alt_base if alt_base == "-": # frameshift # self.edited_nt.pop(rel_pos) - self.edited_aa_pos.update(list(range(rel_pos, len(self.nt) // 3))) + if self.strand == 1: + self.edited_aa_pos.update(list(range(rel_pos, len(self.nt) // 3))) + else: + self.edited_aa_pos.update(list(range(0, rel_pos))) def edit_allele(self, allele_str): if isinstance(allele_str, Allele): @@ -301,7 +315,13 @@ def edit_allele(self, allele_str): for edit_str in edit_strs: self.edit_single(edit_str) if "-" in self.edited_nt: - self.edited_nt.remove("-") + self.edited_nt = [nt for nt in self.edited_nt if nt != "-"] + if self.strand == -1: + # Reverse logged edited positions as it was in the sense direction. + # rev_pos = self.edited_aa_pos[::-1] + self.edited_aa_pos = {len(self.nt) // 3 - 1 - r for r in self.edited_aa_pos} + self.nt = revcomp(self.nt) + self.edited_nt = revcomp(self.edited_nt) def get_aa_change( self, allele_str, include_synonymous=True @@ -311,10 +331,27 @@ def get_aa_change( mutations = CodingNoncodingAllele() mutations.nt_allele.update(self.edits_noncoding) for edited_aa_pos in self.edited_aa_pos: - ref_aa = _translate_single_codon(self.nt, edited_aa_pos) - mt_aa = _translate_single_codon(self.edited_nt, edited_aa_pos) - if mt_aa == "_": - return "translation error" + try: + # print("".join(self.nt)) + # print((3 * edited_aa_pos), (3 * edited_aa_pos + 3)) + ref_aa = _translate_single_codon( + self.nt[(3 * edited_aa_pos) : (3 * edited_aa_pos + 3)] + ) + except ValueError as e: + print(f"Translation mismatch in translating ref for {allele_str}: {e}") + try: + # print("".join(self.nt)) + print(self.edited_nt[(3 * edited_aa_pos) : (3 * edited_aa_pos + 3)]) + mt_aa = _translate_single_codon( + self.edited_nt[(3 * edited_aa_pos) : (3 * edited_aa_pos + 3)] + ) + except ValueError as e: + print( + f"Translation mismatch in translating mutated gene for {allele_str}: {e}" + ) + except IndexError as e: + print(f"End of gene reached by frameshift {allele_str}: {e}") + mt_aa = ">" if not include_synonymous and ref_aa == mt_aa: continue mutations.aa_allele.add( diff --git a/bean/annotate/utils.py b/bean/annotate/utils.py index 436a012..97fe49b 100644 --- a/bean/annotate/utils.py +++ b/bean/annotate/utils.py @@ -20,7 +20,7 @@ debug = logging.debug info = logging.info -complement_base = {"A": "T", "T": "A", "C": "G", "G": "C"} +complement_base = {"A": "T", "T": "A", "C": "G", "G": "C", "-": "-"} def revcomp(nt_list: List[str]): @@ -97,7 +97,9 @@ def get_mane_transcript_id(gene_name: str): return mane_transcript_id, id_version -def get_exons_from_transcript_id(transcript_id: str, id_version: int, ref_version: str = "GRCh38"): +def get_exons_from_transcript_id( + transcript_id: str, id_version: int, ref_version: str = "GRCh38" +): """ Retrieves the exons and the start position of the coding sequence (CDS) for a given transcript ID and version. @@ -114,7 +116,9 @@ def get_exons_from_transcript_id(transcript_id: str, id_version: int, ref_versio if transcript_json["count"] != 1: if transcript_json["count"] > 1: api_url = f"http://tark.ensembl.org/api/transcript/?stable_id={transcript_id}&stable_id_version={id_version}&assembly_name={ref_version}&expand=exons" - response = requests.get(api_url, headers={"Content-Type": "application/json"}) + response = requests.get( + api_url, headers={"Content-Type": "application/json"} + ) transcript_json = response.json() if transcript_json["count"] != 1: raise ValueError( diff --git a/bean/framework/AminoAcidEdit.py b/bean/framework/AminoAcidEdit.py index b1d7c36..57e573c 100644 --- a/bean/framework/AminoAcidEdit.py +++ b/bean/framework/AminoAcidEdit.py @@ -244,6 +244,9 @@ def get_jaccards(self, allele_list: Iterable[CodingNoncodingAllele]): nt_jaccards = np.array(nt_jaccards) return (aa_jaccards, nt_jaccards) + def has_coding(self): + return len(self.aa_allele.edits) > 0 + def map_to_closest( self, allele_list, diff --git a/bean/mapping/GuideEditCounter.py b/bean/mapping/GuideEditCounter.py index f1d60e8..b0d3cbe 100644 --- a/bean/mapping/GuideEditCounter.py +++ b/bean/mapping/GuideEditCounter.py @@ -349,7 +349,7 @@ def _count_guide_edits( if "chrom" in self.screen.guides.columns.tolist(): chrom = self.screen.guides.chrom.iloc[matched_guide_idx] else: - chrom = None + raise ValueError("'chrom' column denoting gRNA chromosome location column not present in the input. Please check the input file.") read_guide_seq, read_guide_qual = self.get_guide_seq_qual( R1_record, len(ref_guide_seq) ) diff --git a/bin/bean-filter b/bin/bean-filter index 786c2b8..7e6f013 100644 --- a/bin/bean-filter +++ b/bin/bean-filter @@ -137,6 +137,8 @@ if __name__ == "__main__": get_mismatch_df().to_csv(f"{args.output_prefix}.translation_ref_mismatches.csv") allele_df_keys.append(filtered_key) + if not any(bdata.uns[filtered_key].aa_allele.map(lambda a: a.has_coding)): + warn("WARNING: No alleles have been translated. Check your gene input(s).") info(f"Filtered down to {len(bdata.uns[filtered_key])} alleles.") if ( diff --git a/tests/test_filter.py b/tests/test_filter.py index 2baaf55..e5e3f59 100644 --- a/tests/test_filter.py +++ b/tests/test_filter.py @@ -1,5 +1,6 @@ import pytest import subprocess +from bean.annotate.translate_allele import CDS @pytest.mark.order(11) @@ -65,3 +66,13 @@ def test_filter_tiling_screen_translate_genenames(): ) except subprocess.CalledProcessError as exc: raise exc + + +def test_translate_aa(): + abca1 = CDS.from_gene_name("ABCA1") + allele_str = "chr9:104903664:0:+:G>A" + assert str(abca1.get_aa_change(allele_str)) == "ABCA1:6:Q>*|", allele_str + + abca1 = CDS.from_gene_name("ABCA1") + allele_str = "chr9:104903664:0:-:C>T" + assert str(abca1.get_aa_change(allele_str)) == "ABCA1:6:Q>*|", allele_str From 755816a8df0de7056222d4f2e07b2c107dbb7063 Mon Sep 17 00:00:00 2001 From: jykr Date: Wed, 27 Mar 2024 11:56:17 -0400 Subject: [PATCH 009/107] require column for tiling screens only --- bean/annotate/translate_allele.py | 16 +++++++-- bean/mapping/GuideEditCounter.py | 57 ++++++++++++++++++++++--------- 2 files changed, 54 insertions(+), 19 deletions(-) diff --git a/bean/annotate/translate_allele.py b/bean/annotate/translate_allele.py index 927e395..842ccc8 100644 --- a/bean/annotate/translate_allele.py +++ b/bean/annotate/translate_allele.py @@ -1,5 +1,6 @@ +from doctest import set_unittest_reportflags import os -from typing import List, Iterable, Dict, Tuple, Collection +from typing import List, Iterable, Dict, Tuple, Collection, Sequence from copy import deepcopy import numpy as np import pandas as pd @@ -199,6 +200,11 @@ def __init__(self): self.edited_nt: List[str] = [] self.nt: List[str] = [] self.strand: int = 1 + self.gene_name: str = "" + self.chrom: str = "" + self.translated_seq: List[str] = [] + self.pos: np.ndarray = None + self.genomic_pos: Sequence = [] @classmethod def from_fasta(cls, fasta_file_name, gene_name, suppressMessage=True): @@ -338,7 +344,10 @@ def get_aa_change( self.nt[(3 * edited_aa_pos) : (3 * edited_aa_pos + 3)] ) except ValueError as e: - print(f"Translation mismatch in translating ref for {allele_str}: {e}") + print( + f"Translation mismatch in translating ref for {allele_str}: {e}. Check the input .fasta or genome version used for the reporter. Ignoring this allele." + ) + continue try: # print("".join(self.nt)) print(self.edited_nt[(3 * edited_aa_pos) : (3 * edited_aa_pos + 3)]) @@ -347,8 +356,9 @@ def get_aa_change( ) except ValueError as e: print( - f"Translation mismatch in translating mutated gene for {allele_str}: {e}" + f"Translation mismatch in translating mutated gene for {allele_str}: {e}. Check the input .fasta or genome version used for the reporter. Ignoring this allele." ) + continue except IndexError as e: print(f"End of gene reached by frameshift {allele_str}: {e}") mt_aa = ">" diff --git a/bean/mapping/GuideEditCounter.py b/bean/mapping/GuideEditCounter.py index b0d3cbe..5345922 100644 --- a/bean/mapping/GuideEditCounter.py +++ b/bean/mapping/GuideEditCounter.py @@ -47,7 +47,8 @@ class NoReadsAfterQualityFiltering(Exception): strand_str_to_int = {"neg": -1, "pos": 1, "-": -1, "+": 1} -base_revcomp = {"A":"T", "T":"A", "G":"C", "C":"G"} +base_revcomp = {"A": "T", "T": "A", "G": "C", "C": "G"} + def _get_stranded_guide_offset(strand: int, start_pos: int, guide_len: int) -> int: if strand == -1: @@ -334,9 +335,6 @@ def _write_guide_self_alleles(self): self.screen.uns["guide_edit_counts"].guide ] - def _get_guide_counts_bcmatch(self): - NotImplemented - def _count_guide_edits( self, matched_guide_idx, R1_record: SeqIO.SeqRecord, single_base_qual_cutoff=30 ): @@ -349,7 +347,12 @@ def _count_guide_edits( if "chrom" in self.screen.guides.columns.tolist(): chrom = self.screen.guides.chrom.iloc[matched_guide_idx] else: - raise ValueError("'chrom' column denoting gRNA chromosome location column not present in the input. Please check the input file.") + if self.screen.tiling: + raise ValueError( + "'chrom' column denoting gRNA chromosome location column not present in the input. Please check the input file." + ) + else: + chrom = None read_guide_seq, read_guide_qual = self.get_guide_seq_qual( R1_record, len(ref_guide_seq) ) @@ -455,7 +458,9 @@ def _count_reporter_edits( guide_allele: Allele from baseedit in gRNA spacer sequence when paired with guide allele. """ ref_reporter_seq = self.screen.guides.reporter.iloc[matched_guide_idx] - read_reporter_seq, read_reporter_qual = self.get_reporter_seq_qual(R2_record, R2_start) + read_reporter_seq, read_reporter_qual = self.get_reporter_seq_qual( + R2_record, R2_start + ) guide_strand, offset = self._get_strand_offset_from_guide_index( matched_guide_idx @@ -516,9 +521,11 @@ def _get_guide_counts_bcmatch_semimatch( R1_seq = str(r1.seq) R2_seq = str(r2.seq) - bc_match, semimatch, R2_start = self._match_read_to_sgRNA_bcmatch_semimatch( - R1_seq, R2_seq - ) + ( + bc_match, + semimatch, + R2_start, + ) = self._match_read_to_sgRNA_bcmatch_semimatch(R1_seq, R2_seq) if len(bc_match) == 0: if len(semimatch) == 0: # no guide match if self.keep_intermediate: @@ -556,10 +563,19 @@ def _get_guide_counts_bcmatch_semimatch( # TODO: what if reporter seq doesn't match barcode & guide? if self.count_guide_reporter_alleles: self._count_reporter_edits( - matched_guide_idx, R1_seq, r2, R2_start = R2_start, guide_allele=guide_allele + matched_guide_idx, + R1_seq, + r2, + R2_start=R2_start, + guide_allele=guide_allele, ) else: - self._count_reporter_edits(matched_guide_idx, R1_seq, r2, R2_start = R2_start, ) + self._count_reporter_edits( + matched_guide_idx, + R1_seq, + r2, + R2_start=R2_start, + ) tqdm_reads.postfix = f"n_read={self.bcmatch}" tqdm_reads.update() @@ -644,9 +660,11 @@ def get_reporter_seq(self, R1_seq, R2_seq): R2_seq[self.guide_bc_len : (self.guide_bc_len + self.reporter_length)] ) - def get_reporter_seq_qual(self, R2_record: SeqIO.SeqRecord, R2_start = 0): + def get_reporter_seq_qual(self, R2_record: SeqIO.SeqRecord, R2_start=0): seq = R2_record[ - (R2_start + self.guide_bc_len) : (R2_start + self.guide_bc_len + self.reporter_length) + (R2_start + self.guide_bc_len) : ( + R2_start + self.guide_bc_len + self.reporter_length + ) ].reverse_complement() return (str(seq.seq), seq.letter_annotations["phred_quality"]) @@ -654,13 +672,20 @@ def get_barcode(self, R1_seq, R2_seq): if self.barcode_start_seq != "": barcode_start_idx = R2_seq.replace( base_revcomp[self.base_edited_from], base_revcomp[self.base_edited_to] - ).find(revcomp(self.barcode_start_seq).replace(base_revcomp[self.base_edited_from], base_revcomp[self.base_edited_to])) + ).find( + revcomp(self.barcode_start_seq).replace( + base_revcomp[self.base_edited_from], + base_revcomp[self.base_edited_to], + ) + ) if barcode_start_idx == -1: return -1, "" barcode_start_idx += len(self.barcode_start_seq) - else: + else: barcode_start_idx = 0 - return barcode_start_idx, revcomp(R2_seq[barcode_start_idx: (barcode_start_idx + self.guide_bc_len)]) + return barcode_start_idx, revcomp( + R2_seq[barcode_start_idx : (barcode_start_idx + self.guide_bc_len)] + ) def _match_read_to_sgRNA_bcmatch_semimatch(self, R1_seq: str, R2_seq: str): # This should be adjusted for each experimental recipes. From 657d595552325c0b029bb6e480f1c674849947f4 Mon Sep 17 00:00:00 2001 From: jykr Date: Wed, 27 Mar 2024 11:57:23 -0400 Subject: [PATCH 010/107] require chrom column for tiling screens only --- bean/annotate/translate_allele.py | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/bean/annotate/translate_allele.py b/bean/annotate/translate_allele.py index 842ccc8..b82414a 100644 --- a/bean/annotate/translate_allele.py +++ b/bean/annotate/translate_allele.py @@ -350,7 +350,7 @@ def get_aa_change( continue try: # print("".join(self.nt)) - print(self.edited_nt[(3 * edited_aa_pos) : (3 * edited_aa_pos + 3)]) + # print(self.edited_nt[(3 * edited_aa_pos) : (3 * edited_aa_pos + 3)]) mt_aa = _translate_single_codon( self.edited_nt[(3 * edited_aa_pos) : (3 * edited_aa_pos + 3)] ) From 87a35232d9129c9813005b68d1476bb1982244e6 Mon Sep 17 00:00:00 2001 From: jykr Date: Thu, 28 Mar 2024 15:30:23 -0400 Subject: [PATCH 011/107] fix translation steps --- bean/annotate/_supporting_fn.py | 17 ++++++++++------- bean/annotate/translate_allele.py | 6 +++--- bean/annotate/utils.py | 6 ++++++ bean/framework/ReporterScreen.py | 6 +++--- bin/bean-filter | 12 ++++++++++++ 5 files changed, 34 insertions(+), 13 deletions(-) diff --git a/bean/annotate/_supporting_fn.py b/bean/annotate/_supporting_fn.py index 127964b..e3b34f5 100644 --- a/bean/annotate/_supporting_fn.py +++ b/bean/annotate/_supporting_fn.py @@ -1,5 +1,5 @@ from copy import deepcopy -from typing import List, Tuple +from typing import List, Tuple, Union from tqdm.auto import tqdm from ..framework.Edit import Edit, Allele from ..framework.AminoAcidEdit import CodingNoncodingAllele @@ -43,14 +43,18 @@ def filter_allele_by_pos( def filter_allele_by_base( allele: Allele, allowed_base_changes: List[Tuple] = None, - allowed_ref_base: str = None, - allowed_alt_base: str = None, + allowed_ref_base: Union[List, str] = None, + allowed_alt_base: Union[List, str] = None, ): """ Filter alleles based on position and return the filtered allele and number of filtered edits. """ filtered_edits = 0 + if isinstance(allowed_ref_base, str): + allowed_ref_base = [allowed_ref_base] + if isinstance(allowed_alt_base, str): + allowed_alt_base = [allowed_alt_base] if ( not (allowed_ref_base is None and allowed_alt_base is None) + (allowed_base_changes is None) @@ -64,15 +68,15 @@ def filter_allele_by_base( allele.edits.remove(edit) elif not allowed_ref_base is None: for edit in allele.edits.copy(): - if edit.ref_base != allowed_ref_base: + if edit.ref_base not in allowed_ref_base: filtered_edits += 1 allele.edits.remove(edit) - elif not allowed_alt_base is None and edit.alt_base != allowed_alt_base: + elif not allowed_alt_base is None and edit.alt_base not in allowed_alt_base: filtered_edits += 1 allele.edits.remove(edit) else: for edit in allele.edits.copy(): - if edit.alt_base != allowed_alt_base: + if edit.alt_base not in allowed_alt_base: filtered_edits += 1 allele.edits.remove(edit) return (allele, filtered_edits) @@ -145,7 +149,6 @@ def _map_alleles_to_filtered( raw_allele_counts.groupby("guide"), desc="Mapping alleles to closest filtered alleles", ): - guide_filtered_allele_counts = filtered_allele_counts.loc[ filtered_allele_counts.guide == guide, : ].set_index(allele_col) diff --git a/bean/annotate/translate_allele.py b/bean/annotate/translate_allele.py index b82414a..6310717 100644 --- a/bean/annotate/translate_allele.py +++ b/bean/annotate/translate_allele.py @@ -175,8 +175,8 @@ def _translate_single_codon( ) -> str: # nt_seq_string: str, aa_pos: int) -> str: """Translate `aa_pos`-th codon of `nt_seq_string`.""" if len(codon) != 3: - print("reached the end of CDS, frameshift.") - return "/" + raise ValueError("reached the end of CDS, frameshift.") + return ">" try: codon = "".join(codon) return codon_map[codon] @@ -302,7 +302,7 @@ def edit_single(self, edit_str): ) ) raise RefBaseMismatchException( - f"{self.gene_name + ';' if hasattr(self, 'gene_name') else ''}ref:{self.nt[rel_pos]} at pos {rel_pos}, got edit {edit}. Gene sequence: {''.join(self.nt)}" + f"{self.gene_name + ';' if hasattr(self, 'gene_name') else ''}ref:{self.nt[rel_pos]} at pos {rel_pos}, got edit {edit}." ) else: self.edited_nt[rel_pos] = alt_base diff --git a/bean/annotate/utils.py b/bean/annotate/utils.py index 97fe49b..2252187 100644 --- a/bean/annotate/utils.py +++ b/bean/annotate/utils.py @@ -280,6 +280,12 @@ def parse_args(): help="Only consider edit within window provided by (edit-start-pos, edit-end-pos). If this flag is not provided, `--edit-start-pos` and `--edit-end-pos` flags are ignored.", action="store_true", ) + parser.add_argument( + "--keep-indels", + "-i", + help="Include indels.", + action="store_true", + ) parser.add_argument( "--filter-target-basechange", "-b", diff --git a/bean/framework/ReporterScreen.py b/bean/framework/ReporterScreen.py index 37631d5..ce56061 100644 --- a/bean/framework/ReporterScreen.py +++ b/bean/framework/ReporterScreen.py @@ -333,7 +333,7 @@ def __getitem__(self, index): lambda slist: ".".join(slist) ) new_uns[k] = df.loc[guides_include, adata.var._rc.unique()] - #adata.var.pop("_rc") + # adata.var.pop("_rc") else: new_uns[k] = df.loc[guides_include, adata.var.rep.unique()] if not isinstance(df, pd.DataFrame): @@ -697,8 +697,8 @@ def filter_allele_counts_by_pos( def filter_allele_counts_by_base( self, - ref_base="A", - alt_base="G", + ref_base: Union[List, str] = "A", + alt_base: Union[List, str] = "G", allele_uns_key="allele_counts", map_to_filtered=True, jaccard_threshold: float = 0.5, diff --git a/bin/bean-filter b/bin/bean-filter index 7e6f013..128ef42 100644 --- a/bin/bean-filter +++ b/bin/bean-filter @@ -100,6 +100,18 @@ if __name__ == "__main__": allele_df_keys.append(filtered_key) info(f"Filtered down to {len(bdata.uns[filtered_key])} alleles.") + if len(bdata.uns[allele_df_keys[-1]]) > 0 and not args.keep_indels: + filtered_key = f"{allele_df_keys[-1]}_noindels" + info(f"Filtering out indels...") + bdata.uns[filtered_key] = bdata.filter_allele_counts_by_base( + ["A", "T", "G", "C"], + ["A", "T", "G", "C"], + map_to_filtered=False, + allele_uns_key=allele_df_keys[-1], + ).reset_index(drop=True) + info(f"Filtered down to {len(bdata.uns[filtered_key])} alleles.") + allele_df_keys.append(filtered_key) + if len(bdata.uns[allele_df_keys[-1]]) > 0 and args.filter_target_basechange: filtered_key = ( f"{allele_df_keys[-1]}_{bdata.base_edited_from}.{bdata.base_edited_to}" From 97d464e37a22324afeeb330731f86295cc11807f Mon Sep 17 00:00:00 2001 From: jykr Date: Thu, 28 Mar 2024 15:41:55 -0400 Subject: [PATCH 012/107] fix allele-df-key for added indel fitlering --- tests/test_run.py | 10 +++++----- 1 file changed, 5 insertions(+), 5 deletions(-) diff --git a/tests/test_run.py b/tests/test_run.py index 3c877b1..734b8ff 100644 --- a/tests/test_run.py +++ b/tests/test_run.py @@ -82,7 +82,7 @@ def test_run_variant_uniform_negctrl(): @pytest.mark.order(22) def test_run_tiling_wo_negctrl(): - cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad --scale-by-acc --acc-bw-path tests/data/accessibility_signal.bw -o tests/test_res/tiling/ --allele-df-key allele_counts_spacer_0_19_A.G_translated_prop0.1_0.3 --control-guide-tag None --repguide-mask None --n-iter 10" + cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad --scale-by-acc --acc-bw-path tests/data/accessibility_signal.bw -o tests/test_res/tiling/ --control-guide-tag None --repguide-mask None --n-iter 10" try: subprocess.check_output( cmd, @@ -95,7 +95,7 @@ def test_run_tiling_wo_negctrl(): @pytest.mark.order(23) def test_run_tiling_with_wo_negctrl_noacc(): - cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad -o tests/test_res/tiling/ --allele-df-key allele_counts_spacer_0_19_A.G_translated_prop0.1_0.3 --control-guide-tag None --repguide-mask None --n-iter 10" + cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad -o tests/test_res/tiling/ --control-guide-tag None --repguide-mask None --n-iter 10" try: subprocess.check_output( cmd, @@ -108,7 +108,7 @@ def test_run_tiling_with_wo_negctrl_noacc(): @pytest.mark.order(23) def test_run_tiling_with_wo_negctrl_uniform(): - cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad -o tests/test_res/tiling/ --uniform-edit --allele-df-key allele_counts_spacer_0_19_A.G_translated_prop0.1_0.3 --control-guide-tag None --repguide-mask None --n-iter 10" + cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad -o tests/test_res/tiling/ --uniform-edit --allele-df-key allele_counts_spacer_0_19_noindels_A.G_translated_prop0.1_0.3 --control-guide-tag None --repguide-mask None --n-iter 10" try: subprocess.check_output( cmd, @@ -134,7 +134,7 @@ def test_run_tiling_negctrl_allelekey(): @pytest.mark.order(25) def test_run_tiling_with_negctrl_noacc(): - cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad -o tests/test_res/tiling/ --allele-df-key allele_counts_spacer_0_19_A.G_translated_prop0.1_0.3 --fit-negctrl --negctrl-col strand --negctrl-col-value neg --control-guide-tag neg --repguide-mask None --n-iter 10" + cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad -o tests/test_res/tiling/ --allele-df-key allele_counts_spacer_0_19_noindels_A.G_translated_prop0.1_0.3 --fit-negctrl --negctrl-col strand --negctrl-col-value neg --control-guide-tag neg --repguide-mask None --n-iter 10" try: subprocess.check_output( cmd, @@ -147,7 +147,7 @@ def test_run_tiling_with_negctrl_noacc(): @pytest.mark.order(26) def test_run_tiling_with_negctrl_uniform(): - cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad -o tests/test_res/tiling/ --uniform-edit --allele-df-key allele_counts_spacer_0_19_A.G_translated_prop0.1_0.3 --fit-negctrl --negctrl-col strand --negctrl-col-value neg --control-guide-tag neg --repguide-mask None --n-iter 10" + cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad -o tests/test_res/tiling/ --uniform-edit --allele-df-key allele_counts_spacer_0_19_noindels_A.G_translated_prop0.1_0.3 --fit-negctrl --negctrl-col strand --negctrl-col-value neg --control-guide-tag neg --repguide-mask None --n-iter 10" try: subprocess.check_output( cmd, From 25c0a1b5a5452c1d9492a5c7b6d51fe34cd3ab88 Mon Sep 17 00:00:00 2001 From: jykr Date: Thu, 28 Mar 2024 17:30:30 -0400 Subject: [PATCH 013/107] Unify control condition arguments, add sanity check for control condition samples --- README.md | 11 ++++++----- bean/framework/Edit.py | 6 +++--- bean/framework/ReporterScreen.py | 13 +++++++++---- bean/model/run.py | 20 +++++++++++--------- bean/qc/utils.py | 2 +- bin/bean-qc | 2 +- bin/bean-run | 22 +++++++++++++--------- notebooks/profile_editing_preference.ipynb | 8 ++++++-- setup.py | 2 +- 9 files changed, 51 insertions(+), 35 deletions(-) diff --git a/README.md b/README.md index b78a3f2..9cf9992 100644 --- a/README.md +++ b/README.md @@ -201,9 +201,9 @@ Above command produces `prefix_editing_preference.[html,ipynb]` as editing prefe ### Parameters * `-o`, `--output-prefix` (default: `None`): Output prefix of editing pattern report (prefix.html, prefix.ipynb). If not provided, base name of `bdata_path` is used. * `--replicate-col` (default: `"rep"`): Column name in `bdata.samples` that describes replicate ID. - * `--condition-col` (default: `"bin"`): Column name in `bdata.samples` that describes experimental condition. (sorting bin, time, etc.) + * `--condition-col` (default: `"condition"`): Column name in `bdata.samples` that describes experimental condition. (sorting bin, time, etc.) * `--pam-col` (default: `None`): Column name describing PAM of each gRNA in `bdata.guides`. - * `--control-condition` (default: `"bulk"`): Control condition where editing preference would be profiled at. Pre-filters data where `bdata.samples[condition_col] == control_condition`. + * `--control-condition` (default: `"bulk"`): Control condition where editing preference would be profiled at. Pre-filters data where `bdata.samples[condition_col] == control_condition`. DO NOT use plasmid library as control here where we do not expect editing. * `-w`, `--window-length` (default: `6`): Window length of editing window of maximal editing efficiency to be identified. This window is used to quantify context specificity within the window. @@ -215,7 +215,7 @@ bean-qc \ my_sorting_screen.h5ad `# Input ReporterScreen .h5ad file path` \ -o my_sorting_screen_masked.h5ad `# Output ReporterScreen .h5ad file path` \ -r qc_report_my_sorting_screen `# Prefix for QC report` \ - + --ctrl-cond presort `# "condition" column in the control sample before selection. Mean gRNA editing rates in these samples are reported. ` \ # Inspect the output qc_report_my_sorting_screen.html to tweak QC threshold bean-qc \ @@ -281,9 +281,9 @@ Note that these arguements will change the way the QC metrics are calculated for Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time, etc.) ###### Editing rate calculation - * `--ctrl-cond CTRL_COND` + * `--control-condition CTRL_COND` Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate - to be calculated + to be calculated. Default is `None`, which considers all samples. * `--rel-pos-is-reporter` Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If `False`, those are relative to spacer position. @@ -453,6 +453,7 @@ Above command produces * `--guide-activity-col`: Column in `bdata.guides` DataFrame showing the editing rate estimated via external tools. * Sample annotations (`bdata.samples` column keys) * `--condition-column` (default: `condition`): Column key in `bdata.samples` that describes experimental condition. + * `--control-condition` (default: `bulk`): Value in `bdata.samples[condition_col]` that indicates control experimental condition. * `-uq`, `--sorting-bin-upper-quantile-column` (default: `upper_quantile`): Column name with upper quantile values of each sorting bin in bdata.samples * `-lq`, `--sorting-bin-lower-quantile-column` (default: `lower_quantile`): Column name with lower quantile values of each sorting bin in bdata.samples diff --git a/bean/framework/Edit.py b/bean/framework/Edit.py index 5abba2e..afd0c26 100644 --- a/bean/framework/Edit.py +++ b/bean/framework/Edit.py @@ -68,7 +68,7 @@ def from_str(cls, edit_str): # pos:strand:start>end def match_str(cls, edit_str): if isinstance(edit_str, Edit): return True - pattern = r"(((chr)?\d+|nan):)?-?\d+:-?\d+:[+-]:[A-Z*-]>[A-Z*-]" + pattern = r"(((chr)?\w+|nan):)?-?\d+:-?\d+:[+-]:[A-Z*-]>[A-Z*-]" pattern2 = r"[\w*]!-?\d+:-?\d+:[+-]:[A-Z*-]>[A-Z*-]" return re.fullmatch(pattern, edit_str) or re.fullmatch(pattern2, edit_str) @@ -175,11 +175,11 @@ def get_range(self): min(edit.pos for edit in self.edits), max(edit.pos for edit in self.edits), ) - + def set_uid(self, uid): self.edits = {edit.set_uid(uid) for edit in self.edits} return self - + def get_uid(self): uid = None if ( diff --git a/bean/framework/ReporterScreen.py b/bean/framework/ReporterScreen.py index ce56061..d470de5 100644 --- a/bean/framework/ReporterScreen.py +++ b/bean/framework/ReporterScreen.py @@ -476,7 +476,7 @@ def get_guide_edit_rate( return_result=False, count_layer="X_bcmatch", edit_layer="edits", - unsorted_condition_label="bulk", + unsorted_condition_label=None, ): """ prior_weight: @@ -497,9 +497,14 @@ def get_guide_edit_rate( num_targetable_sites = self.guides.sequence.map( lambda s: s[editable_base_start:editable_base_end].count(edited_base) ) - bulk_idx = np.where( - self.samples.index.astype(str).map(lambda s: unsorted_condition_label in s) - )[0] + if unsorted_condition_label is not None: + bulk_idx = np.where( + self.samples.index.astype(str).map( + lambda s: unsorted_condition_label in s + ) + )[0] + else: + bulk_idx = np.arange(0, len(self.samples)).astype(int) if prior_weight is None: prior_weight = 1 diff --git a/bean/model/run.py b/bean/model/run.py index e09dcd5..26b24a6 100644 --- a/bean/model/run.py +++ b/bean/model/run.py @@ -113,7 +113,7 @@ def parse_args(): help="Column key in `bdata.samples` that describes time elapsed.", ) parser.add_argument( - "--control-condition-label", + "--control-condition", default="bulk", type=str, help="Value in `bdata.samples[condition_col]` that indicates control experimental condition.", @@ -334,19 +334,21 @@ def check_args(args, bdata): raise ValueError( f"Condition column set by `--replicate-col` {args.replicate_col} not in ReporterScreen.samples.columns:{bdata.samples.columns}. Check your input." ) - if ( - args.control_guide_tag is not None - and not bdata.guides.index.map(lambda s: args.control_guide_tag in s).any() - ): - raise ValueError( - f"Negative control guide label {args.control_guide_tag} provided by `--control-guide-tag` doesn't appear in any of the guide names. Check your input." - ) + if args.control_guide_tag is not None: + if args.library_design == "variant": + raise ValueError( + "`--control-guide-tag` is not used for the variant mode. Make sure you provide the separate `target` column for negative control guide that targets different negative control variant." + ) + elif not bdata.guides.index.map(lambda s: args.control_guide_tag in s).any(): + raise ValueError( + f"Negative control guide label {args.control_guide_tag} provided by `--control-guide-tag` doesn't appear in any of the guide names. Check your input." + ) if args.alpha_if_overdispersion_fitting_fails is not None: try: b0, b1 = args.alpha_if_overdispersion_fitting_fails.split(",") args.popt = (float(b0), float(b1)) except TypeError as e: - raise e( + raise ValueError( f"Input --alpha-if-overdispersion-fitting-fails {args.alpha_if_overdispersion_fitting_fails} is malformatted! Provide [float].[float] format." ) else: diff --git a/bean/qc/utils.py b/bean/qc/utils.py index 3297d11..6ed50cf 100644 --- a/bean/qc/utils.py +++ b/bean/qc/utils.py @@ -148,7 +148,7 @@ def parse_args(): default="top,bot", ) input_parser.add_argument( - "--ctrl-cond", + "--control-condition", help="Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate to be calculated", type=str, default="bulk", diff --git a/bin/bean-qc b/bin/bean-qc index 970cb19..a6880bf 100644 --- a/bin/bean-qc +++ b/bin/bean-qc @@ -31,7 +31,7 @@ def main(): condition_label=args.condition_label, comp_cond1=args.lfc_cond1, comp_cond2=args.lfc_cond2, - ctrl_cond=args.ctrl_cond, + ctrl_cond=args.control_condition, exp_id=args.out_report_prefix, recalculate_edits=~args.dont_recalculate_edits, base_edit_data=args.base_edit_data, diff --git a/bin/bean-run b/bin/bean-run index 550d0ec..b4eb5f4 100644 --- a/bin/bean-run +++ b/bin/bean-run @@ -85,7 +85,7 @@ def main(args, bdata): use_const_pi=args.const_pi, condition_column=args.condition_col, time_column=args.time_col, - control_condition=args.control_condition_label, + control_condition=args.control_condition, control_can_be_selected=args.include_control_condition_for_inference, allele_df_key=args.allele_df_key, control_guide_tag=args.control_guide_tag, @@ -101,7 +101,9 @@ def main(args, bdata): if "edit_rate" not in ndata.screen.guides.columns: ndata.screen.get_edit_from_allele() ndata.screen.get_edit_mat_from_uns(rel_pos_is_reporter=True) - ndata.screen.get_guide_edit_rate(unsorted_condition_label=args.control_condition_label) + ndata.screen.get_guide_edit_rate( + unsorted_condition_label=args.control_condition + ) target_info_df = _get_guide_target_info( ndata.screen, args, cols_include=[args.negctrl_col] ) @@ -182,16 +184,18 @@ def main(args, bdata): prefix=f"{prefix}/", suffix=args.result_suffix, guide_index=guide_index, - guide_acc=ndata.guide_accessibility.cpu().numpy() - if hasattr(ndata, "guide_accessibility") - and ndata.guide_accessibility is not None - else None, + guide_acc=( + ndata.guide_accessibility.cpu().numpy() + if hasattr(ndata, "guide_accessibility") + and ndata.guide_accessibility is not None + else None + ), adjust_confidence_by_negative_control=args.adjust_confidence_by_negative_control, adjust_confidence_negatives=adj_negctrl_idx, sd_is_fitted=(args.selection == "sorting"), - sample_covariates=ndata.sample_covariates - if hasattr(ndata, "sample_covariates") - else None, + sample_covariates=( + ndata.sample_covariates if hasattr(ndata, "sample_covariates") else None + ), ) info("Done!") diff --git a/notebooks/profile_editing_preference.ipynb b/notebooks/profile_editing_preference.ipynb index db1eaa5..f806f5c 100644 --- a/notebooks/profile_editing_preference.ipynb +++ b/notebooks/profile_editing_preference.ipynb @@ -52,7 +52,7 @@ "source": [ "bdata_path = \"../../../bean_manuscript/workflow/results/mapped/LDLRCDS/bean_count_LDLRCDS_combined.h5ad\"\n", "replicate_col = \"rep\"\n", - "condition_col = \"bin\"\n", + "condition_col = \"condition\"\n", "control_condition = \"bulk\"\n", "output_prefix = \"editing pattern\"\n", "max_editing_window_length = 6\n", @@ -97,7 +97,11 @@ } ], "source": [ - "cdata_bulk = cdata[:,cdata.samples[condition_col] == control_condition]" + "cdata_bulk = cdata[:,cdata.samples[condition_col] == control_condition]\n", + "if len(cdata_bulk) == 0:\n", + " raise ValueError(\n", + " f\"No samples with bdata.samples['{condition_col}'] == {control_condition}\"\n", + " )" ] }, { diff --git a/setup.py b/setup.py index 4745dcb..5e00fcf 100644 --- a/setup.py +++ b/setup.py @@ -8,7 +8,7 @@ setup( name="crispr-bean", - version="0.3.1", + version="1.0.0", python_requires=">=3.8.0", author="Jayoung Ryu", author_email="jayoung_ryu@g.harvard.edu", From e938d8c7311da793e6586cc97d444cf49f2d90bd Mon Sep 17 00:00:00 2001 From: jykr Date: Thu, 28 Mar 2024 17:44:07 -0400 Subject: [PATCH 014/107] debug argument name --- bean/model/run.py | 4 ++-- bean/plotting/editing_patterns.py | 6 +++--- 2 files changed, 5 insertions(+), 5 deletions(-) diff --git a/bean/model/run.py b/bean/model/run.py index 26b24a6..ae0c536 100644 --- a/bean/model/run.py +++ b/bean/model/run.py @@ -326,9 +326,9 @@ def check_args(args, bdata): raise ValueError( f"Condition column set by `--condition-col` {args.condition_col} not in ReporterScreen.samples.columns:{bdata.samples.columns}. Check your input." ) - if args.control_condition_label not in bdata.samples[args.condition_col].tolist(): + if args.control_condition not in bdata.samples[args.condition_col].tolist(): raise ValueError( - f"No sample has control label (set by `--control-condition-label`) {args.control_condition_label} in ReporterScreen.samples[{args.condition_col}]: {bdata.samples[args.condition_col]}. Check your input." + f"No sample has control label (set by `--control-condition`) {args.control_condition} in ReporterScreen.samples[{args.condition_col}]: {bdata.samples[args.condition_col]}. Check your input." ) if args.replicate_col not in bdata.samples.columns: raise ValueError( diff --git a/bean/plotting/editing_patterns.py b/bean/plotting/editing_patterns.py index 4f1ccd9..6454438 100644 --- a/bean/plotting/editing_patterns.py +++ b/bean/plotting/editing_patterns.py @@ -129,9 +129,9 @@ def plot_by_pos_context( f"C{target_base}": 0, f"T{target_base}": 0.2, } - edit_rates_df[ - "spacer_pos_ctxt" - ] = edit_rates_df.spacer_pos + edit_rates_df.context.map(context_to_offset_map) + edit_rates_df["spacer_pos_ctxt"] = ( + edit_rates_df.spacer_pos + edit_rates_df.context.map(context_to_offset_map) + ) fig, ax = plt.subplots(figsize=(6, 3)) sns.scatterplot( edit_rates_df.loc[ From 82773adb3231d3f585fe863f85033bf7cf75a781 Mon Sep 17 00:00:00 2001 From: jykr Date: Thu, 28 Mar 2024 18:00:14 -0400 Subject: [PATCH 015/107] change arg default value --- README.md | 2 +- bean/model/run.py | 2 +- 2 files changed, 2 insertions(+), 2 deletions(-) diff --git a/README.md b/README.md index 9cf9992..5267c9c 100644 --- a/README.md +++ b/README.md @@ -446,7 +446,7 @@ Above command produces * `--device`: Optionally use GPU if provided valid GPU device name (ex. cuda:0) * `--ignore-bcmatch` (default: `False`): If provided, even if the screen object has .X_bcmatch, ignore the count when fitting. * `--allele-df-key` (default: `allele_counts`): bdata.uns[allele_df_key] will be used as the allele count. - * `--control-guide-tag` (default: `CONTROL`): (Relevant in bean-run *tiling* mode) If this string is in guide name, treat each guide separately not to mix the position. Used for non-targeting negative controls. + * `--control-guide-tag` (default: `None`): (Relevant in bean-run *tiling* mode) If this string is in guide name, treat each guide separately not to mix the position. Used for non-targeting negative controls. * Guide annotations (`bdata.guides` column keys) * `--acc-col`: Column name in bdata.guides that specify raw ATAC-seq signal. * `--target-column` (default: `target`): Column key in `bdata.guides` that describes the target element of each guide. diff --git a/bean/model/run.py b/bean/model/run.py index ae0c536..b8613c4 100644 --- a/bean/model/run.py +++ b/bean/model/run.py @@ -234,7 +234,7 @@ def parse_args(): parser.add_argument( "--control-guide-tag", type=none_or_str, - default="CONTROL", + default=None, help="If this string is in guide name, treat each guide separately not to mix the position. Used for negative controls.", ) parser.add_argument( From 1f64961f66241e56b32b4681ec80c09eb3bdd893 Mon Sep 17 00:00:00 2001 From: jykr Date: Thu, 28 Mar 2024 19:35:27 -0400 Subject: [PATCH 016/107] fix documentation of time --- README.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/README.md b/README.md index 5267c9c..819fdbd 100644 --- a/README.md +++ b/README.md @@ -118,7 +118,7 @@ For FACS sorting screens: * `lower_quantile`: FACS sorting lower quantile For proliferation / survival screens: -* `time`: FACS sorting upper quantile +* `time`: Numeric time following the base editing of each sample. Also see examples for [FACS sorting screen](tests/data/sample_list.csv). From 368ba55b63698b10f422b65841c000a1bfb3b97b Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 15:59:44 -0400 Subject: [PATCH 017/107] change to replicate --- README.md | 6 +- bean/framework/ReporterScreen.py | 11 +- bean/model/run.py | 7 +- bean/plotting/utils.py | 2 +- bean/preprocessing/data_class.py | 63 +- bean/preprocessing/utils.py | 2 +- bean/qc/utils.py | 14 +- docs/ReporterScreen_api.ipynb | 664 ++++++++++----------- docs/sample_quality_report.ipynb | 8 +- notebooks/profile_editing_preference.ipynb | 2 +- notebooks/sample_quality_report.ipynb | 2 +- tests/data/tiling_mini_screen.h5ad | Bin 1295280 -> 1295408 bytes tests/data/tiling_mini_screen_missing.h5ad | Bin 1204704 -> 1204856 bytes tests/data/var_mini_samples.csv | 2 +- tests/data/var_mini_screen.h5ad | Bin 1452160 -> 1452312 bytes tests/data/var_mini_screen_missing.h5ad | Bin 1342328 -> 1342480 bytes 16 files changed, 400 insertions(+), 383 deletions(-) diff --git a/README.md b/README.md index 819fdbd..b459cfb 100644 --- a/README.md +++ b/README.md @@ -110,7 +110,7 @@ File should contain following columns with header. * `R1_filepath`: Path to read 1 `.fastq[.gz]` file * `R2_filepath`: Path to read 1 `.fastq[.gz]` file * `sample_id`: ID of sequencing sample -* `rep`: Replicate # of this sample (Should NOT contain `.`) +* `replicate`: Replicate # of this sample (Should NOT contain `.`) * `condition`: Name of the sorting bin (ex. `top`, `bot`), or label of timepoint (ex. `D5`, `D18`) For FACS sorting screens: @@ -200,7 +200,7 @@ Above command produces `prefix_editing_preference.[html,ipynb]` as editing prefe ### Parameters * `-o`, `--output-prefix` (default: `None`): Output prefix of editing pattern report (prefix.html, prefix.ipynb). If not provided, base name of `bdata_path` is used. - * `--replicate-col` (default: `"rep"`): Column name in `bdata.samples` that describes replicate ID. + * `--replicate-col` (default: `"replicate"`): Column name in `bdata.samples` that describes replicate ID. * `--condition-col` (default: `"condition"`): Column name in `bdata.samples` that describes experimental condition. (sorting bin, time, etc.) * `--pam-col` (default: `None`): Column name describing PAM of each gRNA in `bdata.guides`. * `--control-condition` (default: `"bulk"`): Control condition where editing preference would be profiled at. Pre-filters data where `bdata.samples[condition_col] == control_condition`. DO NOT use plasmid library as control here where we do not expect editing. @@ -402,7 +402,7 @@ bean-run sorting[survival] variant[tiling] my_sorting_screen_filtered.h5ad \ * `target_group [Optional]`: If negative control gRNA will be used, specify as "NegCtrl" in this column. * `sample_info_table.csv` should have following columns. * `sample_id`: ID of sequencing sample - * `rep`: Replicate # of this sample + * `replicate`: Replicate # of this sample * `bin`: Name of the sorting bin * `upper_quantile`: FACS sorting upper quantile * `lower_quantile`: FACS sorting lower quantile diff --git a/bean/framework/ReporterScreen.py b/bean/framework/ReporterScreen.py index d470de5..6ea0fad 100644 --- a/bean/framework/ReporterScreen.py +++ b/bean/framework/ReporterScreen.py @@ -327,7 +327,7 @@ def __getitem__(self, index): if k.startswith("repguide_mask"): if "sample_covariates" in adata.uns: adata.var["_rc"] = adata.var[ - ["rep"] + list(adata.uns["sample_covariates"]) + ["replicate"] + list(adata.uns["sample_covariates"]) ].values.tolist() adata.var["_rc"] = adata.var["_rc"].map( lambda slist: ".".join(slist) @@ -335,7 +335,14 @@ def __getitem__(self, index): new_uns[k] = df.loc[guides_include, adata.var._rc.unique()] # adata.var.pop("_rc") else: - new_uns[k] = df.loc[guides_include, adata.var.rep.unique()] + try: + new_uns[k] = df.loc[ + guides_include, adata.var["replicate"].unique() + ] + except KeyError as e: + raise ValueError( + f"Replicate column should be `replicate`. Modify your ReporterScreen.samples. Current columns: {adata.var.columns.tolist()}" + ) from e if not isinstance(df, pd.DataFrame): if k == "sample_covariates": new_uns[k] = df diff --git a/bean/model/run.py b/bean/model/run.py index b8613c4..d09fe1e 100644 --- a/bean/model/run.py +++ b/bean/model/run.py @@ -8,6 +8,7 @@ from functools import partial import pyro import bean.model.model as sorting_model +import bean.model.survival_model as survival_model # import bean.model.survival_model as survival_model @@ -127,7 +128,7 @@ def parse_args(): ) parser.add_argument( "--replicate-col", - default="rep", + default="replicate", type=str, help="Column key in `bdata.samples` that describes experimental replicates.", ) @@ -431,8 +432,8 @@ def run_inference( def identify_model_guide(args): if args.selection == "sorting": m = sorting_model - # else: - # m = survival_model + else: + m = survival_model if args.library_design == "tiling": info("Using Mixture Normal model...") return ( diff --git a/bean/plotting/utils.py b/bean/plotting/utils.py index 1233cfb..8080d11 100644 --- a/bean/plotting/utils.py +++ b/bean/plotting/utils.py @@ -27,7 +27,7 @@ def parse_args(): "--replicate-col", help="Column name in `bdata.samples` that describes replicate ID.", type=str, - default="rep", + default="replicate", ) parser.add_argument( "--condition-col", diff --git a/bean/preprocessing/data_class.py b/bean/preprocessing/data_class.py index ed4a2c3..1418b39 100644 --- a/bean/preprocessing/data_class.py +++ b/bean/preprocessing/data_class.py @@ -45,7 +45,7 @@ def __init__( accessibility_col: str = None, accessibility_bw_path: str = None, device: str = None, - replicate_column: str = "rep", + replicate_column: str = "replicate", popt: Optional[Tuple[float]] = None, pi_popt: Optional[Tuple[float]] = None, control_can_be_selected: bool = False, @@ -149,7 +149,9 @@ def _post_init(self): if self.repguide_mask is None: self.repguide_mask = ~(self.X == 0).any(axis=1) else: - print(f"Using repguide mask {self.repguide_mask}") + info( + f"Using replicate x guide mask in ReporterScreen.uns['{self.repguide_mask}'] to filter out outlier guides." + ) assert ( self.repguide_mask in self.screen.uns.keys() ), f"{self.repguide_mask} not in screen.uns" @@ -234,12 +236,12 @@ def get_size_factor(self, X: np.array): return size_factor def get_sample_ids_and_sort(self, screen: be.ReporterScreen, condit_id_col: str): - screen.samples["rep_id"] = -1 - for i, rep in enumerate(sorted(screen.samples.rep.unique())): - screen.var.loc[screen.samples.rep == rep, "rep_id"] = i + screen.samples["replicate_id"] = -1 + for i, rep in enumerate(sorted(screen.samples.replicate.unique())): + screen.var.loc[screen.samples.replicate == rep, "replicate_id"] = i if condit_id_col in screen.samples.columns: screen = screen[ - :, screen.samples.sort_values(["rep_id", condit_id_col]).index + :, screen.samples.sort_values(["replicate_id", condit_id_col]).index ] else: raise ValueError("AnnData doesn't have condition id provided.") @@ -567,6 +569,7 @@ def _post_init( else: allele_counts_selected = self.screen.uns[allele_df_key] if control_guide_tag is not None: + def _set_uid_to_row(row): if control_guide_tag in row.guide: row[allele_col].set_uid(row.guide) @@ -633,16 +636,16 @@ def get_allele_to_edit_tensor( Returns allele_edit_assignment: Binary tensor of shape (n_guides, n_max_alleles_per_guide, n_edits. allele_edit_assignment(i, j, k) is 1 if jth allele of ith guide has kth edit. """ - guide_allele_id_to_allele_df[ - "edits" - ] = guide_allele_id_to_allele_df.aa_allele.map( - lambda a: list(a.aa_allele.edits) + list(a.nt_allele.edits) + guide_allele_id_to_allele_df["edits"] = ( + guide_allele_id_to_allele_df.aa_allele.map( + lambda a: list(a.aa_allele.edits) + list(a.nt_allele.edits) + ) ) guide_allele_id_to_allele_df = guide_allele_id_to_allele_df.reset_index() - guide_allele_id_to_allele_df[ - "edit_idx" - ] = guide_allele_id_to_allele_df.edits.map( - lambda es: [edits_to_index[e.get_abs_edit()] for e in es] + guide_allele_id_to_allele_df["edit_idx"] = ( + guide_allele_id_to_allele_df.edits.map( + lambda es: [edits_to_index[e.get_abs_edit()] for e in es] + ) ) guide_allele_id_to_edit_df = guide_allele_id_to_allele_df[ ["guide", "allele_id_for_guide", "edit_idx"] @@ -708,7 +711,7 @@ def transform_allele(self, adata, reindexed_df): for i in range(self.n_reps): for j in range(self.n_condits): condit_idx = np.where( - (adata.samples.rep_id == i) + (adata.samples.replicate_id == i) & (adata.samples[f"{self.condition_column}_id"] == j) )[0] assert len(condit_idx) == 1, print(i, j, condit_idx) @@ -762,7 +765,7 @@ def transform_allele_control(self, adata, reindexed_df): if self.device is not None: allele_tensor = allele_tensor.cuda() for i in range(self.n_reps): - condit_idx = np.where(adata.samples.rep_id == i)[0] + condit_idx = np.where(adata.samples.replicate_id == i)[0] assert len(condit_idx) == 1, print(i, self.control_condition, condit_idx) condit_idx = condit_idx.item() condit_name = adata.samples.index[condit_idx] @@ -844,7 +847,7 @@ def __init__( control_condition: str = "bulk", lower_quantile_column: str = "lower_quantile", upper_quantile_column: str = "upper_quantile", - replicate_column: str = "rep", + replicate_column: str = "replicate", **kwargs, ): """ @@ -955,7 +958,7 @@ def __init__( control_condition: str = "bulk", control_can_be_selected=True, time_column: str = "time", - replicate_column: str = "rep", + replicate_column: str = "replicate", **kwargs, ): self._pre_init(condition_column) @@ -973,10 +976,14 @@ def __init__( def _pre_init(self, time_column: str, condition_column: str): self.time_column = time_column - if not np.issubdtype(self.screen.samples[time_column].dtype, np.number): + try: + self.screen.samples[time_column] = self.screen.samples[time_column].astype( + float + ) + except ValueError as e: raise ValueError( f"Invalid timepoint value({self.screen.samples[time_column]}) in screen.samples[{time_column}]: check input." - ) + ) from e if not ( self.screen.samples.groupby(condition_column).size() @@ -1017,7 +1024,9 @@ def _post_init( self.sample_covariates ].values self.screen_selected = _assign_rep_ids_and_sort( - self.screen_selected, self.replicate_column, self.condition_column + self.screen_selected, + self.replicate_column, + self.time_column, ) self.screen_control = _assign_rep_ids_and_sort( self.screen_control, @@ -1073,7 +1082,7 @@ def __init__( *args, lower_quantile_column="lower_quantile", upper_quantile_column="upper_quantile", - replicate_column="rep", + replicate_column="replicate", condition_column="bin", target_col="target", sample_mask_column="mask", @@ -1158,7 +1167,7 @@ def __init__( *args, lower_quantile_column="lower_quantile", upper_quantile_column="upper_quantile", - replicate_column="rep", + replicate_column="replicate", condition_column="bin", target_col="target", sample_mask_column="mask", @@ -1205,7 +1214,7 @@ def __init__( *args, lower_quantile_column="lower_quantile", upper_quantile_column="upper_quantile", - replicate_column="rep", + replicate_column="replicate", condition_column="bin", sample_mask_column="mask", use_const_pi: bool = False, @@ -1256,7 +1265,7 @@ def __init__( self, screen, *args, - replicate_column="rep", + replicate_column="replicate", condition_column="condition", time_column="time", control_can_be_selected=True, @@ -1326,7 +1335,7 @@ def __init__( self, screen, *args, - replicate_column="rep", + replicate_column="replicate", condition_column="condition", time_column="time", control_can_be_selected=True, @@ -1372,7 +1381,7 @@ def __init__( self, screen, *args, - replicate_column="rep", + replicate_column="replicate", condition_column="condition", time_column="time", control_can_be_selected=True, diff --git a/bean/preprocessing/utils.py b/bean/preprocessing/utils.py index 84e3d97..21bf6e3 100644 --- a/bean/preprocessing/utils.py +++ b/bean/preprocessing/utils.py @@ -37,7 +37,7 @@ def prepare_bdata(bdata: be.ReporterScreen, args, warn, prefix: str): n_no_support_targets, bdata = filter_no_info_target( bdata, condit_col=args.condition_col, - control_condition=args.control_condition_label, + control_condition=args.control_condition, target_col=args.target_col, write_no_support_targets=True, no_support_target_write_path=f"{prefix}/no_support_targets.csv", diff --git a/bean/qc/utils.py b/bean/qc/utils.py index 6ed50cf..82c8bf8 100644 --- a/bean/qc/utils.py +++ b/bean/qc/utils.py @@ -24,9 +24,9 @@ def parse_args(): "bdata_path", help="Path to the ReporterScreen object to run QC on", type=str ) thres_parser = parser.add_argument_group("QC thresholds") - run_parser = parser.add_argument_group("Run options") - input_parser = parser.add_argument_group("Input .h5ad formatting") - + run_parser = parser.add_argument_group("Run options") + input_parser = parser.add_argument_group("Input .h5ad formatting") + thres_parser.add_argument( "--count-correlation-thres", help="Correlation threshold to mask out.", @@ -45,7 +45,7 @@ def parse_args(): type=float, default=-0.1, ) - + parser.add_argument( "-o", "--out-screen-path", @@ -60,7 +60,8 @@ def parse_args(): ) run_parser.add_argument( - "-b", "--remove-bad-replicates", + "-b", + "--remove-bad-replicates", help="Remove replicates with at least two of its samples meet the QC threshold.", action="store_true", ) @@ -91,7 +92,7 @@ def parse_args(): "--replicate-label", help="Label of column in `bdata.samples` that describes replicate ID.", type=str, - default="rep", + default="replicate", ) input_parser.add_argument( "--sample-covariates", @@ -154,7 +155,6 @@ def parse_args(): default="bulk", ) - args = parser.parse_args() if args.out_screen_path is None: args.out_screen_path = f"{args.bdata_path.rsplit('.h5ad', 1)[0]}.filtered.h5ad" diff --git a/docs/ReporterScreen_api.ipynb b/docs/ReporterScreen_api.ipynb index 211bfc6..bda7bd5 100644 --- a/docs/ReporterScreen_api.ipynb +++ b/docs/ReporterScreen_api.ipynb @@ -2,24 +2,27 @@ "cells": [ { "cell_type": "markdown", - "source": [ - "[![Open In Colab](https://colab.research.google.com/assets/colab-badge.svg)](https://colab.research.google.com/drive/1tsbLl_yB9lcC_lo4sjwEoI0r1KAjPkoB)" - ], "metadata": { "id": "OhCQ5Qon-b8L" - } + }, + "source": [ + "[![Open In Colab](https://colab.research.google.com/assets/colab-badge.svg)](https://colab.research.google.com/drive/1tsbLl_yB9lcC_lo4sjwEoI0r1KAjPkoB)" + ] }, { "cell_type": "markdown", - "source": [ - "# CRISPR Reporter Screen analysis with bean" - ], "metadata": { "id": "qETZ0KgbbJIe" - } + }, + "source": [ + "# CRISPR Reporter Screen analysis with bean" + ] }, { "cell_type": "markdown", + "metadata": { + "id": "GQv2gC9IbOak" + }, "source": [ "In this tutorial, we will cover\n", "* Obtaining target variant editing rate from allele count information\n", @@ -27,16 +30,11 @@ "* Allele filtering based on significance\n", "\n", "For the basic API of ReporterScreen object, see [**perturb-tools tutorial of Screen API**](https://github.com/pinellolab/perturb-tools/blob/main/perturb_tools/screen_demo.ipynb)." - ], - "metadata": { - "id": "GQv2gC9IbOak" - } + ] }, { "cell_type": "code", - "source": [ - "! pip install beans==0.1.2" - ], + "execution_count": 2, "metadata": { "colab": { "base_uri": "https://localhost:8080/", @@ -45,11 +43,10 @@ "id": "b_HqduZRy3DY", "outputId": "eec31693-93b0-4266-8c30-a9902e7da4cd" }, - "execution_count": 2, "outputs": [ { - "output_type": "stream", "name": "stdout", + "output_type": "stream", "text": [ "Looking in indexes: https://pypi.org/simple, https://us-python.pkg.dev/colab-wheels/public/simple/\n", "Collecting beans==0.1.2\n", @@ -137,7 +134,6 @@ ] }, { - "output_type": "display_data", "data": { "application/vnd.colab-display-data+json": { "pip_warning": { @@ -148,30 +144,32 @@ } } }, - "metadata": {} + "metadata": {}, + "output_type": "display_data" } + ], + "source": [ + "! pip install beans==0.1.2" ] }, { "cell_type": "code", + "execution_count": 1, + "metadata": { + "id": "q4U4twaAzLPs" + }, + "outputs": [], "source": [ "import numpy as np\n", "import pandas as pd\n", "import seaborn as sns\n", "import matplotlib.pyplot as plt\n", "import bean as be" - ], - "metadata": { - "id": "q4U4twaAzLPs" - }, - "execution_count": 1, - "outputs": [] + ] }, { "cell_type": "code", - "source": [ - "!gdown 18Azb8YmmMvFZo9urc2TxZr540xXWOv_v" - ], + "execution_count": 33, "metadata": { "colab": { "base_uri": "https://localhost:8080/" @@ -179,11 +177,10 @@ "id": "8KhlwSn_2x9P", "outputId": "1fc11a18-6e01-403d-9e08-73da37eee332" }, - "execution_count": 33, "outputs": [ { - "output_type": "stream", "name": "stdout", + "output_type": "stream", "text": [ "Downloading...\n", "From: https://drive.google.com/uc?id=18Azb8YmmMvFZo9urc2TxZr540xXWOv_v\n", @@ -191,6 +188,9 @@ "100% 40.2M/40.2M [00:00<00:00, 160MB/s]\n" ] } + ], + "source": [ + "!gdown 18Azb8YmmMvFZo9urc2TxZr540xXWOv_v" ] }, { @@ -217,63 +217,7 @@ }, "outputs": [ { - "output_type": "execute_result", "data": { - "text/plain": [ - " guide \\\n", - "0 LDLR_SA_3_g5 \n", - "1 LDLR_SA_3_g5 \n", - "2 LDLR_SA_3_g5 \n", - "3 LDLR_SA_3_g5 \n", - "4 LDLR_SA_3_g5 \n", - "... ... \n", - "165506 2:164588224GAG_Maj_ABE_10_g3 \n", - "165507 2:164588224GAG_Maj_ABE_10_g3 \n", - 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" rep4_VPA_bulk rep1_top rep2_top rep3_VPA_top rep4_VPA_top \n", - "0 31 32 26 43 43 \n", - "1 22 27 11 20 13 \n", - "2 4 17 12 7 3 \n", - "3 9 0 0 0 0 \n", - "4 0 0 0 0 0 \n", - "... ... ... ... ... ... \n", - "165506 0 0 0 0 1 \n", - "165507 0 0 0 0 1 \n", - "165508 0 0 0 0 1 \n", - "165509 0 0 0 0 1 \n", - "165510 0 0 0 0 1 \n", - "\n", - "[165511 rows x 14 columns]" - ], "text/html": [ "\n", "

    \n", @@ -580,10 +524,66 @@ "
    \n", " \n", " " + ], + "text/plain": [ + " guide \\\n", + "0 LDLR_SA_3_g5 \n", + "1 LDLR_SA_3_g5 \n", + "2 LDLR_SA_3_g5 \n", + "3 LDLR_SA_3_g5 \n", + "4 LDLR_SA_3_g5 \n", + "... ... \n", + "165506 2:164588224GAG_Maj_ABE_10_g3 \n", + "165507 2:164588224GAG_Maj_ABE_10_g3 \n", + "165508 rs4921914_Min_ABE_501_g4 \n", + "165509 rs191388787_Maj_ABE_121_g2 \n", + "165510 rs113408797_Maj_ABE_41_g1 \n", + "\n", + " allele rep1_bot rep2_bot \\\n", + "0 26 13 \n", + "1 0:13:+:A>G 6 16 \n", + "2 -12:1:+:A>G,0:13:+:A>G 2 16 \n", + "3 0:13:+:A>G,7:20:+:A>G 2 0 \n", + "4 -12:1:+:A>G,-8:5:+:A>G,0:13:+:A>G,10:23:+:A>G 1 0 \n", + "... ... ... ... \n", + "165506 -10:1:+:C>T,-8:3:+:A>T 0 0 \n", + "165507 -8:3:+:A>T,-3:8:+:C>A,-1:10:+:G>A 0 0 \n", + "165508 -9:3:+:A>G,-8:4:+:A>G,-3:9:+:A>G,2:14:+:A>G,4:... 0 0 \n", + "165509 -7:3:+:A>G,-5:5:+:A>G,-3:7:+:A>G 0 0 \n", + "165510 -8:1:+:A>G,-1:8:+:A>G,3:12:+:A>G,5:14:+:A>G,17... 0 0 \n", + "\n", + " rep3_VPA_bot rep4_VPA_bot rep1_bulk rep2_bulk rep3_VPA_bulk \\\n", + "0 9 13 60 21 27 \n", + "1 11 24 29 21 28 \n", + "2 1 24 16 5 9 \n", + "3 0 0 0 0 0 \n", + "4 0 0 0 0 0 \n", + "... ... ... ... ... ... \n", + "165506 0 0 0 0 0 \n", + "165507 0 0 0 0 0 \n", + "165508 0 0 0 0 0 \n", + "165509 0 0 0 0 0 \n", + "165510 0 0 0 0 0 \n", + "\n", + " rep4_VPA_bulk rep1_top rep2_top rep3_VPA_top rep4_VPA_top \n", + "0 31 32 26 43 43 \n", + "1 22 27 11 20 13 \n", + "2 4 17 12 7 3 \n", + "3 9 0 0 0 0 \n", + "4 0 0 0 0 0 \n", + "... ... ... ... ... ... \n", + "165506 0 0 0 0 1 \n", + "165507 0 0 0 0 1 \n", + "165508 0 0 0 0 1 \n", + "165509 0 0 0 0 1 \n", + "165510 0 0 0 0 1 \n", + "\n", + "[165511 rows x 14 columns]" ] }, + "execution_count": 35, "metadata": {}, - "execution_count": 35 + "output_type": "execute_result" } ], "source": [ @@ -620,14 +620,14 @@ }, { "cell_type": "code", - "source": [ - "bdata.samples[\"rep\"], bdata.samples[\"sort\"] = zip(*bdata.samples.index.map(lambda s: s.rsplit(\"_\", 1)))" - ], + "execution_count": 3, "metadata": { "id": "jaUnqqXt3G2P" }, - "execution_count": 3, - "outputs": [] + "outputs": [], + "source": [ + "bdata.samples[\"replicate\"], bdata.samples[\"sort\"] = zip(*bdata.samples.index.map(lambda s: s.rsplit(\"_\", 1)))" + ] }, { "cell_type": "code", @@ -663,9 +663,7 @@ }, { "cell_type": "code", - "source": [ - "bdata.uns['allele_counts']" - ], + "execution_count": 42, "metadata": { "colab": { "base_uri": "https://localhost:8080/", @@ -674,66 +672,9 @@ "id": "GMV9enV35HKu", "outputId": "e8259a37-98f1-408c-9e16-d5315c2ce06c" }, - "execution_count": 42, "outputs": [ { - "output_type": "execute_result", "data": { - "text/plain": [ - " guide \\\n", - "1 LDLR_SA_3_g5 \n", - "2 LDLR_SA_3_g5 \n", - "3 LDLR_SA_3_g5 \n", - "4 LDLR_SA_3_g5 \n", - "5 LDLR_SA_3_g5 \n", - "... ... \n", - "165506 2:164588224GAG_Maj_ABE_10_g3 \n", - "165507 2:164588224GAG_Maj_ABE_10_g3 \n", - "165508 rs4921914_Min_ABE_501_g4 \n", - "165509 rs191388787_Maj_ABE_121_g2 \n", - "165510 rs113408797_Maj_ABE_41_g1 \n", - "\n", - " allele rep1_bot rep2_bot \\\n", - "1 0:13:+:A>G 6 16 \n", - "2 -12:1:+:A>G,0:13:+:A>G 2 16 \n", - "3 0:13:+:A>G,7:20:+:A>G 2 0 \n", - "4 -12:1:+:A>G,-8:5:+:A>G,0:13:+:A>G,10:23:+:A>G 1 0 \n", - "5 -12:1:+:A>G,0:13:+:A>G,7:20:+:A>G 1 0 \n", - "... ... ... ... \n", - "165506 -10:1:+:C>T,-8:3:+:A>T 0 0 \n", - "165507 -8:3:+:A>T,-3:8:+:C>A,-1:10:+:G>A 0 0 \n", - "165508 -9:3:+:A>G,-8:4:+:A>G,-3:9:+:A>G,2:14:+:A>G,4:... 0 0 \n", - "165509 -7:3:+:A>G,-5:5:+:A>G,-3:7:+:A>G 0 0 \n", - "165510 -8:1:+:A>G,-1:8:+:A>G,3:12:+:A>G,5:14:+:A>G,17... 0 0 \n", - "\n", - " rep3_VPA_bot rep4_VPA_bot rep1_bulk rep2_bulk rep3_VPA_bulk \\\n", - "1 11 24 29 21 28 \n", - "2 1 24 16 5 9 \n", - "3 0 0 0 0 0 \n", - "4 0 0 0 0 0 \n", - "5 0 0 0 2 2 \n", - "... ... ... ... ... ... \n", - "165506 0 0 0 0 0 \n", - "165507 0 0 0 0 0 \n", - "165508 0 0 0 0 0 \n", - "165509 0 0 0 0 0 \n", - "165510 0 0 0 0 0 \n", - "\n", - " rep4_VPA_bulk rep1_top rep2_top rep3_VPA_top rep4_VPA_top \n", - "1 22 27 11 20 13 \n", - "2 4 17 12 7 3 \n", - "3 9 0 0 0 0 \n", - "4 0 0 0 0 0 \n", - "5 0 1 0 0 0 \n", - "... ... ... ... ... ... \n", - "165506 0 0 0 0 1 \n", - "165507 0 0 0 0 1 \n", - "165508 0 0 0 0 1 \n", - "165509 0 0 0 0 1 \n", - "165510 0 0 0 0 1 \n", - "\n", - "[162065 rows x 14 columns]" - ], "text/html": [ "\n", "
    \n", @@ -1040,47 +981,102 @@ "
    \n", " \n", " " - ] - }, - "metadata": {}, - "execution_count": 42 - } - ] - }, - { - "cell_type": "code", - "source": [ - "bdata.uns[\"edit_counts\"] = bdata.get_edit_from_allele()" - ], - "metadata": { - "id": "pEX2eOem4uka" - }, - "execution_count": 5, - "outputs": [] - }, - { - "cell_type": "code", - "source": [ - "bdata.get_edit_mat_from_uns(\"A\", \"G\", target_pos_col = \"target_pos\")" - ], - "metadata": { - "colab": { + ], + "text/plain": [ + " guide \\\n", + "1 LDLR_SA_3_g5 \n", + "2 LDLR_SA_3_g5 \n", + "3 LDLR_SA_3_g5 \n", + "4 LDLR_SA_3_g5 \n", + "5 LDLR_SA_3_g5 \n", + "... ... \n", + "165506 2:164588224GAG_Maj_ABE_10_g3 \n", + "165507 2:164588224GAG_Maj_ABE_10_g3 \n", + "165508 rs4921914_Min_ABE_501_g4 \n", + "165509 rs191388787_Maj_ABE_121_g2 \n", + "165510 rs113408797_Maj_ABE_41_g1 \n", + "\n", + " allele rep1_bot rep2_bot \\\n", + "1 0:13:+:A>G 6 16 \n", + "2 -12:1:+:A>G,0:13:+:A>G 2 16 \n", + "3 0:13:+:A>G,7:20:+:A>G 2 0 \n", + "4 -12:1:+:A>G,-8:5:+:A>G,0:13:+:A>G,10:23:+:A>G 1 0 \n", + "5 -12:1:+:A>G,0:13:+:A>G,7:20:+:A>G 1 0 \n", + "... ... ... ... \n", + "165506 -10:1:+:C>T,-8:3:+:A>T 0 0 \n", + "165507 -8:3:+:A>T,-3:8:+:C>A,-1:10:+:G>A 0 0 \n", + "165508 -9:3:+:A>G,-8:4:+:A>G,-3:9:+:A>G,2:14:+:A>G,4:... 0 0 \n", + "165509 -7:3:+:A>G,-5:5:+:A>G,-3:7:+:A>G 0 0 \n", + "165510 -8:1:+:A>G,-1:8:+:A>G,3:12:+:A>G,5:14:+:A>G,17... 0 0 \n", + "\n", + " rep3_VPA_bot rep4_VPA_bot rep1_bulk rep2_bulk rep3_VPA_bulk \\\n", + "1 11 24 29 21 28 \n", + "2 1 24 16 5 9 \n", + "3 0 0 0 0 0 \n", + "4 0 0 0 0 0 \n", + "5 0 0 0 2 2 \n", + "... ... ... ... ... ... \n", + "165506 0 0 0 0 0 \n", + "165507 0 0 0 0 0 \n", + "165508 0 0 0 0 0 \n", + "165509 0 0 0 0 0 \n", + "165510 0 0 0 0 0 \n", + "\n", + " rep4_VPA_bulk rep1_top rep2_top rep3_VPA_top rep4_VPA_top \n", + "1 22 27 11 20 13 \n", + "2 4 17 12 7 3 \n", + "3 9 0 0 0 0 \n", + "4 0 0 0 0 0 \n", + "5 0 1 0 0 0 \n", + "... ... ... ... ... ... \n", + "165506 0 0 0 0 1 \n", + "165507 0 0 0 0 1 \n", + "165508 0 0 0 0 1 \n", + "165509 0 0 0 0 1 \n", + "165510 0 0 0 0 1 \n", + "\n", + "[162065 rows x 14 columns]" + ] + }, + "execution_count": 42, + "metadata": {}, + "output_type": "execute_result" + } + ], + "source": [ + "bdata.uns['allele_counts']" + ] + }, + { + "cell_type": "code", + "execution_count": 5, + "metadata": { + "id": "pEX2eOem4uka" + }, + "outputs": [], + "source": [ + "bdata.uns[\"edit_counts\"] = bdata.get_edit_from_allele()" + ] + }, + { + "cell_type": "code", + "execution_count": 12, + "metadata": { + "colab": { "base_uri": "https://localhost:8080/" }, "id": "FwKNCaIm7JSH", "outputId": "f874adbf-6286-46a5-8b6b-5f80a204da58" }, - "execution_count": 12, "outputs": [ { - "output_type": "stream", "name": "stdout", + "output_type": "stream", "text": [ "New edit matrix saved in .layers['edits']. Returning old edits.\n" ] }, { - "output_type": "execute_result", "data": { "text/plain": [ "array([[0., 0., 0., ..., 0., 0., 0.],\n", @@ -1092,13 +1088,22 @@ " [0., 0., 0., ..., 0., 0., 0.]])" ] }, + "execution_count": 12, "metadata": {}, - "execution_count": 12 + "output_type": "execute_result" } + ], + "source": [ + "bdata.get_edit_mat_from_uns(\"A\", \"G\", target_pos_col = \"target_pos\")" ] }, { "cell_type": "code", + "execution_count": 13, + "metadata": { + "id": "aJXnxwbb4F3G" + }, + "outputs": [], "source": [ "window_edit_rate= bdata.get_guide_edit_rate(normalize_by_editable_base = True,\n", " edited_base = \"A\",\n", @@ -1107,12 +1112,7 @@ " bcmatch_thres = 5,\n", " prior_weight = 1,\n", " return_result = True)" - ], - "metadata": { - "id": "aJXnxwbb4F3G" - }, - "execution_count": 13, - "outputs": [] + ] }, { "cell_type": "code", @@ -1127,7 +1127,6 @@ }, "outputs": [ { - "output_type": "execute_result", "data": { "text/plain": [ "(array([579., 364., 299., 249., 221., 214., 191., 134., 159., 133., 97.,\n", @@ -1144,20 +1143,21 @@ " )" ] }, + "execution_count": 14, "metadata": {}, - "execution_count": 14 + "output_type": "execute_result" }, { - "output_type": "display_data", "data": { + "image/png": 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", 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+ ] }, "metadata": { "needs_background": "light" - } + }, + "output_type": "display_data" } ], "source": [ @@ -1166,27 +1166,25 @@ }, { "cell_type": "markdown", - "source": [ - "# Allele filtering" - ], "metadata": { "id": "YkgDKePa8ldu" - } + }, + "source": [ + "# Allele filtering" + ] }, { "cell_type": "markdown", - "source": [ - "If you have non-edited control data of reporter (e.g., plasmid library), you can filter out the reporter to select the significant base edits per guides. Significance is determined by Fisher's exact test." - ], "metadata": { "id": "KB1B2sqX8qaA" - } + }, + "source": [ + "If you have non-edited control data of reporter (e.g., plasmid library), you can filter out the reporter to select the significant base edits per guides. Significance is determined by Fisher's exact test." + ] }, { "cell_type": "code", - "source": [ - "! gdown 153fKJntS-4vlrodC6xlZ9oINQo1XT_i9" - ], + "execution_count": 16, "metadata": { "colab": { "base_uri": "https://localhost:8080/" @@ -1194,11 +1192,10 @@ "id": "8tDcYNhB8KGj", "outputId": "7f0523e8-01b7-4139-a6a2-9fb9850caf01" }, - "execution_count": 16, "outputs": [ { - "output_type": "stream", "name": "stdout", + "output_type": "stream", "text": [ "Downloading...\n", "From: https://drive.google.com/uc?id=153fKJntS-4vlrodC6xlZ9oINQo1XT_i9\n", @@ -1206,6 +1203,9 @@ "\r 0% 0.00/5.10M [00:00-RZlVIb~440hP+vD<5bVA0Lzo62h1E z=^whJC#r`?rXZK{v7`I<4}ubbt=~%TV0$U^ zDv^TZl4guuZNh&4Pq)CJ{=G-)3~ppD!$4lIEn${}xS;G#m$NzXrhleEa(14V)U|S0 zv&~~PK~H2q)*+rxNt~ao6M-=$q$jK zkj@l^{VN7$j^bpD`RZGmmbG|pM!Fel*Y%cm>eW>+PDT=rKQjLZzFw*#rzYyg75 zk4ws$tAkceY$$88c8Njl_@MUj;p-qMP5mWGm`i7c1 z8wq>0UKYYmQg58BNjfWY%%~#E^}5!kAeZv!`V${Cm!&u(Yi3$j+RUJitn4~`g4Ai6 zBt96>d*~S1sRm<5k5j@Pre7F^PXqgo7#fzb0NeiI_DM2Q)6-|BW~a)Y*>vIhCohxQ zwriUt+SSLFr)FiPE|+q<(%bT+w(Y}w8{7IbGZs%2nx48)S|tc-QEtc6sYT+rv7=jMW@SukpVU4wc|q;M@;XgRPn&HL z>~Z2w>+2M)uWn)Wb&b|nukbuO+d=(8^4hhF*4Lo$yhwcq6qeU9TA$tD?reVBb&Sq$ z!@}z860I+>@VvpeNwbOHoKAK$bEk$FH&EV!s_b~t*>d}d6D{>#m;Nj&8e?> z;dznzS`?PoIXZnU3(t$xcTiz@-JMRFsV%Mj6 zk@2=IB(IyZ92`=3UZg(VCup~m<+)u~XSr`@(qiRB>T6$EUc2agbSOM8(taJuv)gOg zPU=y+-E9TBFV@a($L)Trvnh_0+X1?G1*m)iYGbiFYHbYr+!)6qRfy@yh6_X9Ih zv(sj0WX+wCx*+V+BgSxtdF6KcdwS(|`g>7s_wVhKOv@w}C^vK3+b?ft-r+t4%FR2_ zw?MgR$$ox$d$UCKFHmmY@c~}BY#hRpfd$IVJ2(rE~Xkr$bd zv11EVsVOO5o->=Kwy|Tww=C7m8!PkZSE(t}$%{;%X|hON%x*Blt4}7bK$V&zhn!(& z>(lmkE0d`R`=^m-=eKw7-UX`Elv#eBUz@a`rp)&8Wa0``sVQ^3yzm{6Hq&YBJ60;| zZ5D*Nt0-@7p}DzTT2ymZ#`~s44TkJZHI!)Tt?ED;~%-Ak!77 zOcpckqvsad+3Cx-B%2z;{^{Yflc!5n{)IZs$RN*-H~*3>ts`}6N~V_=_J}k|_#&LX zMPA-mc{j#N^b=QK;R$ZHJvq zHyMlj205oo+np!7>M8Qb?Gxl&t+6}~m&ft)7=R~>3nWMLN6T=}%i}wF-CrKB$|FM_ z$+97RLUNLmn2~u{k_=@?#>< zs(7iyAIkKvkm0@(+iE{~+%B(We8Jb!zl+2*<@M8jgTx#$zF&|l)0RBL^bekqoL(}` zD5w)GE|lr6EO|4fT$h{iGA^kjxt8RgBK>5#a+*sW{UzsoDcAQ@-kV^e zlxdr7lHnedhb&h)?@Ru8>8In-HfkgN?~vEhra7O}`%KAw>NgmbYq3^T2w>fvoaAhSfC-Zl#JH=QL<#IQl(3mDO0v=e0;fb<;z#7P_bgAN|h^@ z)XK=#^Dj3Z>cx7V=k_1s#RI%pcjj*XATQSQNw>eAPrGrl7wg%F+g~ZKymU3M-BO?cSgp7B@5Hrgday~O3cr{<#TcE62j-GWi)18 z)njsZUC?7#w|`ZeO1^G`!k5IABrYotTVG}SlKxdB4zDv1>$JG}dhA_J9(H&gw;OA2 zc>K=riq>iKHCM-JV_oyyd>yxr!}iyH6@z3gk+rgH`1${|x#=_01i{igDRr^Qt-_ip1`xtipOk~35u)f&o)KAkFOJF~@2m^`j@^05Fo4+dye!r1V0MG5z`(Y~+2G*V;wa?<5n? 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z-4C7Sm+rg`o|fxHkItXm>g#&CewGKC`aYEXOfISUo?PnFsU8F6z33MP3Im0K!a!l5 lFi;pM3={?m1BHRYKw+RTP#7o-6b1?dg@M9AVW5``{6BNlQ11W$ From a93bc83ad97b3524e2ee627c739f2f14e750c564 Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 16:21:14 -0400 Subject: [PATCH 018/107] debug survival negctrl model --- bean/model/readwrite.py | 31 ++++++++++++++++++------------- bean/model/run.py | 17 +++++++++++++++++ bean/model/survival_model.py | 10 +++++----- bin/bean-run | 11 +++-------- 4 files changed, 43 insertions(+), 26 deletions(-) diff --git a/bean/model/readwrite.py b/bean/model/readwrite.py index c1c68cd..a36493b 100644 --- a/bean/model/readwrite.py +++ b/bean/model/readwrite.py @@ -102,7 +102,6 @@ def write_result_table( ) fit_df = pd.DataFrame(param_dict) - fit_df["novl"] = get_novl(fit_df, "mu", "mu_sd") if "negctrl" in param_hist_dict.keys(): print("Normalizing with common negative control distribution") mu0 = param_hist_dict["negctrl"]["params"]["mu_loc"].detach().cpu().numpy() @@ -114,6 +113,8 @@ def write_result_table( .cpu() .numpy() ) + else: + sd0 = 1.0 print(f"Fitted mu0={mu0}" + (f", sd0={sd0}." if sd_is_fitted else "")) fit_df["mu_scaled"] = (mu - mu0) / sd0 fit_df["mu_sd_scaled"] = mu_sd / sd0 @@ -154,12 +155,12 @@ def write_result_table( fit_df, std, suffix="_adj", - mu_adjusted_col="mu_scaled" - if "negctrl" in param_hist_dict.keys() - else "mu", - mu_sd_adjusted_col="mu_sd_scaled" - if "negctrl" in param_hist_dict.keys() - else "mu_sd", + mu_adjusted_col=( + "mu_scaled" if "negctrl" in param_hist_dict.keys() else "mu" + ), + mu_sd_adjusted_col=( + "mu_sd_scaled" if "negctrl" in param_hist_dict.keys() else "mu_sd" + ), ) fit_df = add_credible_interval(fit_df, "mu_adj", "mu_sd_adj") if sample_covariates is not None: @@ -168,12 +169,16 @@ def write_result_table( fit_df, std, suffix=f"_{sample_cov}_adj", - mu_adjusted_col=f"mu_{sample_cov}_scaled" - if "negctrl" in param_hist_dict.keys() - else f"mu_{sample_cov}", - mu_sd_adjusted_col=f"mu_sd_{sample_cov}_scaled" - if "negctrl" in param_hist_dict.keys() - else f"mu_sd_{sample_cov}", + mu_adjusted_col=( + f"mu_{sample_cov}_scaled" + if "negctrl" in param_hist_dict.keys() + else f"mu_{sample_cov}" + ), + mu_sd_adjusted_col=( + f"mu_sd_{sample_cov}_scaled" + if "negctrl" in param_hist_dict.keys() + else f"mu_sd_{sample_cov}" + ), ) fit_df = add_credible_interval( fit_df, f"mu_{sample_cov}_adj", f"mu_sd_{sample_cov}_adj" diff --git a/bean/model/run.py b/bean/model/run.py index d09fe1e..f8bde98 100644 --- a/bean/model/run.py +++ b/bean/model/run.py @@ -488,3 +488,20 @@ def identify_model_guide(args): fit_noise=(not args.dont_fit_noise), ), ) + + +def identify_negctrl_model_guide(args, data_has_bcmatch): + if args.selection == "sorting": + m = sorting_model + else: + m = survival_model + negctrl_model = partial( + m.ControlNormalModel, + use_bcmatch=(not args.ignore_bcmatch and data_has_bcmatch), + ) + + negctrl_guide = partial( + m.ControlNormalGuide, + use_bcmatch=(not args.ignore_bcmatch and data_has_bcmatch), + ) + return negctrl_model, negctrl_guide diff --git a/bean/model/survival_model.py b/bean/model/survival_model.py index 9b68e2c..ddfb362 100644 --- a/bean/model/survival_model.py +++ b/bean/model/survival_model.py @@ -123,12 +123,12 @@ def ControlNormalModel(data, mask_thres=10, use_bcmatch=True): mu_alleles = pyro.sample("mu_alleles", dist.Laplace(0, 1)) mu = mu_alleles.repeat(data.n_guides).unsqueeze(-1) r = torch.exp(mu) + with pyro.plate("rep_plate1", data.n_reps, dim=-1): + q_0 = pyro.sample( + "initial_guide_abundance", + dist.Dirichlet(torch.ones((data.n_reps, data.n_guides))), + ) with replicate_plate: - with pyro.plate("guide_plate2", data.n_guides): - q_0 = pyro.sample( - "initial_guide_abundance", - dist.Dirichlet(torch.ones((data.n_reps, data.n_guides))), - ) with time_plate as t: time = data.timepoints[t] assert time.shape == (data.n_condits,) diff --git a/bin/bean-run b/bin/bean-run index b4eb5f4..540bd37 100644 --- a/bin/bean-run +++ b/bin/bean-run @@ -31,6 +31,7 @@ from bean.model.run import ( parse_args, check_args, identify_model_guide, + identify_negctrl_model_guide, ) logging.basicConfig( @@ -144,14 +145,8 @@ def main(args, bdata): run_inference(model, guide, ndata, num_steps=args.n_iter) ) if args.fit_negctrl: - negctrl_model = partial( - m.ControlNormalModel, - use_bcmatch=(not args.ignore_bcmatch and "X_bcmatch" in bdata.layers), - ) - - negctrl_guide = partial( - m.ControlNormalGuide, - use_bcmatch=(not args.ignore_bcmatch and "X_bcmatch" in bdata.layers), + negctrl_model, negctrl_guide = identify_negctrl_model_guide( + args, "X_bcmatch" in bdata.layers ) negctrl_idx = np.where( guide_info_df[args.negctrl_col].map(lambda s: s.lower()) From c4df38a094d1bab7906e09e508b8e4a2976a1251 Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 21:58:54 -0400 Subject: [PATCH 019/107] update documentation --- .github/workflows/documentation.yml | 27 ++ README.md | 438 ++-------------------------- bean/annotate/utils.py | 24 +- bean/framework/read_from_csvs.py | 20 +- bean/mapping/utils.py | 84 +++++- bean/model/run.py | 17 +- bean/plotting/utils.py | 21 +- bean/qc/utils.py | 29 +- bin/bean-count | 0 bin/bean-count-samples | 0 bin/bean-create-screen | 0 bin/bean-filter | 0 bin/bean-profile | 0 bin/bean-qc | 0 bin/bean-run | 0 docs/ReporterScreen_api.rst | 2 +- docs/cds.rst | 5 + docs/commands/count.md | 31 ++ docs/commands/create-screen.md | 12 + docs/commands/filter.md | 37 +++ docs/commands/input.md | 37 +++ docs/commands/profile.md | 8 + docs/commands/qc.md | 94 ++++++ docs/commands/run.md | 64 ++++ docs/conf.py | 29 ++ docs/count.rst | 10 + docs/count_samples.rst | 11 + docs/filter.rst | 11 + docs/gwas.rst | 5 + docs/index_.rst | 38 +++ docs/input.rst | 4 + docs/profile.rst | 10 + docs/qc.rst | 11 + docs/run.rst | 11 + docs/subcommands.rst | 13 + docs/tutorials/ldl_cds.md | 50 +++- docs/tutorials/ldl_var.md | 46 ++- setup.py | 15 +- tests/test_count.py | 12 +- tests/test_create.py | 3 +- tests/test_filter.py | 10 +- tests/test_qc.py | 8 +- tests/test_run.py | 24 +- 43 files changed, 710 insertions(+), 561 deletions(-) create mode 100644 .github/workflows/documentation.yml mode change 100644 => 100755 bin/bean-count mode change 100644 => 100755 bin/bean-count-samples mode change 100644 => 100755 bin/bean-create-screen mode change 100644 => 100755 bin/bean-filter mode change 100644 => 100755 bin/bean-profile mode change 100644 => 100755 bin/bean-qc mode change 100644 => 100755 bin/bean-run create mode 100644 docs/cds.rst create mode 100644 docs/commands/count.md create mode 100644 docs/commands/create-screen.md create mode 100644 docs/commands/filter.md create mode 100644 docs/commands/input.md create mode 100644 docs/commands/profile.md create mode 100644 docs/commands/qc.md create mode 100644 docs/commands/run.md create mode 100644 docs/conf.py create mode 100644 docs/count.rst create mode 100644 docs/count_samples.rst create mode 100644 docs/filter.rst create mode 100644 docs/gwas.rst create mode 100644 docs/index_.rst create mode 100644 docs/input.rst create mode 100644 docs/profile.rst create mode 100644 docs/qc.rst create mode 100644 docs/run.rst create mode 100644 docs/subcommands.rst diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml new file mode 100644 index 0000000..62a2eb6 --- /dev/null +++ b/.github/workflows/documentation.yml @@ -0,0 +1,27 @@ +name: documentation + +on: [push, pull_request, workflow_dispatch] + +permissions: + contents: write + +jobs: + docs: + runs-on: ubuntu-latest + steps: + - uses: actions/checkout@v3 + - uses: actions/setup-python@v3 + - name: Install dependencies + run: | + pip install sphinx sphinx_rtd_theme myst_parser + - name: Sphinx build + run: | + sphinx-build doc _build + - name: Deploy to GitHub Pages + uses: peaceiris/actions-gh-pages@v3 + if: ${{ github.event_name == 'push' && github.ref == 'refs/heads/main' }} + with: + publish_branch: gh-pages + github_token: ${{ secrets.GITHUB_TOKEN }} + publish_dir: _build/ + force_orphan: true \ No newline at end of file diff --git a/README.md b/README.md index b459cfb..945dce5 100644 --- a/README.md +++ b/README.md @@ -4,41 +4,40 @@ [![PyPI version](https://img.shields.io/pypi/v/crispr-bean)](https://pypi.org/project/crispr-bean/) [![Code style](https://img.shields.io/badge/code%20style-black-black)](https://github.com/psf/black) -**CRISPR** **B**ayesian **E**stimation of variant effect (from **B**ase **E**diting reporter screens) with guide **A**ctivity **N**ormalization -This is an analysis toolkit for the pooled CRISPR reporter or sensor data. The reporter technique transfects cells with plasmid with not only sgRNA but with the **target sequence surrogate** which we call **reporter** or **sensor**. +`bean` (**B**ase **E**diting reporter screens with guide **A**ctivity **N**ormalization) is an analysis toolkit for the pooled CRISPR reporter or sensor data. The reporter technique transfects cells with plasmid with not only sgRNA but with the **target sequence surrogate** which we call **reporter** or **sensor**. Reporter construct ## Overview -`crispr-bean` supports end-to-end analysis of pooled sorting screens, with or without reporter. +`bean` supports end-to-end analysis of pooled sorting screens, with or without reporter. dag_bean_v2.svg -1. [`bean-count-sample`](#bean-count-samples-count-reporter-screen-data): Base-editing-aware **mapping** of guide, optionally with reporter from `.fastq` files. - * [`bean-count-create`](#bean-create-screen-create-reporterscreen-object-from-flat-files) creates minimal ReporterScreen object from flat gRNA count file. Note that this way, allele counts are not included and many functionalities involving allele and edit counts are not supported. -2. [`bean-profile`](#bean-profile-profile-editing-patterns): Profile editing preferences of your editor. -3. [`bean-qc`](#bean-qc-qc-of-reporter-screen-data): Quality control report and filtering out / masking of aberrant sample and guides -4. [`bean-filter`](#bean-filter-filtering-and-optionally-translating-alleles): Filter reporter alleles; essential for `tiling` mode that allows for all alleles generated from gRNA. -5. [`bean-run`](#bean-run-quantify-variant-effects): Quantify targeted variants' effect sizes from screen data. +`bean` subcommands include the following: +1. [`count`, `count-samples`](#bean-count-samples-count-reporter-screen-data): Base-editing-aware **mapping** of guide, optionally with reporter from `.fastq` files. + * [`create-screen`](#bean-create-screen-create-reporterscreen-object-from-flat-files) creates minimal ReporterScreen object from flat gRNA count file. Note that this way, allele counts are not included and many functionalities involving allele and edit counts are not supported. +2. [`profile`](#bean-profile-profile-editing-patterns): Profile editing preferences of your editor. +3. [`qc`](#bean-qc-qc-of-reporter-screen-data): Quality control report and filtering out / masking of aberrant sample and guides +4. [`filter`](#bean-filter-filtering-and-optionally-translating-alleles): Filter reporter alleles; essential for `tiling` mode that allows for all alleles generated from gRNA. +5. [`run`](#bean-run-quantify-variant-effects): Quantify targeted variants' effect sizes from screen data. ### Screen data is saved as *ReporterScreen* object in the pipeline. BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). See [Data Structure](#data-structure) section for more information.

    -## Examples -| Tutorial link | [Library design](#pipeline-run-options-by-library-design) | Selection | Reporter | +## Tutorials +| [Library design](#pipeline-run-options-by-library-design) | Selection | Reporter | Tutorial link | |---------------| -------------- | --------- | -------- | -| [LDL-C GWAS](docs/tutorials/ldl_var.md) | GWAS variant library | FACS sorting | Yes/No | -| [LDL-C LDLR CDS](docs/tutorials/ldl_cds.md) | Coding sequence tiling libarary | FACS sorting | Yes/No | -| TKO simulated (Coming soon!) | GWAS variant library | Survival / Proliferation | Yes/No | -| TKO simulated, tiling (Coming soon!) | Coding sequence tiling libarary | Survival / Proliferation | Yes/No | +| GWAS variant library | FACS sorting | Yes/No | [GWAS variant screen](docs/tutorials/ldl_var.md) +| Coding sequence tiling libarary | FACS sorting | Yes/No | [Coding sequence tiling screen](docs/tutorials/ldl_cds.md) +| GWAS variant library | Survival / Proliferation | Yes/No | TKO simulated (Coming soon!) +| Coding sequence tiling libarary | Survival / Proliferation | Yes/No | TKO simulated, tiling (Coming soon!) -We also provide example scripts in `tests/`. Running `pytest --sparse-ordering` will generate example input/output files. ### Pipeline run options by library design -The `bean-filter` and `bean-run` steps depend on the type of gRNA library design, where BEAN supports two modes of running. +The `bean filter` and `bean run` steps depend on the type of gRNA library design, where BEAN supports two modes of running. 1. `variant` library: Several gRNAs tile each of the targeted variants Ex) variant library design @@ -61,403 +60,18 @@ pip install crispr-bean[model] ``` pip install crispr-bean ``` -This wouldn't have variant effect size quantification (`bean-run`) functionality. +This wouldn't have variant effect size quantification (`bean run`) functionality.

    +## Subcommands +See the full detail for each subcommand in the documentation. +* [`count`, `count-samples`](docs/count.md): Count (reporter) screen data +* [`profile`](docs/profile.md) +* [`qc`](docs/qc.md) +* [`filter`](docs/commands/filter.md) +* [`run`](docs/commands/run.md) +* [`create-screen`](docs/commands/create-screen.md) -## `bean-count-samples`: Count (reporter) screen data -`bean-count-samples` (or `bean-count` for a single sample) maps guide into guide counts, **allowing for base transition in spacer sequence**. When the matched reporter information is provided, it can count the **target site edits** and **alleles produced by each guide**. Mapping is efficiently done based on [CRISPResso2](https://github.com/pinellolab/CRISPResso2) modified for base-edit-aware mapping. - - - -```python -bean-count-samples \ - --input sample_list.csv `# sample with lines 'R1_filepath,R2_filepath,sample_name\n'` \ - -b A `# base that is being edited (A/G)` \ - -f sgRNA_info_table.csv `# sgRNA information` \ - -o . `# output directory` \ - -r `# read edit/allele information from reporter` \ - -t 12 `# number of threads` \ - --name my_sorting_screen `# name of this sample run` \ -``` - -By default, `bean-count[-samples]` assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure. - Read structuren -See full detail in [Parameters](#parameters). - -### Input file format -#### 1. gRNA_library.csv -File should contain following columns. -* `name`: gRNA ID column -* `sequence`: gRNA sequence -* `barcode`: R2 barcode to help match reporter to gRNA, written in the sense direction (as in R1) -* In order to use accessibility in the [variant effect quantification](#bean-run-quantify-variant-effects), provide accessibility information in one of two options. (For non-targeting guides, provide NA values (empty cell).) - * Option 1: `chrom` & `genomic_pos`: Chromosome (ex. `chr19`) and genomic position of guide sequence. You will have to provide the path to the bigwig file with matching reference version in `bean-run`. - * Option 2: `accessibility_signal`: ATAC-seq signal value of the target loci of each guide. -* For variant library (gRNAs are designed to target specific variants and ignores bystander edits) - * `target`: This column denotes which target variant/element of each gRNA. This is not used in `bean-count[-samples]` but required to run `bean-run` in later steps. - * `target_group`: If negative/positive control gRNA will be considered in `bean-qc` and/or `bean-run`, specify as "NegCtrl"/"PosCtrl" in this column. - * `target_pos`: If `--match_target_pos` flag is used, input file needs `target_pos` which specifies 0-based relative position of targeted base within Reporter sequence. -* For tiling library (gRNAs tile coding / noncoding sequences) - * `strand`: Specifies gRNA strand information relative to the reference genome. - * `chrom`: Chromosome of gRNA targeted locus. - * `start_pos`: gRNA starting position in the genome. Required when you provide `strand` column. Should specify the smaller coordinate value among start and end position regardless of gRNA strandedness. - -Also see examples for [variant library](tests/data/test_guide_info.csv) and [tiling library](tests/data/test_guide_info_tiling.csv). - -#### 2. sample_list.csv -File should contain following columns with header. -* `R1_filepath`: Path to read 1 `.fastq[.gz]` file -* `R2_filepath`: Path to read 1 `.fastq[.gz]` file -* `sample_id`: ID of sequencing sample -* `replicate`: Replicate # of this sample (Should NOT contain `.`) -* `condition`: Name of the sorting bin (ex. `top`, `bot`), or label of timepoint (ex. `D5`, `D18`) - -For FACS sorting screens: -* `upper_quantile`: FACS sorting upper quantile -* `lower_quantile`: FACS sorting lower quantile - -For proliferation / survival screens: -* `time`: Numeric time following the base editing of each sample. - - -Also see examples for [FACS sorting screen](tests/data/sample_list.csv). - -### Output file format -`count` or `count-samples` produces `.h5ad` and `.xlsx` file with guide and per-guide allele counts. -* `.h5ad`: This output file follows annotated matrix format compatible with `AnnData` and is based on `Screen` object in [purturb_tools](https://github.com/pinellolab/perturb-tools). See [Data Structure](#data-structure) section for more information. -* `.xlsx`: This output file contains `.guides`, `.samples`, `.X[_bcmatch,_edits]`. (`allele_tables` are often too large to write into an Excel!) - -### Parameters -* `-b`, `--edited-base` (`str`, default: `None`): For base editors, the base that should be ignored when matching the gRNA sequence -* `-i`, `--input`: List of fastq and sample ids. See above for the format. -* `-f`, `--sgRNA-filename` (`str`, default: `None`): sgRNA description file. (See above) - -Output formats -* `-n`, `--name`: Name of the output file will be `bean_count_{name}.h5ad`. -* `-o`, `--output-folder`: Output folder -* `--tiling`: Specify that the guide library is tiling library -* `-r`, `--count-reporter` (default: `False`): Count edited alleles in reporters. -* `--string-allele` (default: `False`): Store allele as quality filtered string instead of Allele object -* `-g`, `--count-guide-edits` (default: `False`): count the self editing of guides (default: False) -* `-m`, `--count-guide-reporter-alleles`: count the matched allele of guide and reporter edit -* `--match-target-pos` (default: `False`): Count the edit in the exact target position. -* `--target-pos-col` (default: `target_pos`): Column name specifying the relative target position within *reporter* sequence. -* `--offset` (default: `False`): Guide file has `offest` column that will be added to the relative position of reporters. -* `--align-fasta` (default: ''): gRNA is aligned to this sequence to infer the offset. Can be used when the exact offset is not provided. - - -Read structure -* `--guide-start-seq` (default: ''): Guide starts after this sequence in R1 -* `--guide-end-seq` (default: ''): Guide ends after this sequence in R1 -* `--barcode-start-seq` (default: ''): Barcode + reporter starts after this sequence in R2, denoted as the sense direction (the same sequence direction as R1). -* `--guide-start-seqs-file` (default: `None`): CSV file path with per-sample `guide_start_seq` to be used, if provided. Formatted as `sample_id, guide_start_seq` -* `--guide-end-seqs-file` (default: `None`): CSV file path with per-sample `guide_end_seq` to be used, if provided. Formatted as `sample_id,guide_end_seq` -* `--guide-start-seqs-file` (default: `None`): CSV file path with per-sample `barcode_start_seq` to be used, if provided. Formatted as `sample_id, guide_start_seq` -* `-l`, `--reporter-length` (default: `32`): Length of the reporter sequence. -* `--gstart-reporter` (default: `6`): Start position of the guide sequence in the reporter -* `--guide-bc` (default: `True`): Construct has guide barcode -* `--guide-bc-len` (default: `4`): Guide barcode sequence length at the beginning of the R2 - -Mapping quality filters -* `-q`, `--min-average-read-quality` (default: `30`): Minimum average quality score -(phred33) to keep a read -* `--keep-intermediate` (default: `False`): Keep all intermediate files generated from filtering. -* `-s`, `--min-single-bp-quality` (default: `0`): Minimum single bp score (phred33) to keep a read (default: 0) -* `--qstart-R1` (default: `0`): Start position of the read when filtering for quality score of the read 1 -* `--qend-R1` (default: `47`): End position of the read when filtering for quality score of the read 1 -* `--qstart-R2` (default: `0`): Start position of the read when filtering for quality score of the read 2 -* `--qend-R2` (default: `36`): End position of the read when filtering for quality score of the read 2 - -Run options -* `-t`, `--threads` (default: `10`): Number of threads to use -* `--rerun` (default: `False`): Recount each sample. If `False`, existing count for each sample is taken. - - -## `bean-create-screen`: Create ReporterScreen object from flat files -```bash -bean-create-screen gRNA_library.csv sample_list.csv gRNA_counts_table.csv -``` -### Input - * [gRNA_library.csv](#1-gRNA_librarycsv) - * [sample_list.csv](#2-sample_listcsv) - * gRNA_counts_table.csv: Table with gRNA ID in the first column and sample IDs as the column names (first row) - -### Full Parameters - * `-e`, `--edits` (default: `None`): Path to edit counts .csv table, with index at first column and column names at the first row. - * `-o`, `--output-prefix` (default: `None`): Output file prefix (output will be saved as `output_prefix.h5ad`). If not provided, `gRNA_counts_table_csv` file prefix is used. - -

    - -## `bean-profile`: Profile editing patterns -```bash -bean-profile my_sorting_screen.h5ad -o output_prefix `# Prefix for editing profile report` -``` -### Output -Above command produces `prefix_editing_preference.[html,ipynb]` as editing preferences ([see example](notebooks/profile_editing_preference.ipynb)). - -Allele translation - -### Parameters - * `-o`, `--output-prefix` (default: `None`): Output prefix of editing pattern report (prefix.html, prefix.ipynb). If not provided, base name of `bdata_path` is used. - * `--replicate-col` (default: `"replicate"`): Column name in `bdata.samples` that describes replicate ID. - * `--condition-col` (default: `"condition"`): Column name in `bdata.samples` that describes experimental condition. (sorting bin, time, etc.) - * `--pam-col` (default: `None`): Column name describing PAM of each gRNA in `bdata.guides`. - * `--control-condition` (default: `"bulk"`): Control condition where editing preference would be profiled at. Pre-filters data where `bdata.samples[condition_col] == control_condition`. DO NOT use plasmid library as control here where we do not expect editing. - * `-w`, `--window-length` (default: `6`): Window length of editing window of maximal editing efficiency to be identified. This window is used to quantify context specificity within the window. - - -

    - -## `bean-qc`: QC of reporter screen data -```bash -bean-qc \ - my_sorting_screen.h5ad `# Input ReporterScreen .h5ad file path` \ - -o my_sorting_screen_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r qc_report_my_sorting_screen `# Prefix for QC report` \ - --ctrl-cond presort `# "condition" column in the control sample before selection. Mean gRNA editing rates in these samples are reported. ` \ -# Inspect the output qc_report_my_sorting_screen.html to tweak QC threshold - -bean-qc \ - my_sorting_screen.h5ad \ - -o my_sorting_screen_masked.h5ad \ - -r qc_report_my_sorting_screen \ - #[--count-correlation-thres 0.7 ...]\ - -b -``` - -`bean-qc` supports following quality control and masks samples with low quality. Specifically: - -Allele translation - -* Plots guide coverage and the uniformity of coverage -* Guide count correlation between samples -* Log fold change correlation when positive controls are provided -* Plots editing rate distribution -* Identify samples with low guide coverage/guide count correlation/editing rate and mask the sample in `bdata.samples.mask` -* Identify outlier guides to filter out - -#### Output -Above command produces -* `my_sorting_screen_masked.h5ad` without problematic replicate and guides and with sample masks, and -* `qc_report_my_sorting_screen.[html,ipynb]` as QC report. - - -#### Additional Parameters -##### Optional arguments: -* `-o OUT_SCREEN_PATH`, `--out-screen-path OUT_SCREEN_PATH` - Path where quality-filtered ReporterScreen object to be written to -* `-r OUT_REPORT_PREFIX`, `--out-report-prefix OUT_REPORT_PREFIX` - Output prefix of qc report (prefix.html, prefix.ipynb) - -##### QC thresholds: -* `--count-correlation-thres COUNT_CORRELATION_THRES` - Correlation threshold to mask out. -* `--edit-rate-thres EDIT_RATE_THRES` - Mean editing rate threshold per sample to mask out. -* `--lfc-thres LFC_THRES` - Positive guides' correlation threshold to filter out. - -##### Run options: -* `-b`, `--remove-bad-replicates` - Remove replicates with at least two of its samples meet the QC threshold (bean-run does not support having only one sorting bin sample for a replicate). -* `-i`, `--ignore-missing-samples` - If the flag is not provided, if the ReporterScreen object does not contain all condiitons for - each replicate, make fake empty samples. If the flag is provided, don't add dummy samples. -* `--no-editing` Ignore QC about editing. Can be used for QC of other editing modalities. -* `--dont-recalculate-edits` - When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from - ReporterScreen.uns['allele_count']. - -##### Input `.h5ad` formatting: -Note that these arguements will change the way the QC metrics are calculated for guides, samples, or replicates. -* `--tiling TILING` Specify that the guide library is tiling library without 'n guides per target' design -* `--replicate-label REPLICATE_LABEL` - Label of column in `bdata.samples` that describes replicate ID. -* `--sample-covariates SAMPLE_COVARIATES` - Comma-separated list of column names in `bdata.samples` that describes non-selective - experimental condition. (drug treatment, etc.) -* `--condition-label CONDITION_LABEL` - Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time, - etc.) -###### Editing rate calculation - * `--control-condition CTRL_COND` - Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate - to be calculated. Default is `None`, which considers all samples. - * `--rel-pos-is-reporter` - Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If - `False`, those are relative to spacer position. - Editing rate is calculated with following parameters in - * Variant screens: - * `--target-pos-col TARGET_POS_COL` - Target position column in `bdata.guides` specifying target edit position in reporter - * tiling screens: - * `--edit-start-pos EDIT_START_POS` - Edit start position to quantify editing rate on, 0-based inclusive. - * `--edit-end-pos EDIT_END_POS` - Edit end position to quantify editing rate on, 0-based exclusive. -###### LFC of positive controls - * `--posctrl-col POSCTRL_COL` - Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all - gRNAs, feed empty string ''. - * `--posctrl-val POSCTRL_VAL` - Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the - positive control in calculating log fold change. - * `--lfc-conds LFC_CONDS` - Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated - between, delimited by comma - -

    - - -## `bean-filter`: Filtering (and optionally translating) alleles -As `tiling` mode of `bean-run` accounts for any robustly observed alleles, `bean-filter` filters for such alleles. -```bash -bean-filter my_sorting_screen_masked.h5ad \ --o my_sorting_screen_filtered.h5ad `# Output file path` \ -``` - -### Output -Above command produces -* `my_sorting_screen_filtered.h5ad` with filtered alleles stored in `.uns`, -* `my_sorting_screen_filtered.filtered_allele_stats.pdf`, and `my_sorting_screen_filtered.filter_log.txt` that report allele count stats in each filtering step. - -You may want to adjust the flitering parameters to obtain optimal balance between # guides per variant & # variants that are scored. See example outputs of filtering step [here](docs/example_filtering_output/). - - -### Translating alleles -If you want to obtain **amino acid level variant** for coding sequence tiling screens, provide coding sequence positions which variants occuring within the coding sequence will be translated. *This is optional, but **highly recommended** to increase per-(coding)variant support.* - -Allele translation - - -```bash -bean-filter my_sorting_screen.h5ad \ --o my_sorting_screen_masked.h5ad \ ---translate `# Translate coding variants` \ -[ --translate-gene-name GENE_SYMBOL OR - --translate-genes-list path_to_gene_names_file.txt OR - --translate-fasta gene_exon.fa, OR - --translate-fastas-csv gene_exon_fas.csv] -``` -* When library covers a single gene, do either of the following: - 1. Feed `--translate-gene-name GENE_SYMBOL` if your `genomic_pos` column of `sgRNA_info_tbl` is compatible with [MANE transcript](https://useast.ensembl.org/info/genome/genebuild/mane.html)'s reference genome. (Per 10/23/2023, GRCh38). This will automatically load the exon positions based on MANE transcript annotation. - 2. To use your custom coding sequence and exon positions, feed `--translate-fasta gene_exon.fa` argument where `gene_exon.fa` is the FASTA file with entries of exons. [See full details here](docs/exon_fa_format.md). -* When library covers multiple genes, do either of the following: - 1. Feed `--translate-genes-list path_to_gene_names_file.txt` where `path_to_gene_names_file.txt` is file with one gene symbol per line. - 2. Feed `--translate-fastas-csv gene_exon_fas.csv` where `gene_exon_fas.csv` is the csv file with lines `gene_id,gene_exon_fasta_path` without header. Each FASTA file in `gene_exon_fasta_path` is formatted [as the single-gene FASTA file](docs/exon_fa_format.md). -* Translation will keep the variants outside the coding sequence as nucleotide-level variants, while aggregating variants leading to the same coding sequence variants. - -### Full list of parameters -* `-o`, `--output-prefix` (default: `None`): Output prefix for log and ReporterScreen file with allele assignment. -* `-p`, `--plasmid-path` (default: `None`): Plasmid `ReporterScreen` object path. - * If provided, alleles are filtered based on if a nucleotide edit is more significantly enriched in sample compared to the plasmid data. - * Negative control data where no edit is expected can be fed in instead of plasmid library. -* `-w`, `--filter-window` (default: `False`): "Only consider edit within window provided by (`edit_start_pos`, `edit_end_pos`). If this flag is not provided, `--edit-start-pos` and `--edit-end-pos` flags are ignored. -* `-s`, `--edit-start-pos` (default: `2`): 0-based start posiiton (inclusive) of edit relative to the start of guide spacer. -* `-e`, `--edit-end-pos` (default: `7`): 0-based end position (exclusive) of edit relative to the start of guide spacer. -* `-b`, `--filter-target-basechange` (default: `False`): Filter for target (intended) base change of edits (stored in `bdata.uns['target_base_change']`) -* `-j`, `--jaccard-threshold` (default: `0.3`): Jaccard Index threshold when the alleles are mapped to the most similar alleles. In each filtering step, allele counts of filtered out alleles will be mapped to the most similar allele only if they have Jaccard Index of shared edit higher than this threshold. -* `--translate` (default: `False`): Translate nucleotide-level variants prior to allele proportion filtering. -* `-f`, `--translate-fasta` (defulat: `None`): fasta file path with exon positions. If not provided and `--translate` flag is provided, LDLR hg19 coordinates will be used. -* `-fs`, `--translate-fastas-csv` (defulat: `None`): .csv with two columns with gene IDs and FASTA file path corresponding to each gene. -* `-g`, `--translate-gene-name` (default: `None`): Gene symbol for translation -* `-gs`, `--translate-genes-list` (default: `None`): Path to the text file with gene symbols in each line -* `-ap`, `--filter-allele-proportion` (default: `0.05`): If provided, only the alleles that exceed `filter_allele_proportion` in `filter-sample-proportion` will be retained. -* `-ac`, `--filter-allele-count` (default: `5`): If provided, alleles that exceed `filter_allele_proportion` AND `filter_allele_count` in `filter-sample-proportion` will be retained. -* `sp`, `--filter-sample-proportion` (default: `0.2`): "If `filter_allele_proportion` is provided, alleles that exceed `filter_allele_proportion` in `filter-sample-proportion` will be retained. - - -

    - -## `bean-run`: Quantify variant effects -BEAN uses Bayesian network to incorporate gRNA editing outcome to provide posterior estimate of variant phenotype. The Bayesian network reflects data generation process. Briefly, -1. Cellular phenotype (either for cells are sorted upon for sorting screen, or log(proliferation rate)) is modeled as the Gaussian mixture distribution of wild-type phenotype and variant phenotype. -2. The weight of the mixture components are inferred from the reporter editing outcome and the chromatin accessibility of the loci. -3. Cells with each gRNA, formulated as the mixture distribution, is sorted by the phenotypic quantile to produce the gRNA counts. - -For the full detail, see the method section of the [BEAN manuscript](https://www.medrxiv.org/content/10.1101/2023.09.08.23295253v1). - -model - -

    -```bash -bean-run sorting[survival] variant[tiling] my_sorting_screen_filtered.h5ad \ -[--uniform-edit, --scale-by-acc [--acc-bw-path accessibility_signal.bw, --acc-col accessibility]] \ --o output_prefix/ \ ---fit-negctrl -``` - -### Input -`my_sorting_screen_filtered.h5ad` can be produced by one of the following: -1. [`bean-count-samples`]((#bean-count-samples-count-reporter-screen-data)) when you have raw `.fastq` file -2. (Limited to `bean-run variant` mode) `bean-create-screen` when you have flat `.csv` tables of gRNA metadata table, sample metadata table, gRNA counts table (# guides x # samples), and optionally # edits table. - ```bash - bean-create-screen gRNA_info_table.csv sample_info_table.csv gRNA_counts_table.csv \ - [--edits edit_counts_table.csv -o output.h5ad] - ``` - * `gRNA_info_table.csv` should have following columns. - * `name`: gRNA ID column - * `target`: This column denotes which target variant/element of each gRNA. - * `target_group [Optional]`: If negative control gRNA will be used, specify as "NegCtrl" in this column. - * `sample_info_table.csv` should have following columns. - * `sample_id`: ID of sequencing sample - * `replicate`: Replicate # of this sample - * `bin`: Name of the sorting bin - * `upper_quantile`: FACS sorting upper quantile - * `lower_quantile`: FACS sorting lower quantile - * `gRNA_counts_table.csv` should be formatted as follows. - * Columns include one of `sample_id` columns in `sample_info_table.csv` file. - * 1st row (row index) follows `name` (gRNA ID) in `gRNA_info_table.csv` file. -3. You can manually create the `AnnData` object with more annotations including allele counts: see [API tutorial](#using-bean-as-python-module) for full detail. - - -### Output -model - -Above command produces -* `output_prefix/bean_element_result.[model_type].csv` with following columns: - * Estimated variant effect sizes - * `mu` (Effect size): Mean of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. - * `mu_sd`: Mean of variant phenotype `mu` is modeled as normal distribution. The column shows fitted standard deviation of `mu` that quantify the uncertainty of the variant effect. - * `mu_z`: z-score of `mu` - * `sd`: Standard deviation of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. - * `CI[0.025`, `0.975]`: Credible interval of `mu` - * When negative control is provided, above columns with `_adj` suffix are provided, which are the corresponding values adjusted for negative control. - * Metrics on per-variant evidence provided in input (provided in `tiling` mode) - * `effective_edit_rate`: Sum of per-variant editing rates over all alleles observed in the input. Allele-level editing rate is divided by the number of variants observed in the allele prior to summing up. - * `n_guides`: # of guides covering the variant. - * `n_coocc`: # of cooccurring variants with a given variant in any alleles observed in the input. -* `output_prefix/bean_sgRNA_result.[model_type].csv`: - * `edit_rate`: Estimated editing rate at the target loci. - -### Optional parameters -* Run options - * `-o`, `--outdir`: Directory to save the run result. - * `--result-suffix` (default: ""): Suffix of the output files. - * `--uniform-edit` (default: `False`): Ignores variable editing - * `--scale-by-acc` (default: `False`): Scale guide editing efficiency by the target loci accessibility. Needs one of `acc_bw_path` or `acc_col` that provides per-guide accessibility information. - * `--acc-bw-path` : Accessibility .bigWig file to be used to assign accessibility of guides. To read accessibility information from the .bigWig file, input `.h5ad` file needs `chr` and `genomic_pos` column in `.guides` DataFrame. - * `--fit-negctrl` (default: `False`): Fit the shared negative control distribution to normalize the fitted parameters. - * `--negctrl-col` (default: `target_group`): Column in bdata.obs specifying if a guide is negative control. If the `bdata.guides[negctrl_col].tolower() == negctrl_col_value`, it is treated as negative control guide. - * `--negctrl-col-value` (default: `negctrl`): Column value in bdata.guides specifying if a guide is negative control. If the `bdata.guides[negctrl_col].tolower() == negctrl_col_value`, it is treated as negative control guide. - * `--repguide-mask` (default: `repguide_mask`): n_replicate x n_guide mask to mask the outlier guides. bdata.uns[repguide_mask] will be used. This is calculated with `bean-qc`. - * `--cuda` (default: `False`): Run on GPU - * `--device`: Optionally use GPU if provided valid GPU device name (ex. cuda:0) - * `--ignore-bcmatch` (default: `False`): If provided, even if the screen object has .X_bcmatch, ignore the count when fitting. - * `--allele-df-key` (default: `allele_counts`): bdata.uns[allele_df_key] will be used as the allele count. - * `--control-guide-tag` (default: `None`): (Relevant in bean-run *tiling* mode) If this string is in guide name, treat each guide separately not to mix the position. Used for non-targeting negative controls. -* Guide annotations (`bdata.guides` column keys) - * `--acc-col`: Column name in bdata.guides that specify raw ATAC-seq signal. - * `--target-column` (default: `target`): Column key in `bdata.guides` that describes the target element of each guide. - * `--guide-activity-col`: Column in `bdata.guides` DataFrame showing the editing rate estimated via external tools. -* Sample annotations (`bdata.samples` column keys) - * `--condition-column` (default: `condition`): Column key in `bdata.samples` that describes experimental condition. - * `--control-condition` (default: `bulk`): Value in `bdata.samples[condition_col]` that indicates control experimental condition. - * `-uq`, `--sorting-bin-upper-quantile-column` (default: `upper_quantile`): Column name with upper quantile values of each sorting bin in bdata.samples - * `-lq`, `--sorting-bin-lower-quantile-column` (default: `lower_quantile`): Column name with lower quantile values of each sorting bin in bdata.samples - -

    ## Data Structure ### ReporterScreen object @@ -484,7 +98,7 @@ Python package `bean` supports multiple data wrangling functionalities for `Repo ## Run time * Installation takes 14.4 mins after pytorch installation with pytorch in Dell XPS 13 Ubuntu WSL. -* `bean-run` takes 4.6 mins with `--scale-by-acc` tag in Dell XPS 13 Ubuntu WSL for variant screen dataset with 3455 guides and 6 replicates with 4 sorting bins. +* `bean run` takes 4.6 mins with `--scale-by-acc` tag in Dell XPS 13 Ubuntu WSL for variant screen dataset with 3455 guides and 6 replicates with 4 sorting bins. * Full pipeline takes 90.1s in GitHub Action for toy dataset of 2 replicates and 30 guides. ## Citation diff --git a/bean/annotate/utils.py b/bean/annotate/utils.py index 2252187..dd1358f 100644 --- a/bean/annotate/utils.py +++ b/bean/annotate/utils.py @@ -218,22 +218,12 @@ def get_cds_seq_pos_from_gene_name(gene_name: str, ref_version: str = "GRCh38"): return cds_chrom, cds_seq, cds_pos, strand -def parse_args(): - """Get the input arguments""" - print( - r""" - _ _ - / \ '\ __ _ _ _ - | \ \ / _(_) | |_ ___ _ _ - \ \ | | _| | | _/ -_) '_| - `.__|/ |_| |_|_|\__\___|_| - """ - ) - print("bean-filter: filter alleles") - parser = argparse.ArgumentParser( - prog="allele_filter", - description="Filter alleles based on edit position in spacer and frequency across samples.", - ) +def parse_args(parser=None): + if parser is None: + parser = argparse.ArgumentParser( + prog="allele_filter", + description="Filter alleles based on edit position in spacer and frequency across samples.", + ) parser.add_argument( "bdata_path", type=str, @@ -349,7 +339,7 @@ def parse_args(): action="store_true", help="Load temporary file and work from there.", ) - return parser.parse_args() + return parser def check_args(args): diff --git a/bean/framework/read_from_csvs.py b/bean/framework/read_from_csvs.py index 8331a3f..24667e0 100644 --- a/bean/framework/read_from_csvs.py +++ b/bean/framework/read_from_csvs.py @@ -4,21 +4,11 @@ from perturb_tools import Screen -def get_input_parser() -> argparse.Namespace: - """Add multi-sample specific arguments to the base parser.""" - print( - r""" - _ _ - / \ '\ _ - | \ \ __ _ _ ___ __ _| |_ ___ - \ \ | / _| '_/ -_) _` | _/ -_) - `.__|/ \__|_| \___\__,_|\__\___| - """ - ) - parser = argparse.ArgumentParser( - description="bean-create-screen parameters", - formatter_class=argparse.ArgumentDefaultsHelpFormatter, - ) +def get_input_parser(parser) -> argparse.Namespace: + # parser = argparse.ArgumentParser( + # description="bean-create-screen parameters", + # formatter_class=argparse.ArgumentDefaultsHelpFormatter, + # ) parser.add_argument( "gRNA_info_table_csv", type=str, diff --git a/bean/mapping/utils.py b/bean/mapping/utils.py index 34054ce..4bd87c1 100644 --- a/bean/mapping/utils.py +++ b/bean/mapping/utils.py @@ -30,23 +30,77 @@ def _check_library(library_name): ) from exc -def _get_input_parser(): - """Get the input data""" - print( - r""" - _ _ - / \ '\ _ - | \ \ __ ___ _ _ _ _| |_ - \ \ | / _/ _ \ || | ' \ _| - `.__|/ \__\___/\_,_|_||_\__| - """ - ) - - parser = argparse.ArgumentParser( - description="bean-count parameters", - formatter_class=argparse.ArgumentDefaultsHelpFormatter, +def get_input_parser(parser=None): + """Add multi-sample specific arguments to the base parser.""" + if parser is None: + parser = argparse.ArgumentParser() + parser.add_argument( + "-i", + "--sample-list", + type=str, + help="List of fastq and sample ids. Formatted as `R1_filepath,R2_filepath,sample_id`", + required=True, + ) + parser = _get_input_parser(parser) + parser.add_argument( + "-t", "--threads", type=int, help="Number of threads", default=10 + ) + parser.add_argument( + "--guide-start-seqs-file", + type=str, + help="CSV file path with per-sample `guide_start_seq` to be used." + + "Formatted as `sample_id, guide_start_seq`", + default=None, + ) + parser.add_argument( + "--guide-end-seqs-file", + type=str, + help="CSV file path with per-sample `guide_end_seq` to be used." + + "Formatted as `sample_id,guide_end_seq`", + default=None, + ) + parser.add_argument( + "--barcode-start-seqs-file", + type=str, + help="CSV file path with per-sample `barcode_start_seq` to be used." + + "Formatted as `sample_id,guide_end_seq`", + default=None, + ) + + parser.add_argument( + "--rerun", help="Recount each sample", action="store_true", default=False ) + return parser + + +def get_input_parser_count(parser=None): + """Get single-sample specific argument parser.""" + if parser is None: + parser = argparse.ArgumentParser() + parser.add_argument( + "--R1", + type=str, + help="FASTQ file path for read 1", + required=True, + ) + parser.add_argument( + "--R2", + type=str, + help="FASTQ file path for read 2.", + required=True, + ) + parser = _get_input_parser(parser) + return parser + + +def _get_input_parser(parser=None): + if parser is None: + parser = argparse.ArgumentParser( + description="bean-count parameters", + formatter_class=argparse.ArgumentDefaultsHelpFormatter, + ) + parser.add_argument( "-b", "--edited-base", diff --git a/bean/model/run.py b/bean/model/run.py index f8bde98..39752a9 100644 --- a/bean/model/run.py +++ b/bean/model/run.py @@ -32,18 +32,9 @@ def none_or_str(value): return value -def parse_args(): - print( - r""" - _ _ - / \ '\ - | \ \ _ _ _ _ _ _ - \ \ | | '_| || | ' \ - `.__|/ |_| \_,_|_||_| - """ - ) - print("bean-run: Run model to identify targeted variants and their impact.") - parser = argparse.ArgumentParser(description="Run model on data.") +def parse_args(parser=None): + if parser is None: + parser = argparse.ArgumentParser(description="Run model on data.") parser.add_argument( "selection", type=str, @@ -259,7 +250,7 @@ def parse_args(): help="Load existing .pkl file if present.", ) - return parser.parse_args() + return parser def check_args(args, bdata): diff --git a/bean/plotting/utils.py b/bean/plotting/utils.py index 8080d11..ef84816 100644 --- a/bean/plotting/utils.py +++ b/bean/plotting/utils.py @@ -1,19 +1,9 @@ import argparse -def parse_args(): - print(" \n~~~BEAN Profile~~~") - print("-Profile editing patterns of your editor-") - print( - r""" - _ _ __ _ _ - / \ '\ _ __ _ _ ___ / _(_) |___ - | \ \ | '_ \ '_/ _ \ _| | / -_) - \ \ | | .__/_| \___/_| |_|_\___| - `.__|/ |_| - """ - ) - parser = argparse.ArgumentParser() +def parse_args(parser=None): + if parser is None: + parser = argparse.ArgumentParser() parser.add_argument( "bdata_path", help="Path to the ReporterScreen object to run QC on", type=str ) @@ -55,10 +45,7 @@ def parse_args(): default=6, ) - args = parser.parse_args() - if args.output_prefix is None: - args.output_prefix = f"{args.bdata_path.rsplit('.h5ad', 1)[0]}" - return args + return parser def check_args(args): diff --git a/bean/qc/utils.py b/bean/qc/utils.py index 82c8bf8..036075f 100644 --- a/bean/qc/utils.py +++ b/bean/qc/utils.py @@ -4,22 +4,12 @@ import pandas as pd from copy import deepcopy import argparse -from ..framework.ReporterScreen import ReporterScreen, concat - - -def parse_args(): - print(" \n~~~BEANQC~~~") - print("-Check guide/sample level quality and mask / discard-") - print( - r""" - _ _ - / \ '\ ___ ___ - | \ \ / _ \ / __| - \ \ | | (_) | (__ - `.__|/ \__\_\\___| - """ - ) - parser = argparse.ArgumentParser() +from bean.framework.ReporterScreen import ReporterScreen, concat + + +def parse_args(parser=None): + if parser is None: + parser = argparse.ArgumentParser() parser.add_argument( "bdata_path", help="Path to the ReporterScreen object to run QC on", type=str ) @@ -155,12 +145,7 @@ def parse_args(): default="bulk", ) - args = parser.parse_args() - if args.out_screen_path is None: - args.out_screen_path = f"{args.bdata_path.rsplit('.h5ad', 1)[0]}.filtered.h5ad" - if args.out_report_prefix is None: - args.out_report_prefix = f"{args.bdata_path.rsplit('.h5ad', 1)[0]}.qc_report" - return args + return parser def check_args(args): diff --git a/bin/bean-count b/bin/bean-count old mode 100644 new mode 100755 diff --git a/bin/bean-count-samples b/bin/bean-count-samples old mode 100644 new mode 100755 diff --git a/bin/bean-create-screen b/bin/bean-create-screen old mode 100644 new mode 100755 diff --git a/bin/bean-filter b/bin/bean-filter old mode 100644 new mode 100755 diff --git a/bin/bean-profile b/bin/bean-profile old mode 100644 new mode 100755 diff --git a/bin/bean-qc b/bin/bean-qc old mode 100644 new mode 100755 diff --git a/bin/bean-run b/bin/bean-run old mode 100644 new mode 100755 diff --git a/docs/ReporterScreen_api.rst b/docs/ReporterScreen_api.rst index d333908..d489ffc 100644 --- a/docs/ReporterScreen_api.rst +++ b/docs/ReporterScreen_api.rst @@ -1,4 +1,4 @@ -Tutorial +ReporterScreen API tutorial ================== Load the required packages. (Anndata import isn't required to use the package). diff --git a/docs/cds.rst b/docs/cds.rst new file mode 100644 index 0000000..5dabfde --- /dev/null +++ b/docs/cds.rst @@ -0,0 +1,5 @@ +Coding sequence tiling library +*********************** +.. mdinclude:: tutorials/ldl_cds.md + +See :ref:`subcommands` for the full details. diff --git a/docs/commands/count.md b/docs/commands/count.md new file mode 100644 index 0000000..5f04b4c --- /dev/null +++ b/docs/commands/count.md @@ -0,0 +1,31 @@ +# `bean count[-samples]`: Count (reporter) screen data +`bean count-samples` (or `bean count` for a single sample) maps guide into guide counts, **allowing for base transition in spacer sequence**. When the matched reporter information is provided, it can count the **target site edits** and **alleles produced by each guide**. Mapping is efficiently done based on [CRISPResso2](https://github.com/pinellolab/CRISPResso2) modified for base-edit-aware mapping. + + + +```python +bean count-samples \ + --input sample_list.csv `# sample with lines 'R1_filepath,R2_filepath,sample_name\n'` \ + -b A `# base that is being edited (A/G)` \ + -f sgRNA_info_table.csv `# sgRNA information` \ + -o . `# output directory` \ + -r `# read edit/allele information from reporter` \ + -t 12 `# number of threads` \ + --name my_sorting_screen `# name of this sample run` \ +``` +```python +bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r +``` +By default, `bean count[-samples]` assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure. + + Read structuren + +See full detail [below](#full-parameters). + +# Input file format +See :ref:`input` for input file formats. + +# Output file format +`count` or `count-samples` produces `.h5ad` and `.xlsx` file with guide and per-guide allele counts. +* `.h5ad`: This output file follows annotated matrix format compatible with `AnnData` and is based on `Screen` object in [purturb_tools](https://github.com/pinellolab/perturb-tools). See [Data Structure](#data-structure) section for more information. +* `.xlsx`: This output file contains `.guides`, `.samples`, `.X[_bcmatch,_edits]`. (`allele_tables` are often too large to write into an Excel!) diff --git a/docs/commands/create-screen.md b/docs/commands/create-screen.md new file mode 100644 index 0000000..4cf6964 --- /dev/null +++ b/docs/commands/create-screen.md @@ -0,0 +1,12 @@ +## `bean create-screen`: Create ReporterScreen object from flat files +```bash +bean create-screen gRNA_library.csv sample_list.csv gRNA_counts_table.csv +``` +### Input + * [gRNA_library.csv](#1-gRNA_librarycsv) + * [sample_list.csv](#2-sample_listcsv) + * gRNA_counts_table.csv: Table with gRNA ID in the first column and sample IDs as the column names (first row) + +### Full Parameters + * `-e`, `--edits` (default: `None`): Path to edit counts .csv table, with index at first column and column names at the first row. + * `-o`, `--output-prefix` (default: `None`): Output file prefix (output will be saved as `output_prefix.h5ad`). If not provided, `gRNA_counts_table_csv` file prefix is used. \ No newline at end of file diff --git a/docs/commands/filter.md b/docs/commands/filter.md new file mode 100644 index 0000000..53d6ec1 --- /dev/null +++ b/docs/commands/filter.md @@ -0,0 +1,37 @@ +# `filter`: Filtering (and optionally translating) alleles +As `tiling` mode of `bean run` accounts for any robustly observed alleles, `bean filter` filters for such alleles. +```bash +bean filter my_sorting_screen_masked.h5ad \ +-o my_sorting_screen_filtered.h5ad `# Output file path` \ +``` + +# Output +Above command produces +* `my_sorting_screen_filtered.h5ad` with filtered alleles stored in `.uns`, +* `my_sorting_screen_filtered.filtered_allele_stats.pdf`, and `my_sorting_screen_filtered.filter_log.txt` that report allele count stats in each filtering step. + +You may want to adjust the flitering parameters to obtain optimal balance between # guides per variant & # variants that are scored. See example outputs of filtering step [here](docs/example_filtering_output/). + + +# Translating alleles +If you want to obtain **amino acid level variant** for coding sequence tiling screens, provide coding sequence positions which variants occuring within the coding sequence will be translated. *This is optional, but **highly recommended** to increase per-(coding)variant support.* + +Allele translation + + +```bash +bean filter my_sorting_screen.h5ad \ +-o my_sorting_screen_masked.h5ad \ +--translate `# Translate coding variants` \ +[ --translate-gene-name GENE_SYMBOL OR + --translate-genes-list path_to_gene_names_file.txt OR + --translate-fasta gene_exon.fa, OR + --translate-fastas-csv gene_exon_fas.csv] +``` +* When library covers a single gene, do either of the following: + 1. Feed `--translate-gene-name GENE_SYMBOL` if your `genomic_pos` column of `sgRNA_info_tbl` is compatible with [MANE transcript](https://useast.ensembl.org/info/genome/genebuild/mane.html)'s reference genome. (Per 10/23/2023, GRCh38). This will automatically load the exon positions based on MANE transcript annotation. + 2. To use your custom coding sequence and exon positions, feed `--translate-fasta gene_exon.fa` argument where `gene_exon.fa` is the FASTA file with entries of exons. [See full details here](docs/exon_fa_format.md). +* When library covers multiple genes, do either of the following: + 1. Feed `--translate-genes-list path_to_gene_names_file.txt` where `path_to_gene_names_file.txt` is file with one gene symbol per line. + 2. Feed `--translate-fastas-csv gene_exon_fas.csv` where `gene_exon_fas.csv` is the csv file with lines `gene_id,gene_exon_fasta_path` without header. Each FASTA file in `gene_exon_fasta_path` is formatted [as the single-gene FASTA file](docs/exon_fa_format.md). +* Translation will keep the variants outside the coding sequence as nucleotide-level variants, while aggregating variants leading to the same coding sequence variants. diff --git a/docs/commands/input.md b/docs/commands/input.md new file mode 100644 index 0000000..9fa1de9 --- /dev/null +++ b/docs/commands/input.md @@ -0,0 +1,37 @@ +This document describes the input files of :ref:`count_samples`. +## sgRNA_info_table.csv +File should contain following columns. +* `name`: gRNA ID column +* `sequence`: gRNA sequence +* `barcode`: R2 barcode to help match reporter to gRNA, written in the sense direction (as in R1) +* In order to use accessibility in the [variant effect quantification](#bean-run-quantify-variant-effects), provide accessibility information in one of two options. (For non-targeting guides, provide NA values (empty cell).) + * Option 1: `chrom` & `genomic_pos`: Chromosome (ex. `chr19`) and genomic position of guide sequence. You will have to provide the path to the bigwig file with matching reference version in `bean run`. + * Option 2: `accessibility_signal`: ATAC-seq signal value of the target loci of each guide. +* For variant library (gRNAs are designed to target specific variants and ignores bystander edits) + * `target`: This column denotes which target variant/element of each gRNA. This is not used in `bean count[-samples]` but required to run `bean run` in later steps. + * `target_group`: If negative/positive control gRNA will be considered in `bean qc` and/or `bean run`, specify as "NegCtrl"/"PosCtrl" in this column. + * `target_pos`: If `--match_target_pos` flag is used, input file needs `target_pos` which specifies 0-based relative position of targeted base within Reporter sequence. +* For tiling library (gRNAs tile coding / noncoding sequences) + * `strand`: Specifies gRNA strand information relative to the reference genome. + * `chrom`: Chromosome of gRNA targeted locus. + * `start_pos`: gRNA starting position in the genome. Required when you provide `strand` column. Should specify the smaller coordinate value among start and end position regardless of gRNA strandedness. + +Also see examples for [variant library](tests/data/test_guide_info.csv) and [tiling library](tests/data/test_guide_info_tiling.csv). + +## sample_list.csv +File should contain following columns with header. +* `R1_filepath`: Path to read 1 `.fastq[.gz]` file +* `R2_filepath`: Path to read 1 `.fastq[.gz]` file +* `sample_id`: ID of sequencing sample +* `replicate`: Replicate # of this sample (Should NOT contain `.`) +* `condition`: Name of the sorting bin (ex. `top`, `bot`), or label of timepoint (ex. `D5`, `D18`) + +For FACS sorting screens: +* `upper_quantile`: FACS sorting upper quantile +* `lower_quantile`: FACS sorting lower quantile + +For proliferation / survival screens: +* `time`: Numeric time following the base editing of each sample. + + +Also see examples for [FACS sorting screen](tests/data/sample_list.csv). \ No newline at end of file diff --git a/docs/commands/profile.md b/docs/commands/profile.md new file mode 100644 index 0000000..bbe8a8c --- /dev/null +++ b/docs/commands/profile.md @@ -0,0 +1,8 @@ +# `bean profile`: Profile editing patterns +```bash +bean profile my_sorting_screen.h5ad -o output_prefix `# Prefix for editing profile report` +``` +# Output +Above command produces `prefix_editing_preference.[html,ipynb]` as editing preferences ([see example](../../notebooks/profile_editing_preference.ipynb)). + +Allele translation \ No newline at end of file diff --git a/docs/commands/qc.md b/docs/commands/qc.md new file mode 100644 index 0000000..82b719e --- /dev/null +++ b/docs/commands/qc.md @@ -0,0 +1,94 @@ +# `bean qc`: QC of reporter screen data +```bash +bean qc \ + my_sorting_screen.h5ad `# Input ReporterScreen .h5ad file path` \ + -o my_sorting_screen_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r qc_report_my_sorting_screen `# Prefix for QC report` \ + --ctrl-cond presort `# "condition" column in the control sample before selection. Mean gRNA editing rates in these samples are reported. ` \ +# Inspect the output qc_report_my_sorting_screen.html to tweak QC threshold + +bean qc \ + my_sorting_screen.h5ad \ + -o my_sorting_screen_masked.h5ad \ + -r qc_report_my_sorting_screen \ + #[--count-correlation-thres 0.7 ...]\ + -b +``` + +`bean qc` supports following quality control and masks samples with low quality. Specifically: + +Allele translation + +* Plots guide coverage and the uniformity of coverage +* Guide count correlation between samples +* Log fold change correlation when positive controls are provided +* Plots editing rate distribution +* Identify samples with low guide coverage/guide count correlation/editing rate and mask the sample in `bdata.samples.mask` +* Identify outlier guides to filter out + +# Output +Above command produces +* `my_sorting_screen_masked.h5ad` without problematic replicate and guides and with sample masks, and +* `qc_report_my_sorting_screen.[html,ipynb]` as QC report. +##### Optional arguments: +* `-o OUT_SCREEN_PATH`, `--out-screen-path OUT_SCREEN_PATH` + Path where quality-filtered ReporterScreen object to be written to +* `-r OUT_REPORT_PREFIX`, `--out-report-prefix OUT_REPORT_PREFIX` + Output prefix of qc report (prefix.html, prefix.ipynb) + +##### QC thresholds: +* `--count-correlation-thres COUNT_CORRELATION_THRES` + Correlation threshold to mask out. +* `--edit-rate-thres EDIT_RATE_THRES` + Mean editing rate threshold per sample to mask out. +* `--lfc-thres LFC_THRES` + Positive guides' correlation threshold to filter out. + +##### Run options: +* `-b`, `--remove-bad-replicates` + Remove replicates with at least two of its samples meet the QC threshold (bean run does not support having only one sorting bin sample for a replicate). +* `-i`, `--ignore-missing-samples` + If the flag is not provided, if the ReporterScreen object does not contain all condiitons for + each replicate, make fake empty samples. If the flag is provided, don't add dummy samples. +* `--no-editing` Ignore QC about editing. Can be used for QC of other editing modalities. +* `--dont-recalculate-edits` + When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from + ReporterScreen.uns['allele_count']. + +##### Input `.h5ad` formatting: +Note that these arguements will change the way the QC metrics are calculated for guides, samples, or replicates. +* `--tiling TILING` Specify that the guide library is tiling library without 'n guides per target' design +* `--replicate-label REPLICATE_LABEL` + Label of column in `bdata.samples` that describes replicate ID. +* `--sample-covariates SAMPLE_COVARIATES` + Comma-separated list of column names in `bdata.samples` that describes non-selective + experimental condition. (drug treatment, etc.) +* `--condition-label CONDITION_LABEL` + Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time, + etc.) +###### Editing rate calculation + * `--control-condition CTRL_COND` + Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate + to be calculated. Default is `None`, which considers all samples. + * `--rel-pos-is-reporter` + Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If + `False`, those are relative to spacer position. + Editing rate is calculated with following parameters in + * Variant screens: + * `--target-pos-col TARGET_POS_COL` + Target position column in `bdata.guides` specifying target edit position in reporter + * tiling screens: + * `--edit-start-pos EDIT_START_POS` + Edit start position to quantify editing rate on, 0-based inclusive. + * `--edit-end-pos EDIT_END_POS` + Edit end position to quantify editing rate on, 0-based exclusive. +###### LFC of positive controls + * `--posctrl-col POSCTRL_COL` + Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all + gRNAs, feed empty string ''. + * `--posctrl-val POSCTRL_VAL` + Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the + positive control in calculating log fold change. + * `--lfc-conds LFC_CONDS` + Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated + between, delimited by comma \ No newline at end of file diff --git a/docs/commands/run.md b/docs/commands/run.md new file mode 100644 index 0000000..4c45259 --- /dev/null +++ b/docs/commands/run.md @@ -0,0 +1,64 @@ +# `bean run`: Quantify variant effects +BEAN uses Bayesian network to incorporate gRNA editing outcome to provide posterior estimate of variant phenotype. The Bayesian network reflects data generation process. Briefly, +1. Cellular phenotype (either for cells are sorted upon for sorting screen, or log(proliferation rate)) is modeled as the Gaussian mixture distribution of wild-type phenotype and variant phenotype. +2. The weight of the mixture components are inferred from the reporter editing outcome and the chromatin accessibility of the loci. +3. Cells with each gRNA, formulated as the mixture distribution, is sorted by the phenotypic quantile to produce the gRNA counts. + +For the full detail, see the method section of the [BEAN manuscript](https://www.medrxiv.org/content/10.1101/2023.09.08.23295253v1). + +model + +

    + +# Usage example +```bash +bean run sorting[survival] variant[tiling] my_sorting_screen_filtered.h5ad \ +[--uniform-edit, --scale-by-acc [--acc-bw-path accessibility_signal.bw, --acc-col accessibility]] \ +-o output_prefix/ \ +--fit-negctrl +``` +See full list of parameters [below](#full-parameters). + + +# Input +`my_sorting_screen_filtered.h5ad` can be produced by one of the following: +1. [`bean count-samples`]((#bean-count-samples-count-reporter-screen-data)) when you have raw `.fastq` file +2. (Limited to `bean run variant` mode) `bean create-screen` when you have flat `.csv` tables of gRNA metadata table, sample metadata table, gRNA counts table (# guides x # samples), and optionally # edits table. + ```bash + bean create-screen gRNA_info_table.csv sample_info_table.csv gRNA_counts_table.csv \ + [--edits edit_counts_table.csv -o output.h5ad] + ``` + * `gRNA_info_table.csv` should have following columns. + * `name`: gRNA ID column + * `target`: This column denotes which target variant/element of each gRNA. + * `target_group [Optional]`: If negative control gRNA will be used, specify as "NegCtrl" in this column. + * `sample_info_table.csv` should have following columns. + * `sample_id`: ID of sequencing sample + * `replicate`: Replicate # of this sample + * `bin`: Name of the sorting bin + * `upper_quantile`: FACS sorting upper quantile + * `lower_quantile`: FACS sorting lower quantile + * `gRNA_counts_table.csv` should be formatted as follows. + * Columns include one of `sample_id` columns in `sample_info_table.csv` file. + * 1st row (row index) follows `name` (gRNA ID) in `gRNA_info_table.csv` file. +3. You can manually create the `AnnData` object with more annotations including allele counts: see [API tutorial](#using-bean-as-python-module) for full detail. + + +# Output +model + +Above command produces +* `output_prefix/bean_element_result.[model_type].csv` with following columns: + * Estimated variant effect sizes + * `mu` (Effect size): Mean of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. + * `mu_sd`: Mean of variant phenotype `mu` is modeled as normal distribution. The column shows fitted standard deviation of `mu` that quantify the uncertainty of the variant effect. + * `mu_z`: z-score of `mu` + * `sd`: Standard deviation of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. + * `CI[0.025`, `0.975]`: Credible interval of `mu` + * When negative control is provided, above columns with `_adj` suffix are provided, which are the corresponding values adjusted for negative control. + * Metrics on per-variant evidence provided in input (provided in `tiling` mode) + * `effective_edit_rate`: Sum of per-variant editing rates over all alleles observed in the input. Allele-level editing rate is divided by the number of variants observed in the allele prior to summing up. + * `n_guides`: # of guides covering the variant. + * `n_coocc`: # of cooccurring variants with a given variant in any alleles observed in the input. +* `output_prefix/bean_sgRNA_result.[model_type].csv`: + * `edit_rate`: Estimated editing rate at the target loci. diff --git a/docs/conf.py b/docs/conf.py new file mode 100644 index 0000000..ba6c48a --- /dev/null +++ b/docs/conf.py @@ -0,0 +1,29 @@ +# Configuration file for the Sphinx documentation builder. +# +# For the full list of built-in configuration values, see the documentation: +# https://www.sphinx-doc.org/en/master/usage/configuration.html + +# -- Project information ----------------------------------------------------- +# https://www.sphinx-doc.org/en/master/usage/configuration.html#project-information + +project = "bean" +copyright = "2024, Jayoung Ryu" +author = "Jayoung Ryu" +release = "1.0.0" + +# -- General configuration --------------------------------------------------- +# https://www.sphinx-doc.org/en/master/usage/configuration.html#general-configuration + +extensions = ["sphinxarg.ext", "m2r"] + +templates_path = ["_templates"] +exclude_patterns = ["_build", "Thumbs.db", ".DS_Store"] + +root_doc = "index_" +numpydoc_show_class_members = False + +# -- Options for HTML output ------------------------------------------------- +# https://www.sphinx-doc.org/en/master/usage/configuration.html#options-for-html-output + +html_theme = "alabaster" +html_static_path = ["_static"] diff --git a/docs/count.rst b/docs/count.rst new file mode 100644 index 0000000..206dbd2 --- /dev/null +++ b/docs/count.rst @@ -0,0 +1,10 @@ +`bean count` +*********************** +.. mdinclude:: commands/count.md + +Full parameters +================== +.. argparse:: + :filename: ../bean/mapping/utils.py + :func: get_input_parser_count + :prog: bean count \ No newline at end of file diff --git a/docs/count_samples.rst b/docs/count_samples.rst new file mode 100644 index 0000000..fbbf8f1 --- /dev/null +++ b/docs/count_samples.rst @@ -0,0 +1,11 @@ +.. _count_samples: +`bean count-samples` +*********************** +.. mdinclude:: commands/count.md + +Full parameters +================== +.. argparse:: + :filename: ../bean/mapping/utils.py + :func: get_input_parser + :prog: bean count-samples \ No newline at end of file diff --git a/docs/filter.rst b/docs/filter.rst new file mode 100644 index 0000000..ce485ab --- /dev/null +++ b/docs/filter.rst @@ -0,0 +1,11 @@ +.. _filter: +`bean filter` +*********************** +.. mdinclude:: commands/filter.md + +Full parameters +================== +.. argparse:: + :filename: ../bean/annotate/utils.py + :func: parse_args + :prog: bean filter \ No newline at end of file diff --git a/docs/gwas.rst b/docs/gwas.rst new file mode 100644 index 0000000..e407ad9 --- /dev/null +++ b/docs/gwas.rst @@ -0,0 +1,5 @@ +GWAS variant library +*********************** +.. mdinclude:: tutorials/ldl_var.md + +See :ref:`subcommands` for the full details. diff --git a/docs/index_.rst b/docs/index_.rst new file mode 100644 index 0000000..d78bc9a --- /dev/null +++ b/docs/index_.rst @@ -0,0 +1,38 @@ +.. bean documentation master file, created by + sphinx-quickstart on Fri Mar 29 19:10:46 2024. + You can adapt this file completely to your liking, but it should at least + contain the root `toctree` directive. + +Welcome to `bean`'s documentation! +================================ +=================== +Workflows +=================== +.. toctree:: + :maxdepth: 2 + + gwas + cds + input + +=================== +`bean` subcommands +=================== +.. toctree:: + :maxdepth: 3 + + subcommands + +=================== +Screen data structure +=================== +.. toctree:: + ReporterScreen_api + +================== +Indices and tables +================== + +* :ref:`genindex` +* :ref:`modindex` +* :ref:`search` diff --git a/docs/input.rst b/docs/input.rst new file mode 100644 index 0000000..d63417e --- /dev/null +++ b/docs/input.rst @@ -0,0 +1,4 @@ +.. _input: +Input file format +*********************** +.. mdinclude:: commands/input.md \ No newline at end of file diff --git a/docs/profile.rst b/docs/profile.rst new file mode 100644 index 0000000..3ee525f --- /dev/null +++ b/docs/profile.rst @@ -0,0 +1,10 @@ +`bean profile` +*********************** +.. mdinclude:: commands/profile.md + +Full parameters +================== +.. argparse:: + :filename: ../bean/plotting/utils.py + :func: parse_args + :prog: bean profile \ No newline at end of file diff --git a/docs/qc.rst b/docs/qc.rst new file mode 100644 index 0000000..e0fbb94 --- /dev/null +++ b/docs/qc.rst @@ -0,0 +1,11 @@ +.. _qc: +`bean qc` +*********************** +.. mdinclude:: commands/qc.md + +Full parameters +================== +.. argparse:: + :filename: ../bean/qc/utils.py + :func: parse_args + :prog: bean qc \ No newline at end of file diff --git a/docs/run.rst b/docs/run.rst new file mode 100644 index 0000000..aea11a9 --- /dev/null +++ b/docs/run.rst @@ -0,0 +1,11 @@ +.. _run: +`bean run` +*********************** +.. mdinclude:: commands/run.md + +Full parameters +================== +.. argparse:: + :filename: ../bean/model/run.py + :func: parse_args + :prog: bean run \ No newline at end of file diff --git a/docs/subcommands.rst b/docs/subcommands.rst new file mode 100644 index 0000000..2feb03d --- /dev/null +++ b/docs/subcommands.rst @@ -0,0 +1,13 @@ +.. _subcommands: +=================== +Subcommands +=================== +.. toctree:: + :maxdepth: 2 + + count + count_samples + profile + qc + filter + run diff --git a/docs/tutorials/ldl_cds.md b/docs/tutorials/ldl_cds.md index c3de47a..ba78a00 100644 --- a/docs/tutorials/ldl_cds.md +++ b/docs/tutorials/ldl_cds.md @@ -14,7 +14,45 @@ Tiling screen that tiles gRNA densely across locus or multiple loci, selected ba

    -## 1. Count gRNA & reporter ([`bean-count-samples`](../../README#bean-count-samples-count-reporter-screen-data)) +## Example workflow +```bash +screen_id=my_sorting_tiling_screen + +# 1. Count gRNA & reporter +bean-count-samples \ +--input tests/data/sample_list_tiling.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f tests/data/test_guide_info_tiling_chrom.csv `# Contains gRNA metadata; see test file for example.`\ +-o ./ `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen` \ +--tiling + +# 2. QC samples & guides +bean-qc \ + bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r qc_report_${screen_id} `# Prefix for QC report` \ + +# 3. Filter & translate alleles +bean-filter ./bean_count_${screen_id}_masked.h5ad \ +-o ./bean_count_${screen_id}_alleleFiltered \ +--filter-target-basechange `# Filter based on intended base changes. If -b A was provided in bean-count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\ +--filter-window --edit-start-pos 0 --edit-end-pos 19 `# Filter based on editing window in spacer position within reporter.`\ +--filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \ +--translate --translate-genes-list tests/data/gene_symbols.txt + +# 4. Quantify variant effect +bean-run sorting tiling \ + ./bean_count_${screen_id}_alleleFiltered.h5ad \ + -o tests/test_res/var/ \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility +``` +See more details below. + +## 1. Count gRNA & reporter (:ref:`count_samples`) ``` screen_id=my_sorting_tiling_screen @@ -29,7 +67,7 @@ bean-count-samples \ ``` Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. -## 2. QC ([`bean-qc`](../../README#bean-qc-qc-of-reporter-screen-data)) +## 2. QC (:ref:`qc`) Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean-qc`](../../README#bean-qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) ``` bean-qc \ @@ -43,7 +81,7 @@ bean-qc \ If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. -## 3. Filter alleles ([`bean-filter`](../../README#bean-filter-filtering-and-optionally-translating-alleles)) +## 3. Filter alleles (:ref:`filter`) As tiling library doesn't have designated per-gRNA target variant, any base edit observed in reporter may be the candidate variant, while having too many variants with very low editing rate significantly decreases the power. Variants are filtered based on multiple criteria in `bean-fitler`. If the screen targets coding sequence, it's beneficial to translate edits into coding varaints whenever possible for better power. For translation, provide `--translate` and one of the following: @@ -68,7 +106,7 @@ bean-filter ./bean_count_${screen_id}_masked.h5ad \ Ouptut file `` shows number of alleles per guide and number of guides per variant, where we want high enough values for the latter. See the typical output for dataset with good editing coverage & filtering result [here](../example_filtering_ouptut/). -## 4. Quantify variant effect ([`bean-run`](../../README#bean-run-quantify-variant-effects)) +## 4. Quantify variant effect (:ref:`run`) By default, `bean-run [sorting,survival] tiling` uses most filtered allele counts table for variant identification and quantification of their effects. **Check [allele filtering output](../example_filtering_ouptut/)** and choose alternative filtered allele counts table if necessary. `bean-run` can take 3 run options to quantify editing rate: @@ -107,6 +145,4 @@ By default, `bean-run [sorting,survival] tiling` uses most filtered allele count -o tests/test_res/var/ \ --fit-negctrl \ --uniform-edit - ``` - -See [full argument list](../../README#optional-parameters) to accommodate different input sample & guide metadata columns/values and run options. \ No newline at end of file + ``` \ No newline at end of file diff --git a/docs/tutorials/ldl_var.md b/docs/tutorials/ldl_var.md index 263899a..1542536 100644 --- a/docs/tutorials/ldl_var.md +++ b/docs/tutorials/ldl_var.md @@ -14,10 +14,41 @@ GWAS variant screen with per-variant gRNA tiling design, selected based on FACS

    -## 1. Count gRNA & reporter ([`bean-count-samples`](../../README#bean-count-samples-count-reporter-screen-data)) +## Example workflow +```bash +screen_id=my_sorting_tiling_screen + +# 1. Count gRNA & reporter +bean-count-samples \ +--input tests/data/sample_list.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f tests/data/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ +-o ./ `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen to be counted` + +# 2. QC samples & guides +bean-qc \ + bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r qc_report_${screen_id} `# Prefix for QC report` \ + -b ` # Remove replicates with no good samples. + +# 3. Quantify variant effect +bean-run sorting variant \ + tests/data/bean_count_${screen_id}_masked.h5ad \ + -o tests/test_res/var/ \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility ``` +See more details below. + +## 1. Count gRNA & reporter (:ref:`count_samples`) +```bash screen_id=my_sorting_tiling_screen +# 1. Count gRNA & reporter bean-count-samples \ --input tests/data/sample_list.csv `# Contains fastq file path; see test file for example.`\ -b A `# Base A is edited (into G)` \ @@ -28,7 +59,7 @@ bean-count-samples \ ``` Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. -## 2. QC ([`bean-qc`](../../README#bean-qc-qc-of-reporter-screen-data)) +## 2. QC samples & guides (:ref:`qc`) Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean-qc`](../../README#bean-qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) ``` bean-qc \ @@ -42,7 +73,7 @@ bean-qc \ If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. -## 3. Quantify variant effect ([`bean-run`](../../README#bean-run-quantify-variant-effects)) +## 3. Quantify variant effect (:ref:`run`) `bean-run` can take 3 run options to quantify editing rate: 1. From **reporter + accessibility** @@ -65,7 +96,9 @@ If the data does not include reporter editing data, you can provide `--no-editin --accessibility-bw accessibility.bw ``` -2. From **reporter** +2. From **reporter**, without accessibility + + This assumes the all target sites have the uniform chromatin accessibility. ``` bean-run sorting variant \ tests/data/bean_count_${screen_id}_masked.h5ad \ @@ -73,12 +106,11 @@ If the data does not include reporter editing data, you can provide `--no-editin --fit-negctrl ``` 3. No reporter information, assume the same editing efficiency of all gRNAs. - Use this option if your data don't have editing rate information. + Use this option if your data don't have editing outcome information. ``` bean-run sorting variant \ tests/data/bean_count_${screen_id}_masked.h5ad \ -o tests/test_res/var/ \ --fit-negctrl \ --uniform-edit - ``` -See [full argument list](../../README#optional-parameters) to accommodate different input sample & guide metadata columns/values and run options. \ No newline at end of file + ``` \ No newline at end of file diff --git a/setup.py b/setup.py index 5e00fcf..04a28ce 100644 --- a/setup.py +++ b/setup.py @@ -24,13 +24,14 @@ "Cython", ], scripts=[ - "bin/bean-count", - "bin/bean-count-samples", - "bin/bean-create-screen", - "bin/bean-profile", - "bin/bean-qc", - "bin/bean-filter", - "bin/bean-run", # TODO: prevent error when extra requirements are not met. + "bin/bean" + # "bin/bean-count", + # "bin/bean-count-samples", + # "bin/bean-create-screen", + # "bin/bean-profile", + # "bin/bean-qc", + # "bin/bean-filter", + # "bin/bean-run", # TODO: prevent error when extra requirements are not met. ], install_requires=[ "numpy", diff --git a/tests/test_count.py b/tests/test_count.py index fd9500a..dc5fb89 100644 --- a/tests/test_count.py +++ b/tests/test_count.py @@ -7,7 +7,7 @@ @pytest.mark.order(2) def test_count(): - cmd = "bean-count --R1 tests/data/test_R1.fastq --R2 tests/data/test_R2.fastq -b A -f tests/data/test_guide_info.csv -o tests/test_res/ -r --guide-start-seq=GGAAAGGACGAAACACCG" + cmd = "bean count --R1 tests/data/test_R1.fastq --R2 tests/data/test_R2.fastq -b A -f tests/data/test_guide_info.csv -o tests/test_res/ -r --guide-start-seq=GGAAAGGACGAAACACCG" try: subprocess.check_output( cmd, @@ -20,7 +20,7 @@ def test_count(): @pytest.mark.order(3) def test_guide_count(): - cmd = "bean-count --R1 tests/data/test_R1.fastq --R2 tests/data/test_R2.fastq -b A -f tests/data/test_guide_info.csv -o tests/test_res/ -g --guide-start-seq=GGAAAGGACGAAACACCG" + cmd = "bean count --R1 tests/data/test_R1.fastq --R2 tests/data/test_R2.fastq -b A -f tests/data/test_guide_info.csv -o tests/test_res/ -g --guide-start-seq=GGAAAGGACGAAACACCG" try: subprocess.check_output( cmd, @@ -33,7 +33,7 @@ def test_guide_count(): @pytest.mark.order(4) def test_count_samples(): - cmd = "bean-count-samples --input tests/data/sample_list.csv -b A -f tests/data/test_guide_info.csv -o tests/test_res/ -r --guide-start-seq=GGAAAGGACGAAACACCG" + cmd = "bean count-samples --input tests/data/sample_list.csv -b A -f tests/data/test_guide_info.csv -o tests/test_res/ -r --guide-start-seq=GGAAAGGACGAAACACCG" try: subprocess.check_output( cmd, @@ -45,7 +45,7 @@ def test_count_samples(): @pytest.mark.order(5) def test_count_samples_bcstart(): - cmd = "bean-count-samples --input tests/data/sample_list.csv -b A -f tests/data/test_guide_info.csv -o tests/test_res/ -r --barcode-start-seq=GGAA" + cmd = "bean count-samples --input tests/data/sample_list.csv -b A -f tests/data/test_guide_info.csv -o tests/test_res/ -r --barcode-start-seq=GGAA" try: subprocess.check_output( cmd, @@ -67,7 +67,7 @@ def test_barcode_start_idx(): @pytest.mark.order(6) def test_count_samples_tiling(): - cmd = "bean-count-samples --input tests/data/sample_list_tiling.csv -b A -f tests/data/test_guide_info_tiling_chrom.csv -o tests/test_res/ -r" + cmd = "bean count-samples --input tests/data/sample_list_tiling.csv -b A -f tests/data/test_guide_info_tiling_chrom.csv -o tests/test_res/ -r" try: subprocess.check_output( cmd, @@ -80,7 +80,7 @@ def test_count_samples_tiling(): @pytest.mark.order(7) def test_count_chroms(): - cmd = "bean-count --R1 tests/data/test_tiling_R1.fastq --R2 tests/data/test_tiling_R2.fastq -b A -f tests/data/test_guide_info_tiling_chrom.csv -o tests/test_res/ -r" + cmd = "bean count --R1 tests/data/test_tiling_R1.fastq --R2 tests/data/test_tiling_R2.fastq -b A -f tests/data/test_guide_info_tiling_chrom.csv -o tests/test_res/ -r" try: subprocess.check_output( cmd, diff --git a/tests/test_create.py b/tests/test_create.py index 8adc87c..c9373ef 100755 --- a/tests/test_create.py +++ b/tests/test_create.py @@ -1,8 +1,9 @@ import pytest import subprocess + def test_create_screen(): - cmd = "bean-create-screen tests/data/var_mini_guides.csv tests/data/var_mini_samples.csv tests/data/var_mini_counts.csv" + cmd = "bean create-screen tests/data/var_mini_guides.csv tests/data/var_mini_samples.csv tests/data/var_mini_counts.csv" try: subprocess.check_output( cmd, diff --git a/tests/test_filter.py b/tests/test_filter.py index e5e3f59..06b733c 100644 --- a/tests/test_filter.py +++ b/tests/test_filter.py @@ -5,7 +5,7 @@ @pytest.mark.order(11) def test_filter_varscreen(): - cmd = "bean-filter tests/data/var_mini_screen_masked.h5ad -o tests/data/var_mini_screen_annotated -s 0 -e 19 -w -b -t -ap 0.1 -sp 0.3" + cmd = "bean filter tests/data/var_mini_screen_masked.h5ad -o tests/data/var_mini_screen_annotated -s 0 -e 19 -w -b -t -ap 0.1 -sp 0.3" try: subprocess.check_output( cmd, @@ -18,7 +18,7 @@ def test_filter_varscreen(): @pytest.mark.order(12) def test_filter_tiling_screen(): - cmd = "bean-filter tests/data/tiling_mini_screen_masked.h5ad -o tests/data/tiling_mini_screen_annotated -s 0 -e 19 -w -b -t -ap 0.1 -sp 0.3" + cmd = "bean filter tests/data/tiling_mini_screen_masked.h5ad -o tests/data/tiling_mini_screen_annotated -s 0 -e 19 -w -b -t -ap 0.1 -sp 0.3" try: subprocess.check_output( cmd, @@ -31,7 +31,7 @@ def test_filter_tiling_screen(): @pytest.mark.order(13) def test_filter_tiling_screen_translate_genename(): - cmd = "bean-filter tests/data/tiling_mini_screen_masked.h5ad -o tests/data/tiling_mini_screen_annotated_wrong -s 0 -e 19 -w -b -ap 0.1 -sp 0.3 --translate --translate-gene LDLR" + cmd = "bean filter tests/data/tiling_mini_screen_masked.h5ad -o tests/data/tiling_mini_screen_annotated_wrong -s 0 -e 19 -w -b -ap 0.1 -sp 0.3 --translate --translate-gene LDLR" try: subprocess.check_output( cmd, @@ -44,7 +44,7 @@ def test_filter_tiling_screen_translate_genename(): @pytest.mark.order(14) def test_filter_tiling_screen_translate_fasta(): - cmd = "bean-filter tests/data/tiling_mini_screen_masked.h5ad -o tests/data/tiling_mini_screen_annotated_wrong -s 0 -e 19 -w -b -ap 0.1 -sp 0.3 --translate --translate-fasta tests/data/ldlr_exons.fa" + cmd = "bean filter tests/data/tiling_mini_screen_masked.h5ad -o tests/data/tiling_mini_screen_annotated_wrong -s 0 -e 19 -w -b -ap 0.1 -sp 0.3 --translate --translate-fasta tests/data/ldlr_exons.fa" try: subprocess.check_output( cmd, @@ -57,7 +57,7 @@ def test_filter_tiling_screen_translate_fasta(): @pytest.mark.order(15) def test_filter_tiling_screen_translate_genenames(): - cmd = "bean-filter tests/data/tiling_mini_screen_masked.h5ad -o tests/data/tiling_mini_screen_alleleFiltered -s 0 -e 19 -w -b -t -ap 0.1 -sp 0.3 --translate --translate-genes-list tests/data/gene_symbols.txt" + cmd = "bean filter tests/data/tiling_mini_screen_masked.h5ad -o tests/data/tiling_mini_screen_alleleFiltered -s 0 -e 19 -w -b -t -ap 0.1 -sp 0.3 --translate --translate-genes-list tests/data/gene_symbols.txt" try: subprocess.check_output( cmd, diff --git a/tests/test_qc.py b/tests/test_qc.py index 4ceb76e..b2c0c05 100644 --- a/tests/test_qc.py +++ b/tests/test_qc.py @@ -4,7 +4,7 @@ @pytest.mark.order(7) def test_qc(): - cmd = "bean-qc tests/data/var_mini_screen.h5ad -o tests/data/var_mini_screen_masked.h5ad -r tests/test_res/qc_report_var_mini_screen --count-correlation-thres 0.6" + cmd = "bean qc tests/data/var_mini_screen.h5ad -o tests/data/var_mini_screen_masked.h5ad -r tests/test_res/qc_report_var_mini_screen --count-correlation-thres 0.6" try: subprocess.check_output( cmd, @@ -17,7 +17,7 @@ def test_qc(): @pytest.mark.order(8) def test_qc_tiling(): - cmd = "bean-qc tests/data/tiling_mini_screen.h5ad -o tests/data/tiling_mini_screen_masked.h5ad -r tests/test_res/qc_report_tiling_mini_screen --count-correlation-thres 0.6 --posctrl-col ''" + cmd = "bean qc tests/data/tiling_mini_screen.h5ad -o tests/data/tiling_mini_screen_masked.h5ad -r tests/test_res/qc_report_tiling_mini_screen --count-correlation-thres 0.6 --posctrl-col ''" try: subprocess.check_output( cmd, @@ -30,7 +30,7 @@ def test_qc_tiling(): @pytest.mark.order(9) def test_dummy_insertion_varscreen(): - cmd = "bean-qc tests/data/var_mini_screen_missing.h5ad -o tests/data/var_mini_screen_missing_masked.h5ad -r tests/test_res/qc_report_var_mini_screen_missing --count-correlation-thres 0.6" + cmd = "bean qc tests/data/var_mini_screen_missing.h5ad -o tests/data/var_mini_screen_missing_masked.h5ad -r tests/test_res/qc_report_var_mini_screen_missing --count-correlation-thres 0.6" try: subprocess.check_output( cmd, shell=True, universal_newlines=True, stderr=subprocess.STDOUT @@ -43,7 +43,7 @@ def test_dummy_insertion_varscreen(): @pytest.mark.order(10) def test_dummy_insertion_tilingscreen(): - cmd = "bean-qc tests/data/tiling_mini_screen_missing.h5ad -o tests/data/tiling_mini_screen_missing_masked.h5ad -r tests/test_res/qc_report_tiling_mini_screen_missing --count-correlation-thres 0.6 --posctrl-col ''" + cmd = "bean qc tests/data/tiling_mini_screen_missing.h5ad -o tests/data/tiling_mini_screen_missing_masked.h5ad -r tests/test_res/qc_report_tiling_mini_screen_missing --count-correlation-thres 0.6 --posctrl-col ''" try: subprocess.check_output( cmd, shell=True, universal_newlines=True, stderr=subprocess.STDOUT diff --git a/tests/test_run.py b/tests/test_run.py index 734b8ff..699d1c3 100644 --- a/tests/test_run.py +++ b/tests/test_run.py @@ -4,7 +4,7 @@ @pytest.mark.order(16) def test_run_variant_wacc(): - cmd = "bean-run sorting variant tests/data/var_mini_screen_annotated.h5ad --scale-by-acc --acc-bw-path tests/data/accessibility_signal_chr6.bw -o tests/test_res/var/ --repguide-mask None --n-iter 10" + cmd = "bean run sorting variant tests/data/var_mini_screen_annotated.h5ad --scale-by-acc --acc-bw-path tests/data/accessibility_signal_chr6.bw -o tests/test_res/var/ --repguide-mask None --n-iter 10" try: subprocess.check_output( cmd, @@ -17,7 +17,7 @@ def test_run_variant_wacc(): @pytest.mark.order(17) def test_run_variant_noacc(): - cmd = "bean-run sorting variant tests/data/var_mini_screen_annotated.h5ad -o tests/test_res/var/ --n-iter 10" + cmd = "bean run sorting variant tests/data/var_mini_screen_annotated.h5ad -o tests/test_res/var/ --n-iter 10" try: subprocess.check_output( cmd, @@ -30,7 +30,7 @@ def test_run_variant_noacc(): @pytest.mark.order(18) def test_run_variant_wo_negctrl_uniform(): - cmd = "bean-run sorting variant tests/data/var_mini_screen_annotated.h5ad -o tests/test_res/var/ --uniform-edit --n-iter 10" + cmd = "bean run sorting variant tests/data/var_mini_screen_annotated.h5ad -o tests/test_res/var/ --uniform-edit --n-iter 10" try: subprocess.check_output( cmd, @@ -43,7 +43,7 @@ def test_run_variant_wo_negctrl_uniform(): @pytest.mark.order(19) def test_run_variant_wacc_negctrl(): - cmd = "bean-run sorting variant tests/data/var_mini_screen_annotated.h5ad --scale-by-acc --acc-bw-path tests/data/accessibility_signal_chr6.bw -o tests/test_res/var/ --repguide-mask None --n-iter 10 --fit-negctrl " + cmd = "bean run sorting variant tests/data/var_mini_screen_annotated.h5ad --scale-by-acc --acc-bw-path tests/data/accessibility_signal_chr6.bw -o tests/test_res/var/ --repguide-mask None --n-iter 10 --fit-negctrl " try: subprocess.check_output( cmd, @@ -56,7 +56,7 @@ def test_run_variant_wacc_negctrl(): @pytest.mark.order(20) def test_run_variant_noacc_negctrl(): - cmd = "bean-run sorting variant tests/data/var_mini_screen_annotated.h5ad -o tests/test_res/var/ --fit-negctrl --n-iter 10" + cmd = "bean run sorting variant tests/data/var_mini_screen_annotated.h5ad -o tests/test_res/var/ --fit-negctrl --n-iter 10" try: subprocess.check_output( cmd, @@ -69,7 +69,7 @@ def test_run_variant_noacc_negctrl(): @pytest.mark.order(21) def test_run_variant_uniform_negctrl(): - cmd = "bean-run sorting variant tests/data/var_mini_screen_annotated.h5ad -o tests/test_res/var/ --uniform-edit --fit-negctrl --n-iter 10" + cmd = "bean run sorting variant tests/data/var_mini_screen_annotated.h5ad -o tests/test_res/var/ --uniform-edit --fit-negctrl --n-iter 10" try: subprocess.check_output( cmd, @@ -82,7 +82,7 @@ def test_run_variant_uniform_negctrl(): @pytest.mark.order(22) def test_run_tiling_wo_negctrl(): - cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad --scale-by-acc --acc-bw-path tests/data/accessibility_signal.bw -o tests/test_res/tiling/ --control-guide-tag None --repguide-mask None --n-iter 10" + cmd = "bean run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad --scale-by-acc --acc-bw-path tests/data/accessibility_signal.bw -o tests/test_res/tiling/ --control-guide-tag None --repguide-mask None --n-iter 10" try: subprocess.check_output( cmd, @@ -95,7 +95,7 @@ def test_run_tiling_wo_negctrl(): @pytest.mark.order(23) def test_run_tiling_with_wo_negctrl_noacc(): - cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad -o tests/test_res/tiling/ --control-guide-tag None --repguide-mask None --n-iter 10" + cmd = "bean run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad -o tests/test_res/tiling/ --control-guide-tag None --repguide-mask None --n-iter 10" try: subprocess.check_output( cmd, @@ -108,7 +108,7 @@ def test_run_tiling_with_wo_negctrl_noacc(): @pytest.mark.order(23) def test_run_tiling_with_wo_negctrl_uniform(): - cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad -o tests/test_res/tiling/ --uniform-edit --allele-df-key allele_counts_spacer_0_19_noindels_A.G_translated_prop0.1_0.3 --control-guide-tag None --repguide-mask None --n-iter 10" + cmd = "bean run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad -o tests/test_res/tiling/ --uniform-edit --allele-df-key allele_counts_spacer_0_19_noindels_A.G_translated_prop0.1_0.3 --control-guide-tag None --repguide-mask None --n-iter 10" try: subprocess.check_output( cmd, @@ -121,7 +121,7 @@ def test_run_tiling_with_wo_negctrl_uniform(): @pytest.mark.order(24) def test_run_tiling_negctrl_allelekey(): - cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad --scale-by-acc --acc-bw-path tests/data/accessibility_signal.bw -o tests/test_res/tiling/ --fit-negctrl --negctrl-col strand --negctrl-col-value neg --control-guide-tag neg --repguide-mask None --n-iter 10" + cmd = "bean run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad --scale-by-acc --acc-bw-path tests/data/accessibility_signal.bw -o tests/test_res/tiling/ --fit-negctrl --negctrl-col strand --negctrl-col-value neg --control-guide-tag neg --repguide-mask None --n-iter 10" try: subprocess.check_output( cmd, @@ -134,7 +134,7 @@ def test_run_tiling_negctrl_allelekey(): @pytest.mark.order(25) def test_run_tiling_with_negctrl_noacc(): - cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad -o tests/test_res/tiling/ --allele-df-key allele_counts_spacer_0_19_noindels_A.G_translated_prop0.1_0.3 --fit-negctrl --negctrl-col strand --negctrl-col-value neg --control-guide-tag neg --repguide-mask None --n-iter 10" + cmd = "bean run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad -o tests/test_res/tiling/ --allele-df-key allele_counts_spacer_0_19_noindels_A.G_translated_prop0.1_0.3 --fit-negctrl --negctrl-col strand --negctrl-col-value neg --control-guide-tag neg --repguide-mask None --n-iter 10" try: subprocess.check_output( cmd, @@ -147,7 +147,7 @@ def test_run_tiling_with_negctrl_noacc(): @pytest.mark.order(26) def test_run_tiling_with_negctrl_uniform(): - cmd = "bean-run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad -o tests/test_res/tiling/ --uniform-edit --allele-df-key allele_counts_spacer_0_19_noindels_A.G_translated_prop0.1_0.3 --fit-negctrl --negctrl-col strand --negctrl-col-value neg --control-guide-tag neg --repguide-mask None --n-iter 10" + cmd = "bean run sorting tiling tests/data/tiling_mini_screen_annotated.h5ad -o tests/test_res/tiling/ --uniform-edit --allele-df-key allele_counts_spacer_0_19_noindels_A.G_translated_prop0.1_0.3 --fit-negctrl --negctrl-col strand --negctrl-col-value neg --control-guide-tag neg --repguide-mask None --n-iter 10" try: subprocess.check_output( cmd, From ce22f2d526401751557762a9b07029141e541a3c Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 22:05:53 -0400 Subject: [PATCH 020/107] test doc --- .github/workflows/documentation.yml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 62a2eb6..261cb97 100644 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -16,7 +16,7 @@ jobs: pip install sphinx sphinx_rtd_theme myst_parser - name: Sphinx build run: | - sphinx-build doc _build + sphinx-build bean/doc _build - name: Deploy to GitHub Pages uses: peaceiris/actions-gh-pages@v3 if: ${{ github.event_name == 'push' && github.ref == 'refs/heads/main' }} From 4f11b82ceba9422fb7e4bf07b30735a48c0e9aed Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 22:07:39 -0400 Subject: [PATCH 021/107] test doc --- .github/workflows/documentation.yml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 261cb97..bca2a17 100644 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -16,7 +16,7 @@ jobs: pip install sphinx sphinx_rtd_theme myst_parser - name: Sphinx build run: | - sphinx-build bean/doc _build + sphinx-build docs _build - name: Deploy to GitHub Pages uses: peaceiris/actions-gh-pages@v3 if: ${{ github.event_name == 'push' && github.ref == 'refs/heads/main' }} From d7eaa055eaaf54edd88fa2ede64f73dcdf4b0823 Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 22:09:17 -0400 Subject: [PATCH 022/107] move files --- bean/cli/__init__.py | 0 bin/bean-count => bean/cli/count.py | 195 ++++++++---------- .../cli/count_samples.py | 73 ++----- .../cli/create_screen.py | 20 +- bean/cli/execute.py | 60 ++++++ bin/bean-filter => bean/cli/filter.py | 41 ++-- bin/bean-profile => bean/cli/profile.py | 18 +- bin/bean-qc => bean/cli/qc.py | 102 ++++----- bin/bean-run => bean/cli/run.py | 21 +- bin/bean | 5 + 10 files changed, 290 insertions(+), 245 deletions(-) create mode 100755 bean/cli/__init__.py rename bin/bean-count => bean/cli/count.py (77%) mode change 100755 => 100644 rename bin/bean-count-samples => bean/cli/count_samples.py (77%) mode change 100755 => 100644 rename bin/bean-create-screen => bean/cli/create_screen.py (65%) mode change 100755 => 100644 create mode 100755 bean/cli/execute.py rename bin/bean-filter => bean/cli/filter.py (87%) mode change 100755 => 100644 rename bin/bean-profile => bean/cli/profile.py (70%) mode change 100755 => 100644 rename bin/bean-qc => bean/cli/qc.py (73%) mode change 100755 => 100644 rename bin/bean-run => bean/cli/run.py (93%) mode change 100755 => 100644 create mode 100755 bin/bean diff --git a/bean/cli/__init__.py b/bean/cli/__init__.py new file mode 100755 index 0000000..e69de29 diff --git a/bin/bean-count b/bean/cli/count.py old mode 100755 new mode 100644 similarity index 77% rename from bin/bean-count rename to bean/cli/count.py index 49a7153..73cf1a9 --- a/bin/bean-count +++ b/bean/cli/count.py @@ -1,105 +1,90 @@ -#!/usr/bin/env python -# -*- coding: utf-8 -*- -"""Count guides, optionally with reporter and alleles of a single sequencing sample.""" - -import logging -import os -import sys - -import bean -from bean.mapping.utils import ( - _check_arguments, - _get_input_parser, - _check_file, - _get_first_read_length, - _check_read_length, -) - -logging.basicConfig( - level=logging.INFO, - format="%(levelname)-5s @ %(asctime)s:\n\t %(message)s \n", - datefmt="%a, %d %b %Y %H:%M:%S", - stream=sys.stderr, - filemode="w", -) -error = logging.critical -warn = logging.warning -debug = logging.debug -info = logging.info - - -_ROOT = os.path.abspath(os.path.dirname(__file__)) - - -def get_input_parser(): - """Get single-sample specific argument parser.""" - parser = _get_input_parser() - parser.add_argument( - "--R1", - type=str, - help="fastq file for read 1", - required=True, - default="Fastq filename", - ) - parser.add_argument( - "--R2", - type=str, - help="fastq file for read 2, sorted as the same name order as in --R1 file.", - required=True, - default="Fastq filename", - ) - return parser - - -def check_arguments(args, info_logger, warn_logger, error_logger): - args = _check_arguments( - args, info_logger=info, warn_logger=warn, error_logger=error - ) - _check_file(args.R1) - _check_file(args.R2) - read_length = _get_first_read_length(args.R1) - _check_read_length(args, read_length, warn) - return args - - -def main(): - parser = get_input_parser() - args = parser.parse_args() - - args = check_arguments(args, info_logger=info, warn_logger=warn, error_logger=error) - args_dict = vars(args) - - edited_from = args_dict["edited_base"] - match_target_pos = args_dict["match_target_pos"] - - counter = bean.mp.GuideEditCounter(**args_dict) - counter.check_filter_fastq() - - counter.get_counts() - if counter.count_reporter_edits: - counter.screen.uns["allele_counts"] = counter.screen.uns["allele_counts"].loc[ - counter.screen.uns["allele_counts"].allele.map(str) != "", : - ] - if match_target_pos: - base_editing_map = {"A": "G", "C": "T"} - edited_to = base_editing_map[edited_from] - counter.screen.get_edit_mat_from_uns( - edited_from, edited_to, match_target_pos - ) - counter.screen.write(f"{counter.output_dir}.h5ad") - counter.screen.to_Excel(f"{counter.output_dir}.xlsx") - info(f"Output written at:\n {counter.output_dir}.h5ad,\n {counter.output_dir}.xlsx") - info("All Done!") - print( - r""" - _ _ - / \ '\ _ - | \ \ __ ___ _ _ _ _| |_ - \ \ | / _/ _ \ || | ' \ _| - `.__|/ \__\___/\_,_|_||_\__| - """ - ) - - -if __name__ == "__main__": - main() +#!/usr/bin/env python +# -*- coding: utf-8 -*- +"""Count guides, optionally with reporter and alleles of a single sequencing sample.""" + +import logging +import os +import sys + +import bean +from bean.mapping.utils import ( + _check_arguments, + _check_file, + _get_first_read_length, + _check_read_length, +) + +logging.basicConfig( + level=logging.INFO, + format="%(levelname)-5s @ %(asctime)s:\n\t %(message)s \n", + datefmt="%a, %d %b %Y %H:%M:%S", + stream=sys.stderr, + filemode="w", +) +error = logging.critical +warn = logging.warning +debug = logging.debug +info = logging.info + + +_ROOT = os.path.abspath(os.path.dirname(__file__)) + + + + + +def check_arguments(args, info_logger, warn_logger, error_logger): + args = _check_arguments( + args, info_logger=info, warn_logger=warn, error_logger=error + ) + _check_file(args.R1) + _check_file(args.R2) + read_length = _get_first_read_length(args.R1) + _check_read_length(args, read_length, warn) + return args + + +def main(args): + """Get the input data""" + print( + r""" + _ _ + / \ '\ _ + | \ \ __ ___ _ _ _ _| |_ + \ \ | / _/ _ \ || | ' \ _| + `.__|/ \__\___/\_,_|_||_\__| + """ + ) + args = check_arguments(args, info_logger=info, warn_logger=warn, error_logger=error) + args_dict = vars(args) + + edited_from = args_dict["edited_base"] + match_target_pos = args_dict["match_target_pos"] + + counter = bean.mp.GuideEditCounter(**args_dict) + counter.check_filter_fastq() + + counter.get_counts() + if counter.count_reporter_edits: + counter.screen.uns["allele_counts"] = counter.screen.uns["allele_counts"].loc[ + counter.screen.uns["allele_counts"].allele.map(str) != "", : + ] + if match_target_pos: + base_editing_map = {"A": "G", "C": "T"} + edited_to = base_editing_map[edited_from] + counter.screen.get_edit_mat_from_uns( + edited_from, edited_to, match_target_pos + ) + counter.screen.write(f"{counter.output_dir}.h5ad") + counter.screen.to_Excel(f"{counter.output_dir}.xlsx") + info(f"Output written at:\n {counter.output_dir}.h5ad,\n {counter.output_dir}.xlsx") + info("All Done!") + print( + r""" + _ _ + / \ '\ _ + | \ \ __ ___ _ _ _ _| |_ + \ \ | / _/ _ \ || | ' \ _| + `.__|/ \__\___/\_,_|_||_\__| + """ + ) diff --git a/bin/bean-count-samples b/bean/cli/count_samples.py old mode 100755 new mode 100644 similarity index 77% rename from bin/bean-count-samples rename to bean/cli/count_samples.py index 06c3cdb..fcec874 --- a/bin/bean-count-samples +++ b/bean/cli/count_samples.py @@ -14,7 +14,6 @@ from bean.mapping.utils import ( InputFileError, _check_arguments, - _get_input_parser, _get_first_read_length, _check_read_length, ) @@ -32,48 +31,6 @@ info = logging.info -def get_input_parser() -> argparse.Namespace: - """Add multi-sample specific arguments to the base parser.""" - parser = _get_input_parser() - parser.add_argument( - "-i", - "--input", - type=str, - help="List of fastq and sample ids. Formatted as `R1_filepath,R2_filepath,sample_id`", - required=True, - ) - parser.add_argument( - "-t", "--threads", type=int, help="Number of threads", default=10 - ) - parser.add_argument( - "--guide-start-seqs-file", - type=str, - help="CSV file path with per-sample `guide_start_seq` to be used." - + "Formatted as `sample_id, guide_start_seq`", - default=None, - ) - parser.add_argument( - "--guide-end-seqs-file", - type=str, - help="CSV file path with per-sample `guide_end_seq` to be used." - + "Formatted as `sample_id,guide_end_seq`", - default=None, - ) - parser.add_argument( - "--barcode-start-seqs-file", - type=str, - help="CSV file path with per-sample `barcode_start_seq` to be used." - + "Formatted as `sample_id,guide_end_seq`", - default=None, - ) - - parser.add_argument( - "--rerun", help="Recount each sample", action="store_true", default=False - ) - - return parser - - def count_sample(R1: str, R2: str, sample_id: str, args: argparse.Namespace): """Count single sample given R1 and R2 paths. Arguments are modified accordingly to the provided sample_id before being passed to GuideEditCounter. @@ -102,7 +59,9 @@ def count_sample(R1: str, R2: str, sample_id: str, args: argparse.Namespace): "barcode_start_seqs_tbl" in args_dict and args_dict["barcode_start_seqs_tbl"] is not None ): - args_dict["barcode_start_seq"] = str(args_dict["barcode_start_seqs_tbl"][sample_id]) + args_dict["barcode_start_seq"] = str( + args_dict["barcode_start_seqs_tbl"][sample_id] + ) counter = bean.mp.GuideEditCounter(**args_dict) if os.path.exists(f"{counter.output_dir}.h5ad") and not args_dict["rerun"]: screen = bean.read_h5ad(f"{counter.output_dir}.h5ad") @@ -147,10 +106,10 @@ def count_sample(R1: str, R2: str, sample_id: str, args: argparse.Namespace): def check_arguments(args: argparse.Namespace) -> argparse.Namespace: """Checks the validity of the argument.""" args = _check_arguments(args, info, warn, error) - sample_tbl = pd.read_csv(args.input) + sample_tbl = pd.read_csv(args.sample_list) if len(sample_tbl.iloc[:, 2].unique()) != len(sample_tbl.iloc[:, 2]): raise InputFileError( - f"Sample ID not unique. Please check your input file {args.input}." + f"Sample ID not unique. Please check your input file {args.sample_list}." ) first_read_lengths = [ _get_first_read_length(fastq_R1) for fastq_R1 in sample_tbl.iloc[:, 0] @@ -187,11 +146,19 @@ def _check_return_guide_seqs_tbl(guide_seqs_file, sample_tbl, label): return args -def main(): - parser = get_input_parser() - args = parser.parse_args() +def main(args): + """Get the input data""" + print( + r""" + _ _ + / \ '\ _ + | \ \ __ ___ _ _ _ _| |_ + \ \ | / _/ _ \ || | ' \ _| + `.__|/ \__\___/\_,_|_||_\__| + """ + ) args = check_arguments(args) - sample_tbl = pd.read_csv(args.input) # R1_filepath, R2_filepath, sample_name + sample_tbl = pd.read_csv(args.sample_list) # R1_filepath, R2_filepath, sample_name sample_tbl_input = sample_tbl.iloc[:, :3] sample_info_tbl = sample_tbl.iloc[:, 2:].set_index(sample_tbl.columns[2]) with Pool(processes=args.threads, maxtasksperchild=1) as p: @@ -205,7 +172,7 @@ def main(): # result = p.starmap(count_sample, sample_tbl[0], sample_tbl[1], sample_tbl[2]) screen = bean.concat(result, axis=1) - database_id = args.name or os.path.basename(args.input).split(".")[0] + database_id = args.name or os.path.basename(args.sample_list).split(".")[0] output_path = os.path.join( os.path.abspath(args.output_folder), f"bean_count_{database_id}" ) @@ -230,7 +197,3 @@ def main(): `.__|/ \__\___/\_,_|_||_\__| """ ) - - -if __name__ == "__main__": - main() diff --git a/bin/bean-create-screen b/bean/cli/create_screen.py old mode 100755 new mode 100644 similarity index 65% rename from bin/bean-create-screen rename to bean/cli/create_screen.py index 596b5c9..d0c03de --- a/bin/bean-create-screen +++ b/bean/cli/create_screen.py @@ -4,7 +4,7 @@ import os import sys import logging -from bean.framework.read_from_csvs import get_input_parser, create_screen +from bean.framework.read_from_csvs import create_screen logging.basicConfig( level=logging.INFO, @@ -19,15 +19,19 @@ info = logging.info -def main(): - parser = get_input_parser() - args = parser.parse_args() +def main(args): + """Add multi-sample specific arguments to the base parser.""" + print( + r""" + _ _ + / \ '\ _ + | \ \ __ _ _ ___ __ _| |_ ___ + \ \ | / _| '_/ -_) _` | _/ -_) + `.__|/ \__|_| \___\__,_|\__\___| + """ + ) screen = create_screen(args) info(f"Done obtaining screen:\n{screen}\nWriting result...") output_path = f"{args.output_prefix if args.output_prefix else os.path.splitext(args.gRNA_counts_table_csv)[0]}.h5ad" screen.write(output_path) info(f"Done writing screen object to {output_path}.") - - -if __name__ == "__main__": - main() diff --git a/bean/cli/execute.py b/bean/cli/execute.py new file mode 100755 index 0000000..8932e5e --- /dev/null +++ b/bean/cli/execute.py @@ -0,0 +1,60 @@ +import argparse +from bean.mapping.utils import get_input_parser_count as get_count_parser +from bean.mapping.utils import get_input_parser as get_count_samples_parser +from bean.plotting.utils import parse_args as get_profile_parser +from bean.qc.utils import parse_args as get_qc_parser +from bean.annotate.utils import parse_args as get_filter_parser +from bean.model.run import parse_args as get_run_parser +from bean.framework.read_from_csvs import get_input_parser as get_create_screen_parser +from bean.cli.count import main as count +from bean.cli.count_samples import main as count_samples +from bean.cli.profile import main as profile +from bean.cli.qc import main as qc +from bean.cli.filter import main as filter +from bean.cli.run import main as run +from bean.cli.create_screen import main as create_screen + + +def get_base_parser(): + parser = argparse.ArgumentParser(prog="bean") + subparsers = parser.add_subparsers(help="Subcommands", dest="subcommand") + count_parser = subparsers.add_parser("count", help="count") + count_parser = get_count_parser(count_parser) + count_samples_parser = subparsers.add_parser("count-samples", help="count samples") + count_samples_parser = get_count_samples_parser(count_samples_parser) + profile_parser = subparsers.add_parser("profile", help="profile") + profile_parser = get_profile_parser(profile_parser) + qc_parser = subparsers.add_parser("qc", help="qc") + qc_parser = get_qc_parser(qc_parser) + filter_parser = subparsers.add_parser("filter", help="filter") + filter_parser = get_filter_parser(filter_parser) + run_parser = subparsers.add_parser("run", help="run") + run_parser = get_run_parser(run_parser) + create_screen_parser = subparsers.add_parser("create_screen", help="create") + create_screen_parser = get_create_screen_parser(create_screen_parser) + return parser + + +global_parser = None + + +def main() -> None: + parser = get_parser() + global_parser = parser + args = parser.parse_args() + if args.subcommand == "count": + count(args) + elif args.subcommand == "count-samples": + count_samples(args) + elif args.subcommand == "profile": + profile(args) + elif args.subcommand == "qc": + qc(args) + elif args.subcommand == "filter": + filter(args) + elif args.subcommand == "run": + run(args) + elif args.subcommand == "create-screen": + create_screen(args) + else: + parser.print_help() diff --git a/bin/bean-filter b/bean/cli/filter.py old mode 100755 new mode 100644 similarity index 87% rename from bin/bean-filter rename to bean/cli/filter.py index 128ef42..bfbbdc7 --- a/bin/bean-filter +++ b/bean/cli/filter.py @@ -5,13 +5,12 @@ import logging import pandas as pd import bean as be +import bean.annotate.filter_alleles as filter_alleles from bean.plotting.allele_stats import ( - plot_n_alleles_per_guide, - plot_n_guides_per_edit, plot_allele_stats, ) from bean.annotate.translate_allele import get_mismatch_df -from bean.annotate.utils import parse_args, check_args +from bean.annotate.utils import check_args import matplotlib.pyplot as plt plt.style.use("default") @@ -28,8 +27,18 @@ info = logging.info -if __name__ == "__main__": - args = parse_args() +def main(args): + """Get the input arguments""" + print( + r""" + _ _ + / \ '\ __ _ _ _ + | \ \ / _(_) | |_ ___ _ _ + \ \ | | _| | | _/ -_) '_| + `.__|/ |_| |_|_|\__\___|_| + """ + ) + print("bean-filter: filter alleles") args = check_args(args) if not args.load_tmp: bdata = be.read_h5ad(args.bdata_path) @@ -49,7 +58,7 @@ ( q_val_each, sig_allele_df, - ) = be.an.filter_alleles.filter_alleles( + ) = filter_alleles.filter_alleles( bdata, plasmid_adata, filter_each_sample=True, run_parallel=True ) bdata.uns["sig_allele_counts"] = sig_allele_df.reset_index(drop=True) @@ -59,17 +68,15 @@ print(len(bdata.uns[allele_df_keys[-1]])) if len(bdata.uns[allele_df_keys[-1]]) >= 1: info("Filtering out edits outside spacer position...") - bdata.uns[ - f"{allele_df_keys[-1]}_spacer" - ] = bdata.filter_allele_counts_by_pos( - rel_pos_start=0, - rel_pos_end=20, - rel_pos_is_reporter=False, - map_to_filtered=True, - allele_uns_key=allele_df_keys[-1], - jaccard_threshold=0.2, - ).reset_index( - drop=True + bdata.uns[f"{allele_df_keys[-1]}_spacer"] = ( + bdata.filter_allele_counts_by_pos( + rel_pos_start=0, + rel_pos_end=20, + rel_pos_is_reporter=False, + map_to_filtered=True, + allele_uns_key=allele_df_keys[-1], + jaccard_threshold=0.2, + ).reset_index(drop=True) ) info( f"Filtered down to {len(bdata.uns[f'{allele_df_keys[-1]}_spacer'])} alleles." diff --git a/bin/bean-profile b/bean/cli/profile.py old mode 100755 new mode 100644 similarity index 70% rename from bin/bean-profile rename to bean/cli/profile.py index 8b48daa..abb1c87 --- a/bin/bean-profile +++ b/bean/cli/profile.py @@ -5,8 +5,18 @@ from bean.plotting.utils import parse_args, check_args -def main(): - args = parse_args() +def main(args): + print(" \n~~~BEAN Profile~~~") + print("-Profile editing patterns of your editor-") + print( + r""" + _ _ __ _ _ + / \ '\ _ __ _ _ ___ / _(_) |___ + | \ \ | '_ \ '_/ _ \ _| | / -_) + \ \ | | .__/_| \___/_| |_|_\___| + `.__|/ |_| + """ + ) args = check_args(args) os.system( "python -m ipykernel install --user --name bean_python3 --display-name bean_python3" @@ -28,7 +38,3 @@ def main(): os.system( f"jupyter nbconvert --to html {args.output_prefix}_editing_preference.ipynb" ) - - -if __name__ == "__main__": - main() diff --git a/bin/bean-qc b/bean/cli/qc.py old mode 100755 new mode 100644 similarity index 73% rename from bin/bean-qc rename to bean/cli/qc.py index a6880bf..4102ac8 --- a/bin/bean-qc +++ b/bean/cli/qc.py @@ -1,46 +1,56 @@ -#!/usr/bin/env python -import os -import papermill as pm -import bean as be -from bean.qc.utils import parse_args, check_args - - -def main(): - args = parse_args() - args = check_args(args) - os.system( - "python -m ipykernel install --user --name bean_python3 --display-name bean_python3" - ) - pm.execute_notebook( - f"{os.path.dirname(be.__file__)}/../notebooks/sample_quality_report.ipynb", - f"{args.out_report_prefix}.ipynb", - parameters=dict( - bdata_path=args.bdata_path, - out_bdata_path=args.out_screen_path, - tiling=args.tiling, - edit_quantification_start_pos=args.edit_start_pos, - edit_quantification_end_pos=args.edit_end_pos, - target_pos_col=args.target_pos_col, - rel_pos_is_reporter=args.rel_pos_is_reporter, - corr_X_thres=args.count_correlation_thres, - edit_rate_thres=args.edit_rate_thres, - posctrl_col=args.posctrl_col, - posctrl_val=args.posctrl_val, - lfc_thres=args.lfc_thres, - replicate_label=args.replicate_label, - condition_label=args.condition_label, - comp_cond1=args.lfc_cond1, - comp_cond2=args.lfc_cond2, - ctrl_cond=args.control_condition, - exp_id=args.out_report_prefix, - recalculate_edits=~args.dont_recalculate_edits, - base_edit_data=args.base_edit_data, - remove_bad_replicates=args.remove_bad_replicates, - ), - kernel_name="bean_python3", - ) - os.system(f"jupyter nbconvert --to html {args.out_report_prefix}.ipynb") - - -if __name__ == "__main__": - main() +#!/usr/bin/env python +import os +import papermill as pm +import bean as be +from bean.qc.utils import parse_args, check_args + + +def main(args): + print(" \n~~~BEANQC~~~") + print("-Check guide/sample level quality and mask / discard-") + print( + r""" + _ _ + / \ '\ ___ ___ + | \ \ / _ \ / __| + \ \ | | (_) | (__ + `.__|/ \__\_\\___| + """ + ) + if args.out_screen_path is None: + args.out_screen_path = f"{args.bdata_path.rsplit('.h5ad', 1)[0]}.filtered.h5ad" + if args.out_report_prefix is None: + args.out_report_prefix = f"{args.bdata_path.rsplit('.h5ad', 1)[0]}.qc_report" + args = check_args(args) + os.system( + "python -m ipykernel install --user --name bean_python3 --display-name bean_python3" + ) + pm.execute_notebook( + f"{os.path.dirname(be.__file__)}/../notebooks/sample_quality_report.ipynb", + f"{args.out_report_prefix}.ipynb", + parameters=dict( + bdata_path=args.bdata_path, + out_bdata_path=args.out_screen_path, + tiling=args.tiling, + edit_quantification_start_pos=args.edit_start_pos, + edit_quantification_end_pos=args.edit_end_pos, + target_pos_col=args.target_pos_col, + rel_pos_is_reporter=args.rel_pos_is_reporter, + corr_X_thres=args.count_correlation_thres, + edit_rate_thres=args.edit_rate_thres, + posctrl_col=args.posctrl_col, + posctrl_val=args.posctrl_val, + lfc_thres=args.lfc_thres, + replicate_label=args.replicate_label, + condition_label=args.condition_label, + comp_cond1=args.lfc_cond1, + comp_cond2=args.lfc_cond2, + ctrl_cond=args.control_condition, + exp_id=args.out_report_prefix, + recalculate_edits=~args.dont_recalculate_edits, + base_edit_data=args.base_edit_data, + remove_bad_replicates=args.remove_bad_replicates, + ), + kernel_name="bean_python3", + ) + os.system(f"jupyter nbconvert --to html {args.out_report_prefix}.ipynb") diff --git a/bin/bean-run b/bean/cli/run.py old mode 100755 new mode 100644 similarity index 93% rename from bin/bean-run rename to bean/cli/run.py index 540bd37..064cf46 --- a/bin/bean-run +++ b/bean/cli/run.py @@ -60,7 +60,19 @@ ) -def main(args, bdata): +def main(args): + print( + r""" + _ _ + / \ '\ + | \ \ _ _ _ _ _ _ + \ \ | | '_| || | ' \ + `.__|/ |_| \_,_|_||_| + """ + ) + print("bean-run: Run model to identify targeted variants and their impact.") + bdata = be.read_h5ad(args.bdata_path) + args, bdata = check_args(args, bdata) if args.cuda: os.environ["CUDA_VISIBLE_DEVICES"] = "1" torch.set_default_tensor_type(torch.cuda.FloatTensor) @@ -193,10 +205,3 @@ def main(args, bdata): ), ) info("Done!") - - -if __name__ == "__main__": - args = parse_args() - bdata = be.read_h5ad(args.bdata_path) - args, bdata = check_args(args, bdata) - main(args, bdata) diff --git a/bin/bean b/bin/bean new file mode 100755 index 0000000..4591905 --- /dev/null +++ b/bin/bean @@ -0,0 +1,5 @@ +#!/usr/bin/env python +from bean.cli.execute import main + +if __name__ == "__main__": + main() From 2bf56a7f618ee966640ebcf7acc7375f669a3ec8 Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 22:10:38 -0400 Subject: [PATCH 023/107] add Sphinx dependencies --- setup.py | 2 ++ 1 file changed, 2 insertions(+) diff --git a/setup.py b/setup.py index 04a28ce..c5b7f13 100644 --- a/setup.py +++ b/setup.py @@ -53,6 +53,8 @@ "pytest-order", "nbconvert", "logomaker", + "sphinx-argparse>=0.4", + "m2r", ], extras_require={"model": ["pyBigWig", "pyro-ppl<=1.8.1", "statsmodels", "torch<2"]}, include_package_data=True, From f5ee597884773cfbcf1c596d471a15ecbb9b8d09 Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 22:13:57 -0400 Subject: [PATCH 024/107] add Sphinx dependencies --- setup.py | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/setup.py b/setup.py index c5b7f13..9b59969 100644 --- a/setup.py +++ b/setup.py @@ -53,7 +53,7 @@ "pytest-order", "nbconvert", "logomaker", - "sphinx-argparse>=0.4", + "sphinx-argparse", "m2r", ], extras_require={"model": ["pyBigWig", "pyro-ppl<=1.8.1", "statsmodels", "torch<2"]}, From 4be0796461cc15e01c03eea4cafec07d016d39f1 Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 22:15:16 -0400 Subject: [PATCH 025/107] fix main parser --- bean/cli/execute.py | 3 +-- 1 file changed, 1 insertion(+), 2 deletions(-) diff --git a/bean/cli/execute.py b/bean/cli/execute.py index 8932e5e..4300431 100755 --- a/bean/cli/execute.py +++ b/bean/cli/execute.py @@ -15,7 +15,7 @@ from bean.cli.create_screen import main as create_screen -def get_base_parser(): +def get_parser(): parser = argparse.ArgumentParser(prog="bean") subparsers = parser.add_subparsers(help="Subcommands", dest="subcommand") count_parser = subparsers.add_parser("count", help="count") @@ -40,7 +40,6 @@ def get_base_parser(): def main() -> None: parser = get_parser() - global_parser = parser args = parser.parse_args() if args.subcommand == "count": count(args) From 998289695baee1ebaf98d91856036bb39a12c93f Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 22:18:00 -0400 Subject: [PATCH 026/107] work under docs/ --- .github/workflows/documentation.yml | 4 +++- 1 file changed, 3 insertions(+), 1 deletion(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index bca2a17..6909420 100644 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -15,9 +15,11 @@ jobs: run: | pip install sphinx sphinx_rtd_theme myst_parser - name: Sphinx build + working-directory: docs/ run: | - sphinx-build docs _build + sphinx-build . _build - name: Deploy to GitHub Pages + working-directory: docs/ uses: peaceiris/actions-gh-pages@v3 if: ${{ github.event_name == 'push' && github.ref == 'refs/heads/main' }} with: From 3cf9777d4f9ae2ffe496423e63a96e03f4621ac7 Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 22:20:17 -0400 Subject: [PATCH 027/107] work under docs/ --- .github/workflows/documentation.yml | 5 ++--- 1 file changed, 2 insertions(+), 3 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 6909420..0ae4b86 100644 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -15,15 +15,14 @@ jobs: run: | pip install sphinx sphinx_rtd_theme myst_parser - name: Sphinx build - working-directory: docs/ + working-directory: ./docs run: | sphinx-build . _build - name: Deploy to GitHub Pages - working-directory: docs/ uses: peaceiris/actions-gh-pages@v3 if: ${{ github.event_name == 'push' && github.ref == 'refs/heads/main' }} with: publish_branch: gh-pages github_token: ${{ secrets.GITHUB_TOKEN }} - publish_dir: _build/ + publish_dir: docs/_build/ force_orphan: true \ No newline at end of file From 995faed549df223619336575eec2acc39f3d7b1b Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 22:24:51 -0400 Subject: [PATCH 028/107] move requirements --- docs/requirements.txt | 2 ++ setup.py | 2 -- 2 files changed, 2 insertions(+), 2 deletions(-) create mode 100644 docs/requirements.txt diff --git a/docs/requirements.txt b/docs/requirements.txt new file mode 100644 index 0000000..7979724 --- /dev/null +++ b/docs/requirements.txt @@ -0,0 +1,2 @@ +sphinx-argparse +m2r \ No newline at end of file diff --git a/setup.py b/setup.py index 9b59969..04a28ce 100644 --- a/setup.py +++ b/setup.py @@ -53,8 +53,6 @@ "pytest-order", "nbconvert", "logomaker", - "sphinx-argparse", - "m2r", ], extras_require={"model": ["pyBigWig", "pyro-ppl<=1.8.1", "statsmodels", "torch<2"]}, include_package_data=True, From e7e22c7497f64f583401ba35598bcebaaa0340a3 Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 22:27:57 -0400 Subject: [PATCH 029/107] update test arguments --- tests/test_count.py | 29 +++++++++++++++++++++++------ 1 file changed, 23 insertions(+), 6 deletions(-) diff --git a/tests/test_count.py b/tests/test_count.py index dc5fb89..c375492 100644 --- a/tests/test_count.py +++ b/tests/test_count.py @@ -33,7 +33,7 @@ def test_guide_count(): @pytest.mark.order(4) def test_count_samples(): - cmd = "bean count-samples --input tests/data/sample_list.csv -b A -f tests/data/test_guide_info.csv -o tests/test_res/ -r --guide-start-seq=GGAAAGGACGAAACACCG" + cmd = "bean count-samples -i tests/data/sample_list.csv -b A -f tests/data/test_guide_info.csv -o tests/test_res/ -r --guide-start-seq=GGAAAGGACGAAACACCG" try: subprocess.check_output( cmd, @@ -42,10 +42,11 @@ def test_count_samples(): ) except subprocess.CalledProcessError as exc: raise exc - + + @pytest.mark.order(5) def test_count_samples_bcstart(): - cmd = "bean count-samples --input tests/data/sample_list.csv -b A -f tests/data/test_guide_info.csv -o tests/test_res/ -r --barcode-start-seq=GGAA" + cmd = "bean count-samples -i tests/data/sample_list.csv -b A -f tests/data/test_guide_info.csv -o tests/test_res/ -r --barcode-start-seq=GGAA" try: subprocess.check_output( cmd, @@ -55,19 +56,35 @@ def test_count_samples_bcstart(): except subprocess.CalledProcessError as exc: raise exc + def test_barcode_start_idx(): - args = vars(_get_input_parser().parse_args(["-b", "A", "-f", "tests/data/test_guide_info.csv",])) + args = vars( + _get_input_parser().parse_args( + [ + "-b", + "A", + "-f", + "tests/data/test_guide_info.csv", + ] + ) + ) args["barcode_start_seq"] = "gtttgaattcgctagctaggtcttg" args["R1"] = "tests/data/test_R1.fastq" args["R2"] = "tests/data/test_R2.fastq" gc = GuideEditCounter(**args) - sidx, bc = gc.get_barcode("", revcomp("agtggcaccgagtcggtgcttttttTAACAGTGTAATCTGGCGAGCCACTGTTCTTTGTACCAGAAgtttgaattcgctagctaggtcttgctgg".upper())) + sidx, bc = gc.get_barcode( + "", + revcomp( + "agtggcaccgagtcggtgcttttttTAACAGTGTAATCTGGCGAGCCACTGTTCTTTGTACCAGAAgtttgaattcgctagctaggtcttgctgg".upper() + ), + ) assert sidx == 29 assert bc == "AGAA" + @pytest.mark.order(6) def test_count_samples_tiling(): - cmd = "bean count-samples --input tests/data/sample_list_tiling.csv -b A -f tests/data/test_guide_info_tiling_chrom.csv -o tests/test_res/ -r" + cmd = "bean count-samples -i tests/data/sample_list_tiling.csv -b A -f tests/data/test_guide_info_tiling_chrom.csv -o tests/test_res/ -r" try: subprocess.check_output( cmd, From 932dba44e4f7541e39b45adb754ecde5723b032e Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 22:31:37 -0400 Subject: [PATCH 030/107] try sphinx doc --- .github/workflows/documentation.yml | 42 +++++++++++++---------------- docs/requirements.txt | 3 ++- 2 files changed, 21 insertions(+), 24 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 0ae4b86..2814be5 100644 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -1,28 +1,24 @@ -name: documentation +name: "Sphinx: Render docs" -on: [push, pull_request, workflow_dispatch] - -permissions: - contents: write +on: push jobs: - docs: + build: runs-on: ubuntu-latest + permissions: + contents: write steps: - - uses: actions/checkout@v3 - - uses: actions/setup-python@v3 - - name: Install dependencies - run: | - pip install sphinx sphinx_rtd_theme myst_parser - - name: Sphinx build - working-directory: ./docs - run: | - sphinx-build . _build - - name: Deploy to GitHub Pages - uses: peaceiris/actions-gh-pages@v3 - if: ${{ github.event_name == 'push' && github.ref == 'refs/heads/main' }} - with: - publish_branch: gh-pages - github_token: ${{ secrets.GITHUB_TOKEN }} - publish_dir: docs/_build/ - force_orphan: true \ No newline at end of file + - uses: actions/checkout@v4 + - name: Build HTML + uses: ammaraskar/sphinx-action@master + - name: Upload artifacts + uses: actions/upload-artifact@v4 + with: + name: html-docs + path: docs/build/html/ + - name: Deploy + uses: peaceiris/actions-gh-pages@v3 + if: github.ref == 'refs/heads/main' + with: + github_token: ${{ secrets.GITHUB_TOKEN }} + publish_dir: docs/build/html \ No newline at end of file diff --git a/docs/requirements.txt b/docs/requirements.txt index 7979724..b8c9f1d 100644 --- a/docs/requirements.txt +++ b/docs/requirements.txt @@ -1,2 +1,3 @@ sphinx-argparse -m2r \ No newline at end of file +m2r +furo==2021.11.16 \ No newline at end of file From 4338d5ea0a3c3959a1c022cd730e232eee6cae9a Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 22:36:14 -0400 Subject: [PATCH 031/107] try sphinx doc edit --- .github/workflows/documentation.yml | 25 ++++++++++++------------- 1 file changed, 12 insertions(+), 13 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 2814be5..4cd6cf2 100644 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -5,20 +5,19 @@ on: push jobs: build: runs-on: ubuntu-latest - permissions: - contents: write steps: - uses: actions/checkout@v4 - - name: Build HTML - uses: ammaraskar/sphinx-action@master - - name: Upload artifacts - uses: actions/upload-artifact@v4 + - name: Set up Python + uses: actions/setup-python@v3 with: - name: html-docs - path: docs/build/html/ - - name: Deploy - uses: peaceiris/actions-gh-pages@v3 - if: github.ref == 'refs/heads/main' + python-version: '3.x' + + - name: Install Sphinx & Dependencies + run: pip install sphinx sphinx_markdown_builder sphinx_rtd_theme + - name: Build Documentation + run: cd "$GITHUB_WORKSPACE/doc" && make html + + - uses: actions/upload-artifact@v3 with: - github_token: ${{ secrets.GITHUB_TOKEN }} - publish_dir: docs/build/html \ No newline at end of file + name: Documentation + path: "$GITHUB_WORKSPACE/doc/_build/html/" \ No newline at end of file From c488dd46289496d8db0dab552fbf975ca3207abc Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 22:40:33 -0400 Subject: [PATCH 032/107] fix doc --- .github/workflows/documentation.yml | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 4cd6cf2..fc62d84 100644 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -15,9 +15,9 @@ jobs: - name: Install Sphinx & Dependencies run: pip install sphinx sphinx_markdown_builder sphinx_rtd_theme - name: Build Documentation - run: cd "$GITHUB_WORKSPACE/doc" && make html + run: cd "$GITHUB_WORKSPACE/docs" && make html - uses: actions/upload-artifact@v3 with: name: Documentation - path: "$GITHUB_WORKSPACE/doc/_build/html/" \ No newline at end of file + path: "$GITHUB_WORKSPACE/docs/_build/html/" \ No newline at end of file From 426a4f8dbc2919e09403b872dd33e327cd10029f Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 22:41:53 -0400 Subject: [PATCH 033/107] fix command --- bean/cli/execute.py | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/bean/cli/execute.py b/bean/cli/execute.py index 4300431..046f08d 100755 --- a/bean/cli/execute.py +++ b/bean/cli/execute.py @@ -30,7 +30,7 @@ def get_parser(): filter_parser = get_filter_parser(filter_parser) run_parser = subparsers.add_parser("run", help="run") run_parser = get_run_parser(run_parser) - create_screen_parser = subparsers.add_parser("create_screen", help="create") + create_screen_parser = subparsers.add_parser("create-screen", help="create") create_screen_parser = get_create_screen_parser(create_screen_parser) return parser From 232a25bfb0ef201d3afad8141e6f2fdcbc919198 Mon Sep 17 00:00:00 2001 From: Jayoung Kim Ryu Date: Fri, 29 Mar 2024 22:43:44 -0400 Subject: [PATCH 034/107] Update documentation.yml --- .github/workflows/documentation.yml | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index fc62d84..cc24aa3 100644 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -15,9 +15,9 @@ jobs: - name: Install Sphinx & Dependencies run: pip install sphinx sphinx_markdown_builder sphinx_rtd_theme - name: Build Documentation - run: cd "$GITHUB_WORKSPACE/docs" && make html + run: cd "$GITHUB_WORKSPACE/docs" && sphinx-build . _build - uses: actions/upload-artifact@v3 with: name: Documentation - path: "$GITHUB_WORKSPACE/docs/_build/html/" \ No newline at end of file + path: "$GITHUB_WORKSPACE/docs/_build/html/" From 085fee2b9325bfa3af27d553c69ce5ecb74f2ca4 Mon Sep 17 00:00:00 2001 From: Jayoung Kim Ryu Date: Fri, 29 Mar 2024 22:45:18 -0400 Subject: [PATCH 035/107] Update documentation.yml --- .github/workflows/documentation.yml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index cc24aa3..e28c0ee 100644 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -13,7 +13,7 @@ jobs: python-version: '3.x' - name: Install Sphinx & Dependencies - run: pip install sphinx sphinx_markdown_builder sphinx_rtd_theme + run: pip install sphinx sphinx_markdown_builder sphinx_rtd_theme sphinx-argparse m2r - name: Build Documentation run: cd "$GITHUB_WORKSPACE/docs" && sphinx-build . _build From 6dd11e1b042796bc7976a2f7ea416a73e5856eb8 Mon Sep 17 00:00:00 2001 From: Jayoung Kim Ryu Date: Fri, 29 Mar 2024 22:47:40 -0400 Subject: [PATCH 036/107] Update documentation.yml --- .github/workflows/documentation.yml | 6 ++++++ 1 file changed, 6 insertions(+) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index e28c0ee..23543ff 100644 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -14,6 +14,12 @@ jobs: - name: Install Sphinx & Dependencies run: pip install sphinx sphinx_markdown_builder sphinx_rtd_theme sphinx-argparse m2r + - name: Install bean + run: | + python -m pip install --upgrade pip + pip install torch==1.12.1+cpu torchvision==0.13.1+cpu torchaudio==0.12.1 --extra-index-url https://download.pytorch.org/whl/cpu + pip install -r requirements.txt + pip install -e . - name: Build Documentation run: cd "$GITHUB_WORKSPACE/docs" && sphinx-build . _build From 882271ad51e689b86cc66d17909098bb580f5ff6 Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 22:50:12 -0400 Subject: [PATCH 037/107] changed --- .github/workflows/documentation.yml | 2 +- requirements.txt | 2 +- 2 files changed, 2 insertions(+), 2 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index fc62d84..b3d5986 100644 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -13,7 +13,7 @@ jobs: python-version: '3.x' - name: Install Sphinx & Dependencies - run: pip install sphinx sphinx_markdown_builder sphinx_rtd_theme + run: pip install sphinx sphinx_markdown_builder sphinx_rtd_theme sphinx-argparse m2r - name: Build Documentation run: cd "$GITHUB_WORKSPACE/docs" && make html diff --git a/requirements.txt b/requirements.txt index 1ef9d71..04bdbca 100644 --- a/requirements.txt +++ b/requirements.txt @@ -11,7 +11,7 @@ liftover openpyxl>=3.0.10 papermill>=2.4.0 pyBigWig>=0.3.18 -pyro-ppl==1.8.1 +pyro-ppl scikit-learn statsmodels>=0.12.1 ipykernel From 31a79f72f161541bfb5aa5dbca15eaaebdbdd43a Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 22:54:59 -0400 Subject: [PATCH 038/107] try torch 2 --- .github/workflows/documentation.yml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 23543ff..6a71792 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -17,7 +17,7 @@ jobs: - name: Install bean run: | python -m pip install --upgrade pip - pip install torch==1.12.1+cpu torchvision==0.13.1+cpu torchaudio==0.12.1 --extra-index-url https://download.pytorch.org/whl/cpu + pip install torch torchvision torchaudio pip install -r requirements.txt pip install -e . - name: Build Documentation From 2d983fca3b6cae0ee341b26112c17f27e11a659b Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 23:07:38 -0400 Subject: [PATCH 039/107] move parser to not require import --- bean/cli/execute.py | 6 +- bean/cli/qc.py | 2 +- bean/framework/parser.py | 39 ++++++ bean/framework/read_from_csvs.py | 35 ----- bean/model/parser.py | 228 ++++++++++++++++++++++++++++++ bean/model/run.py | 229 +------------------------------ bean/qc/parser.py | 143 +++++++++++++++++++ bean/qc/utils.py | 143 ------------------- docs/requirements.txt | 6 +- 9 files changed, 419 insertions(+), 412 deletions(-) create mode 100644 bean/framework/parser.py create mode 100644 bean/model/parser.py create mode 100644 bean/qc/parser.py diff --git a/bean/cli/execute.py b/bean/cli/execute.py index 046f08d..08b5959 100755 --- a/bean/cli/execute.py +++ b/bean/cli/execute.py @@ -2,10 +2,10 @@ from bean.mapping.utils import get_input_parser_count as get_count_parser from bean.mapping.utils import get_input_parser as get_count_samples_parser from bean.plotting.utils import parse_args as get_profile_parser -from bean.qc.utils import parse_args as get_qc_parser +from bean.qc.parser import parse_args as get_qc_parser from bean.annotate.utils import parse_args as get_filter_parser -from bean.model.run import parse_args as get_run_parser -from bean.framework.read_from_csvs import get_input_parser as get_create_screen_parser +from bean.model.parser import parse_args as get_run_parser +from bean.framework.parser import get_input_parser as get_create_screen_parser from bean.cli.count import main as count from bean.cli.count_samples import main as count_samples from bean.cli.profile import main as profile diff --git a/bean/cli/qc.py b/bean/cli/qc.py index 4102ac8..b9f8f6c 100644 --- a/bean/cli/qc.py +++ b/bean/cli/qc.py @@ -2,7 +2,7 @@ import os import papermill as pm import bean as be -from bean.qc.utils import parse_args, check_args +from bean.qc.utils import check_args def main(args): diff --git a/bean/framework/parser.py b/bean/framework/parser.py new file mode 100644 index 0000000..318ec26 --- /dev/null +++ b/bean/framework/parser.py @@ -0,0 +1,39 @@ +import argparse + + +def get_input_parser(parser=None): + if parser is None: + parser = argparse.ArgumentParser( + description="bean-create-screen parameters", + formatter_class=argparse.ArgumentDefaultsHelpFormatter, + ) + parser.add_argument( + "gRNA_info_table_csv", + type=str, + help="Path to gRNA info .csv table, with index at first column and column names at the first row.", + ) + parser.add_argument( + "samples_info_table_csv", + type=str, + help="Path to sample info .csv table, with index at first column and column names at the first row.", + ) + parser.add_argument( + "gRNA_counts_table_csv", + type=str, + help="Path to gRNA counts .csv table, with index at first column and column names at the first row.", + ) + parser.add_argument( + "-e", + "--edits", + type=str, + help="Path to edit counts .csv table, with index at first column and column names at the first row.", + default=None, + ) + parser.add_argument( + "-o", + "--output-prefix", + type=str, + help="Output file prefix (output will be saved as `output_prefix.h5ad`). If not provided, `gRNA_counts_table_csv` file prefix is used.", + default=None, + ) + return parser diff --git a/bean/framework/read_from_csvs.py b/bean/framework/read_from_csvs.py index 24667e0..2a3bd9a 100644 --- a/bean/framework/read_from_csvs.py +++ b/bean/framework/read_from_csvs.py @@ -4,41 +4,6 @@ from perturb_tools import Screen -def get_input_parser(parser) -> argparse.Namespace: - # parser = argparse.ArgumentParser( - # description="bean-create-screen parameters", - # formatter_class=argparse.ArgumentDefaultsHelpFormatter, - # ) - parser.add_argument( - "gRNA_info_table_csv", - type=str, - help="Path to gRNA info .csv table, with index at first column and column names at the first row.", - ) - parser.add_argument( - "samples_info_table_csv", - type=str, - help="Path to sample info .csv table, with index at first column and column names at the first row.", - ) - parser.add_argument( - "gRNA_counts_table_csv", - type=str, - help="Path to gRNA counts .csv table, with index at first column and column names at the first row.", - ) - parser.add_argument( - "-e", - "--edits", - type=str, - help="Path to edit counts .csv table, with index at first column and column names at the first row.", - default=None, - ) - parser.add_argument( - "-o", - "--output-prefix", - type=str, - help="Output file prefix (output will be saved as `output_prefix.h5ad`). If not provided, `gRNA_counts_table_csv` file prefix is used.", - default=None, - ) - return parser def create_screen(args: argparse.Namespace) -> Screen: diff --git a/bean/model/parser.py b/bean/model/parser.py new file mode 100644 index 0000000..db79c14 --- /dev/null +++ b/bean/model/parser.py @@ -0,0 +1,228 @@ +import argparse + + +def none_or_str(value): + if value == "None": + return None + return value + + +def parse_args(parser=None): + if parser is None: + parser = argparse.ArgumentParser(description="Run model on data.") + parser.add_argument( + "selection", + type=str, + choices=["sorting", "survival"], + help="Screen selection type whether cells are sorted based on continuous phenotype ('sorting') or proliferated based on their viability ('survival').", + ) + parser.add_argument( + "library_design", + type=str, + choices=["variant", "tiling"], + help="Library design type whether to run variant or tiling screen model.\nVariant library design assumes gRNA has specific target variant and bystander edits are ignored. Tiling library design considers all alleles generated by gRNA in reporter.", + ) + parser.add_argument("bdata_path", type=str, help="Path of an ReporterScreen object") + parser.add_argument( + "--rep-pi", + "-r", + action="store_true", + default=False, + help="Fit replicate specific scaling factor. Recommended to set as True if you expect variable editing activity across biological replicates.", + ) + parser.add_argument( + "--uniform-edit", + "-p", + action="store_true", + default=False, + help="Assume uniform editing rate for all guides.", + ) + parser.add_argument( + "--scale-by-acc", + action="store_true", + default=False, + help="Scale guide editing efficiency by the target loci accessibility", + ) + parser.add_argument( + "--acc-bw-path", + type=str, + default=None, + help="Accessibility .bigWig file to be used to assign accessibility of guides.", + ) + parser.add_argument( + "--acc-col", + type=str, + default=None, + help="Column name in bdata.guides that specify raw ATAC-seq signal.", + ) + parser.add_argument( + "--const-pi", + default=False, + action="store_true", + help="Use constant pi provided in --guide-activity-col (instead of fitting from reporter data)", + ) + parser.add_argument( + "--shrink-alpha", + default=False, + action="store_true", + help="Instead of using the trend-fitted alpha values, use estimated alpha values for each gRNA that are shrunk towards the fitted trend.", + ) + parser.add_argument( + "--condition-col", + default="condition", + type=str, + help="Column key in `bdata.samples` that describes experimental condition.", + ) + parser.add_argument( + "--time-col", + default="time", + type=str, + help="Column key in `bdata.samples` that describes time elapsed.", + ) + parser.add_argument( + "--control-condition", + default="bulk", + type=str, + help="Value in `bdata.samples[condition_col]` that indicates control experimental condition.", + ) + parser.add_argument( + "--include-control-condition-for-inference", + "-ic", + default=False, + action="store_true", + help="Include control conditions for inference. Currently only supported for survival screens.", + ) + parser.add_argument( + "--replicate-col", + default="replicate", + type=str, + help="Column key in `bdata.samples` that describes experimental replicates.", + ) + parser.add_argument( + "--target-col", + default="target", + type=str, + help="Column key in `bdata.guides` that describes the target element of each guide.", + ) + parser.add_argument( + "--guide-activity-col", + "-a", + type=str, + default=None, + help="Column in ReporterScreen.guides DataFrame showing the editing rate estimated via external tools", + ) + parser.add_argument( + "--outdir", + "-o", + default=".", + type=str, + help="Directory to save the run result.", + ) + parser.add_argument( + "--result-suffix", + default="", + type=str, + help="Suffix of the output files", + ) + parser.add_argument( + "--sorting-bin-upper-quantile-col", + "-uq", + help="Column name with upper quantile values of each sorting bin in [Reporter]Screen.samples (or AnnData.var)", + default="upper_quantile", + ) + parser.add_argument( + "--sorting-bin-lower-quantile-col", + "-lq", + help="Column name with lower quantile values of each sorting bin in [Reporter]Screen.samples (or AnnData var)", + default="lower_quantile", + ) + parser.add_argument( + "--alpha-if-overdispersion-fitting-fails", + "-af", + default=None, + type=str, + help="Comma-separated regression coefficient (b0, b1) of log(a0) ~ log(q) that will be used if fitting dispersion on the data fails.", + ) + parser.add_argument("--cuda", action="store_true", default=False, help="run on GPU") + parser.add_argument( + "--sample-mask-col", + type=str, + default=None, + help="Name of the column indicating the sample mask in [Reporter]Screen.samples (or AnnData.var). Sample is ignored if the value in this column is 0. This can be used to mask out low-quality samples.", + ) + parser.add_argument( + "--fit-negctrl", + action="store_true", + default=False, + help="Fit the shared negative control distribution to normalize the fitted parameters", + ) + parser.add_argument( + "--negctrl-col", + type=str, + default="target_group", + help="Column in bdata.obs specifying if a guide is negative control. If the `bdata.guides[negctrl_col].lower() == negctrl_col_value`, it is treated as negative control guide.", + ) + parser.add_argument( + "--negctrl-col-value", + type=str, + default="negctrl", + help="Column value in bdata.guides specifying if a guide is negative control. If the `bdata.guides[negctrl_col].lower() == negctrl_col_value`, it is treated as negative control guide.", + ) + parser.add_argument( + "--repguide-mask", + type=none_or_str, + default="repguide_mask", + help="n_replicate x n_guide mask to mask the outlier guides. screen.uns[repguide_mask] will be used.", + ) + parser.add_argument( + "--device", + type=str, + default=None, + help="Optionally use GPU if provided valid GPU device name (ex. cuda:0)", + ) + parser.add_argument( + "--ignore-bcmatch", + action="store_true", + default=False, + help="If provided, even if the screen object has .X_bcmatch, ignore the count when fitting.", + ) + parser.add_argument( + "--allele-df-key", + type=str, + default=None, + help="screen.uns[allele_df_key] will be used as the allele count.", + ) + parser.add_argument( + "--splice-site-path", + type=str, + default=None, + help="Path to splicing site", + ) + parser.add_argument( + "--control-guide-tag", + type=none_or_str, + default=None, + help="If this string is in guide name, treat each guide separately not to mix the position. Used for negative controls.", + ) + parser.add_argument( + "--dont-fit-noise", # TODO: add check args + action="store_true", + ) + parser.add_argument( + "--dont-adjust-confidence-by-negative-control", + action="store_true", + help="Adjust confidence by negative controls. For variant library_design, this uses negative control variants. For tiling library_design, adjusts confidence by synonymous edits.", + ) + parser.add_argument( + "--n-iter", # TODO: add check args + type=int, + default=2000, + help="# of SVI steps taken for inference.", + ) + parser.add_argument( + "--load-existing", # TODO: add check args + action="store_true", + help="Load existing .pkl file if present.", + ) + + return parser diff --git a/bean/model/run.py b/bean/model/run.py index 39752a9..74c73ff 100644 --- a/bean/model/run.py +++ b/bean/model/run.py @@ -1,6 +1,6 @@ import os import sys -import argparse + from tqdm import tqdm import pickle as pkl import pandas as pd @@ -26,233 +26,6 @@ pyro.set_rng_seed(101) -def none_or_str(value): - if value == "None": - return None - return value - - -def parse_args(parser=None): - if parser is None: - parser = argparse.ArgumentParser(description="Run model on data.") - parser.add_argument( - "selection", - type=str, - choices=["sorting", "survival"], - help="Screen selection type whether cells are sorted based on continuous phenotype ('sorting') or proliferated based on their viability ('survival').", - ) - parser.add_argument( - "library_design", - type=str, - choices=["variant", "tiling"], - help="Library design type whether to run variant or tiling screen model.\nVariant library design assumes gRNA has specific target variant and bystander edits are ignored. Tiling library design considers all alleles generated by gRNA in reporter.", - ) - parser.add_argument("bdata_path", type=str, help="Path of an ReporterScreen object") - parser.add_argument( - "--rep-pi", - "-r", - action="store_true", - default=False, - help="Fit replicate specific scaling factor. Recommended to set as True if you expect variable editing activity across biological replicates.", - ) - parser.add_argument( - "--uniform-edit", - "-p", - action="store_true", - default=False, - help="Assume uniform editing rate for all guides.", - ) - parser.add_argument( - "--scale-by-acc", - action="store_true", - default=False, - help="Scale guide editing efficiency by the target loci accessibility", - ) - parser.add_argument( - "--acc-bw-path", - type=str, - default=None, - help="Accessibility .bigWig file to be used to assign accessibility of guides.", - ) - parser.add_argument( - "--acc-col", - type=str, - default=None, - help="Column name in bdata.guides that specify raw ATAC-seq signal.", - ) - parser.add_argument( - "--const-pi", - default=False, - action="store_true", - help="Use constant pi provided in --guide-activity-col (instead of fitting from reporter data)", - ) - parser.add_argument( - "--shrink-alpha", - default=False, - action="store_true", - help="Instead of using the trend-fitted alpha values, use estimated alpha values for each gRNA that are shrunk towards the fitted trend.", - ) - parser.add_argument( - "--condition-col", - default="condition", - type=str, - help="Column key in `bdata.samples` that describes experimental condition.", - ) - parser.add_argument( - "--time-col", - default="time", - type=str, - help="Column key in `bdata.samples` that describes time elapsed.", - ) - parser.add_argument( - "--control-condition", - default="bulk", - type=str, - help="Value in `bdata.samples[condition_col]` that indicates control experimental condition.", - ) - parser.add_argument( - "--include-control-condition-for-inference", - "-ic", - default=False, - action="store_true", - help="Include control conditions for inference. Currently only supported for survival screens.", - ) - parser.add_argument( - "--replicate-col", - default="replicate", - type=str, - help="Column key in `bdata.samples` that describes experimental replicates.", - ) - parser.add_argument( - "--target-col", - default="target", - type=str, - help="Column key in `bdata.guides` that describes the target element of each guide.", - ) - parser.add_argument( - "--guide-activity-col", - "-a", - type=str, - default=None, - help="Column in ReporterScreen.guides DataFrame showing the editing rate estimated via external tools", - ) - parser.add_argument( - "--outdir", - "-o", - default=".", - type=str, - help="Directory to save the run result.", - ) - parser.add_argument( - "--result-suffix", - default="", - type=str, - help="Suffix of the output files", - ) - parser.add_argument( - "--sorting-bin-upper-quantile-col", - "-uq", - help="Column name with upper quantile values of each sorting bin in [Reporter]Screen.samples (or AnnData.var)", - default="upper_quantile", - ) - parser.add_argument( - "--sorting-bin-lower-quantile-col", - "-lq", - help="Column name with lower quantile values of each sorting bin in [Reporter]Screen.samples (or AnnData var)", - default="lower_quantile", - ) - parser.add_argument( - "--alpha-if-overdispersion-fitting-fails", - "-af", - default=None, - type=str, - help="Comma-separated regression coefficient (b0, b1) of log(a0) ~ log(q) that will be used if fitting dispersion on the data fails.", - ) - parser.add_argument("--cuda", action="store_true", default=False, help="run on GPU") - parser.add_argument( - "--sample-mask-col", - type=str, - default=None, - help="Name of the column indicating the sample mask in [Reporter]Screen.samples (or AnnData.var). Sample is ignored if the value in this column is 0. This can be used to mask out low-quality samples.", - ) - parser.add_argument( - "--fit-negctrl", - action="store_true", - default=False, - help="Fit the shared negative control distribution to normalize the fitted parameters", - ) - parser.add_argument( - "--negctrl-col", - type=str, - default="target_group", - help="Column in bdata.obs specifying if a guide is negative control. If the `bdata.guides[negctrl_col].lower() == negctrl_col_value`, it is treated as negative control guide.", - ) - parser.add_argument( - "--negctrl-col-value", - type=str, - default="negctrl", - help="Column value in bdata.guides specifying if a guide is negative control. If the `bdata.guides[negctrl_col].lower() == negctrl_col_value`, it is treated as negative control guide.", - ) - parser.add_argument( - "--repguide-mask", - type=none_or_str, - default="repguide_mask", - help="n_replicate x n_guide mask to mask the outlier guides. screen.uns[repguide_mask] will be used.", - ) - parser.add_argument( - "--device", - type=str, - default=None, - help="Optionally use GPU if provided valid GPU device name (ex. cuda:0)", - ) - parser.add_argument( - "--ignore-bcmatch", - action="store_true", - default=False, - help="If provided, even if the screen object has .X_bcmatch, ignore the count when fitting.", - ) - parser.add_argument( - "--allele-df-key", - type=str, - default=None, - help="screen.uns[allele_df_key] will be used as the allele count.", - ) - parser.add_argument( - "--splice-site-path", - type=str, - default=None, - help="Path to splicing site", - ) - parser.add_argument( - "--control-guide-tag", - type=none_or_str, - default=None, - help="If this string is in guide name, treat each guide separately not to mix the position. Used for negative controls.", - ) - parser.add_argument( - "--dont-fit-noise", # TODO: add check args - action="store_true", - ) - parser.add_argument( - "--dont-adjust-confidence-by-negative-control", - action="store_true", - help="Adjust confidence by negative controls. For variant library_design, this uses negative control variants. For tiling library_design, adjusts confidence by synonymous edits.", - ) - parser.add_argument( - "--n-iter", # TODO: add check args - type=int, - default=2000, - help="# of SVI steps taken for inference.", - ) - parser.add_argument( - "--load-existing", # TODO: add check args - action="store_true", - help="Load existing .pkl file if present.", - ) - - return parser - - def check_args(args, bdata): args.adjust_confidence_by_negative_control = ( not args.dont_adjust_confidence_by_negative_control diff --git a/bean/qc/parser.py b/bean/qc/parser.py new file mode 100644 index 0000000..2207e83 --- /dev/null +++ b/bean/qc/parser.py @@ -0,0 +1,143 @@ +import argparse +import distutils + + +def parse_args(parser=None): + if parser is None: + parser = argparse.ArgumentParser() + parser.add_argument( + "bdata_path", help="Path to the ReporterScreen object to run QC on", type=str + ) + thres_parser = parser.add_argument_group("QC thresholds") + run_parser = parser.add_argument_group("Run options") + input_parser = parser.add_argument_group("Input .h5ad formatting") + + thres_parser.add_argument( + "--count-correlation-thres", + help="Correlation threshold to mask out.", + type=float, + default=0.7, + ) + thres_parser.add_argument( + "--edit-rate-thres", + help="Mean editing rate threshold per sample to mask out.", + type=float, + default=0.1, + ) + thres_parser.add_argument( + "--lfc-thres", + help="Positive guides' correlation threshold to filter out.", + type=float, + default=-0.1, + ) + + parser.add_argument( + "-o", + "--out-screen-path", + help="Path where quality-filtered ReporterScreen object to be written to", + type=str, + ) + parser.add_argument( + "-r", + "--out-report-prefix", + help="Output prefix of qc report (prefix.html, prefix.ipynb)", + type=str, + ) + + run_parser.add_argument( + "-b", + "--remove-bad-replicates", + help="Remove replicates with at least two of its samples meet the QC threshold.", + action="store_true", + ) + run_parser.add_argument( + "-i", + "--ignore-missing-samples", + help="If the flag is not provided, if the ReporterScreen object does not contain all condiitons for each replicate, make fake empty samples. If the flag is provided, don't add dummy samples.", + action="store_true", + ) + run_parser.add_argument( + "--no-editing", + help="Ignore QC about editing. Can be used for QC of other editing modalities.", + action="store_true", + ) + run_parser.add_argument( + "--dont-recalculate-edits", + help="When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from ReporterScreen.uns['allele_count'].", + action="store_true", + ) + + input_parser.add_argument( + "--tiling", + dest="tiling", + type=lambda x: bool(distutils.util.strtobool(x)), + help="Specify that the guide library is tiling library without 'n guides per target' design", + ) + input_parser.add_argument( + "--replicate-label", + help="Label of column in `bdata.samples` that describes replicate ID.", + type=str, + default="replicate", + ) + input_parser.add_argument( + "--sample-covariates", + help="Comma-separated list of column names in `bdata.samples` that describes non-selective experimental condition. (drug treatment, etc.)", + type=str, + default=None, + ) + input_parser.add_argument( + "--condition-label", + help="Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time, etc.)", + type=str, + default="condition", + ) + input_parser.add_argument( + "--target-pos-col", + help="Target position column in `bdata.guides` specifying target edit position in reporter", + type=str, + default="target_pos", + ) + input_parser.add_argument( + "--rel-pos-is-reporter", + help="Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If `False`, those are relative to spacer position.", + action="store_true", + default=False, + ) + input_parser.add_argument( + "--edit-start-pos", + help="Edit start position to quantify editing rate on, 0-based inclusive.", + default=2, + ) + input_parser.add_argument( + "--edit-end-pos", + help="Edit end position to quantify editing rate on, 0-based exclusive.", + default=7, + ) + + input_parser.add_argument( + "--posctrl-col", + help="Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all gRNAs, feed empty string ''.", + type=str, + default="target_group", + ) + input_parser.add_argument( + "--posctrl-val", + help="Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the positive control in calculating log fold change.", + type=str, + default="PosCtrl", + ) + + input_parser.add_argument( + "--lfc-conds", + help="Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated between, delimited by comma", + type=str, + default="top,bot", + ) + input_parser.add_argument( + "--control-condition", + help="Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate to be calculated", + type=str, + default="bulk", + ) + + return parser diff --git a/bean/qc/utils.py b/bean/qc/utils.py index 036075f..b56738a 100644 --- a/bean/qc/utils.py +++ b/bean/qc/utils.py @@ -1,153 +1,10 @@ -import distutils from typing import Union, List import numpy as np import pandas as pd from copy import deepcopy -import argparse from bean.framework.ReporterScreen import ReporterScreen, concat -def parse_args(parser=None): - if parser is None: - parser = argparse.ArgumentParser() - parser.add_argument( - "bdata_path", help="Path to the ReporterScreen object to run QC on", type=str - ) - thres_parser = parser.add_argument_group("QC thresholds") - run_parser = parser.add_argument_group("Run options") - input_parser = parser.add_argument_group("Input .h5ad formatting") - - thres_parser.add_argument( - "--count-correlation-thres", - help="Correlation threshold to mask out.", - type=float, - default=0.7, - ) - thres_parser.add_argument( - "--edit-rate-thres", - help="Mean editing rate threshold per sample to mask out.", - type=float, - default=0.1, - ) - thres_parser.add_argument( - "--lfc-thres", - help="Positive guides' correlation threshold to filter out.", - type=float, - default=-0.1, - ) - - parser.add_argument( - "-o", - "--out-screen-path", - help="Path where quality-filtered ReporterScreen object to be written to", - type=str, - ) - parser.add_argument( - "-r", - "--out-report-prefix", - help="Output prefix of qc report (prefix.html, prefix.ipynb)", - type=str, - ) - - run_parser.add_argument( - "-b", - "--remove-bad-replicates", - help="Remove replicates with at least two of its samples meet the QC threshold.", - action="store_true", - ) - run_parser.add_argument( - "-i", - "--ignore-missing-samples", - help="If the flag is not provided, if the ReporterScreen object does not contain all condiitons for each replicate, make fake empty samples. If the flag is provided, don't add dummy samples.", - action="store_true", - ) - run_parser.add_argument( - "--no-editing", - help="Ignore QC about editing. Can be used for QC of other editing modalities.", - action="store_true", - ) - run_parser.add_argument( - "--dont-recalculate-edits", - help="When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from ReporterScreen.uns['allele_count'].", - action="store_true", - ) - - input_parser.add_argument( - "--tiling", - dest="tiling", - type=lambda x: bool(distutils.util.strtobool(x)), - help="Specify that the guide library is tiling library without 'n guides per target' design", - ) - input_parser.add_argument( - "--replicate-label", - help="Label of column in `bdata.samples` that describes replicate ID.", - type=str, - default="replicate", - ) - input_parser.add_argument( - "--sample-covariates", - help="Comma-separated list of column names in `bdata.samples` that describes non-selective experimental condition. (drug treatment, etc.)", - type=str, - default=None, - ) - input_parser.add_argument( - "--condition-label", - help="Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time, etc.)", - type=str, - default="condition", - ) - input_parser.add_argument( - "--target-pos-col", - help="Target position column in `bdata.guides` specifying target edit position in reporter", - type=str, - default="target_pos", - ) - input_parser.add_argument( - "--rel-pos-is-reporter", - help="Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If `False`, those are relative to spacer position.", - action="store_true", - default=False, - ) - input_parser.add_argument( - "--edit-start-pos", - help="Edit start position to quantify editing rate on, 0-based inclusive.", - default=2, - ) - input_parser.add_argument( - "--edit-end-pos", - help="Edit end position to quantify editing rate on, 0-based exclusive.", - default=7, - ) - - input_parser.add_argument( - "--posctrl-col", - help="Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all gRNAs, feed empty string ''.", - type=str, - default="target_group", - ) - input_parser.add_argument( - "--posctrl-val", - help="Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the positive control in calculating log fold change.", - type=str, - default="PosCtrl", - ) - - input_parser.add_argument( - "--lfc-conds", - help="Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated between, delimited by comma", - type=str, - default="top,bot", - ) - input_parser.add_argument( - "--control-condition", - help="Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate to be calculated", - type=str, - default="bulk", - ) - - return parser - - def check_args(args): lfc_conds = args.lfc_conds.split(",") if not len(lfc_conds) == 2: diff --git a/docs/requirements.txt b/docs/requirements.txt index b8c9f1d..3e4a1a4 100644 --- a/docs/requirements.txt +++ b/docs/requirements.txt @@ -1,3 +1,5 @@ +pandas +bio + sphinx-argparse -m2r -furo==2021.11.16 \ No newline at end of file +m2r \ No newline at end of file From 22858cd9bf18356ba2e61a29e985ae84b50837a1 Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 23:08:36 -0400 Subject: [PATCH 040/107] remove bean dependency --- .github/workflows/documentation.yml | 8 +------- 1 file changed, 1 insertion(+), 7 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 6a71792..19a808c 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -13,13 +13,7 @@ jobs: python-version: '3.x' - name: Install Sphinx & Dependencies - run: pip install sphinx sphinx_markdown_builder sphinx_rtd_theme sphinx-argparse m2r - - name: Install bean - run: | - python -m pip install --upgrade pip - pip install torch torchvision torchaudio - pip install -r requirements.txt - pip install -e . + run: pip install sphinx sphinx_markdown_builder sphinx_rtd_theme sphinx-argparse m2r pandas bio - name: Build Documentation run: cd "$GITHUB_WORKSPACE/docs" && sphinx-build . _build From f026a07239a196ba13fa701fba5a7cebd45e5515 Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 23:17:33 -0400 Subject: [PATCH 041/107] update dependency --- .github/workflows/CI.yml | 2 +- docs/commands/create-screen.md | 13 +++++-------- docs/qc.rst | 2 +- docs/run.rst | 2 +- docs/subcommands.rst | 1 + 5 files changed, 9 insertions(+), 11 deletions(-) diff --git a/.github/workflows/CI.yml b/.github/workflows/CI.yml index 0da743b..0e6ba20 100755 --- a/.github/workflows/CI.yml +++ b/.github/workflows/CI.yml @@ -27,7 +27,7 @@ jobs: - name: Install dependencies run: | python -m pip install --upgrade pip - pip install torch==1.12.1+cpu torchvision==0.13.1+cpu torchaudio==0.12.1 --extra-index-url https://download.pytorch.org/whl/cpu + pip install torch torchvision torchaudio pip install -r requirements.txt pip install -e . - name: Test with pytest diff --git a/docs/commands/create-screen.md b/docs/commands/create-screen.md index 4cf6964..d9200b7 100644 --- a/docs/commands/create-screen.md +++ b/docs/commands/create-screen.md @@ -1,12 +1,9 @@ -## `bean create-screen`: Create ReporterScreen object from flat files +# `bean create-screen`: Create ReporterScreen object from flat files ```bash bean create-screen gRNA_library.csv sample_list.csv gRNA_counts_table.csv ``` -### Input - * [gRNA_library.csv](#1-gRNA_librarycsv) - * [sample_list.csv](#2-sample_listcsv) +## Input + * gRNA_library.csv + * sample_list.csv * gRNA_counts_table.csv: Table with gRNA ID in the first column and sample IDs as the column names (first row) - -### Full Parameters - * `-e`, `--edits` (default: `None`): Path to edit counts .csv table, with index at first column and column names at the first row. - * `-o`, `--output-prefix` (default: `None`): Output file prefix (output will be saved as `output_prefix.h5ad`). If not provided, `gRNA_counts_table_csv` file prefix is used. \ No newline at end of file +`gRNA_library.csv` and `sample_list.csv` should be formatted as :ref:`input`. \ No newline at end of file diff --git a/docs/qc.rst b/docs/qc.rst index e0fbb94..604e77a 100644 --- a/docs/qc.rst +++ b/docs/qc.rst @@ -6,6 +6,6 @@ Full parameters ================== .. argparse:: - :filename: ../bean/qc/utils.py + :filename: ../bean/qc/parser.py :func: parse_args :prog: bean qc \ No newline at end of file diff --git a/docs/run.rst b/docs/run.rst index aea11a9..a6dc1fc 100644 --- a/docs/run.rst +++ b/docs/run.rst @@ -6,6 +6,6 @@ Full parameters ================== .. argparse:: - :filename: ../bean/model/run.py + :filename: ../bean/model/parser.py :func: parse_args :prog: bean run \ No newline at end of file diff --git a/docs/subcommands.rst b/docs/subcommands.rst index 2feb03d..56319ca 100644 --- a/docs/subcommands.rst +++ b/docs/subcommands.rst @@ -11,3 +11,4 @@ Subcommands qc filter run + create_screen \ No newline at end of file From 90683be72cb34b435dd7aa56aba761ad9e12097b Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 23:19:00 -0400 Subject: [PATCH 042/107] update dependency --- .github/workflows/documentation.yml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 19a808c..f9b97a8 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -13,7 +13,7 @@ jobs: python-version: '3.x' - name: Install Sphinx & Dependencies - run: pip install sphinx sphinx_markdown_builder sphinx_rtd_theme sphinx-argparse m2r pandas bio + run: pip install sphinx sphinx_markdown_builder sphinx_rtd_theme sphinx-argparse m2r pandas bio distutils - name: Build Documentation run: cd "$GITHUB_WORKSPACE/docs" && sphinx-build . _build From b529340fa7c6ba842674d17c71aa732ddf7bbfea Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 23:20:45 -0400 Subject: [PATCH 043/107] update dependency --- .github/workflows/documentation.yml | 4 +++- 1 file changed, 3 insertions(+), 1 deletion(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index f9b97a8..f6aad42 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -13,7 +13,9 @@ jobs: python-version: '3.x' - name: Install Sphinx & Dependencies - run: pip install sphinx sphinx_markdown_builder sphinx_rtd_theme sphinx-argparse m2r pandas bio distutils + run: | + pip install sphinx sphinx_markdown_builder sphinx_rtd_theme sphinx-argparse m2r pandas bio + sudo apt-get install python3-distutils - name: Build Documentation run: cd "$GITHUB_WORKSPACE/docs" && sphinx-build . _build From 920d937de8f5d96be4a14d0678fd86012d26e780 Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 23:24:28 -0400 Subject: [PATCH 044/107] remove dependency of distutils --- bean/qc/parser.py | 14 ++++++++++++-- 1 file changed, 12 insertions(+), 2 deletions(-) diff --git a/bean/qc/parser.py b/bean/qc/parser.py index 2207e83..dd41f66 100644 --- a/bean/qc/parser.py +++ b/bean/qc/parser.py @@ -1,5 +1,15 @@ import argparse -import distutils + + +def str2bool(v): + if isinstance(v, bool): + return v + if v.lower() in ("yes", "true", "t", "y", "1"): + return True + elif v.lower() in ("no", "false", "f", "n", "0"): + return False + else: + raise argparse.ArgumentTypeError("Boolean value expected.") def parse_args(parser=None): @@ -70,7 +80,7 @@ def parse_args(parser=None): input_parser.add_argument( "--tiling", dest="tiling", - type=lambda x: bool(distutils.util.strtobool(x)), + type=str2bool, help="Specify that the guide library is tiling library without 'n guides per target' design", ) input_parser.add_argument( From e241fa94cba3e8896dcc2966455c4af787ec8470 Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 23:27:14 -0400 Subject: [PATCH 045/107] remove 3.8 requirement --- .github/workflows/CI.yml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/.github/workflows/CI.yml b/.github/workflows/CI.yml index 0e6ba20..1044580 100755 --- a/.github/workflows/CI.yml +++ b/.github/workflows/CI.yml @@ -23,7 +23,7 @@ jobs: - name: Set up Python uses: actions/setup-python@v3 with: - python-version: '3.8' + python-version: '3.x' - name: Install dependencies run: | python -m pip install --upgrade pip From 59dc74e0d0c204445fb056afb7eb38b4b7816b53 Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 23:37:13 -0400 Subject: [PATCH 046/107] deploy --- .github/workflows/documentation.yml | 7 ++++--- 1 file changed, 4 insertions(+), 3 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index f6aad42..5b84678 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -19,7 +19,8 @@ jobs: - name: Build Documentation run: cd "$GITHUB_WORKSPACE/docs" && sphinx-build . _build - - uses: actions/upload-artifact@v3 + - name: Deploy + uses: peaceiris/actions-gh-pages@v3 with: - name: Documentation - path: "$GITHUB_WORKSPACE/docs/_build/html/" + github_token: ${{ secrets.GITHUB_TOKEN }} + publish_dir: docs/_build/html From affb80532d798675649d4e41f5985956cf9259d9 Mon Sep 17 00:00:00 2001 From: jykr Date: Fri, 29 Mar 2024 23:44:59 -0400 Subject: [PATCH 047/107] change deploy dif --- .github/workflows/documentation.yml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 5b84678..bd348e0 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -23,4 +23,4 @@ jobs: uses: peaceiris/actions-gh-pages@v3 with: github_token: ${{ secrets.GITHUB_TOKEN }} - publish_dir: docs/_build/html + publish_dir: _build/html From c778ac05ec2312263d4d88a495b693c0c2372091 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 00:00:43 -0400 Subject: [PATCH 048/107] update directory --- .github/workflows/documentation.yml | 7 +++---- LICENSE | 0 MANIFEST.in | 0 README.md | 0 bean/__init__.py | 0 bean/annotate/__init__.py | 0 bean/annotate/_supporting_fn.py | 0 bean/annotate/filter_alleles.py | 0 bean/annotate/ldlr_exons.fa | 0 bean/annotate/translate_allele.py | 0 bean/annotate/utils.py | 0 bean/cli/count.py | 0 bean/cli/count_samples.py | 0 bean/cli/create_screen.py | 0 bean/cli/filter.py | 0 bean/cli/profile.py | 0 bean/cli/qc.py | 0 bean/cli/run.py | 0 bean/framework/AminoAcidEdit.py | 0 bean/framework/Edit.py | 0 bean/framework/ReporterScreen.py | 0 bean/framework/__init__.py | 0 bean/framework/__pycache__/ReporterScreen_.py | 0 bean/framework/allele_counts.py | 0 bean/framework/parser.py | 0 bean/framework/read_from_csvs.py | 0 bean/mapping/CRISPResso2Align.pyx | 0 bean/mapping/GuideEditCounter.py | 0 bean/mapping/__init__.py | 0 bean/mapping/_supporting_fn.py | 0 bean/mapping/utils.py | 0 bean/model/__init__.py | 0 bean/model/model.py | 0 bean/model/parser.py | 0 bean/model/readwrite.py | 0 bean/model/run.py | 0 bean/model/survival_model.py | 0 bean/model/utils.py | 0 bean/plotting/__init__.py | 0 bean/plotting/allele_stats.py | 0 bean/plotting/editing_patterns.py | 0 bean/plotting/utils.py | 0 bean/preprocessing/__init__.py | 0 bean/preprocessing/data_class.py | 0 bean/preprocessing/get_alpha0.py | 0 bean/preprocessing/get_pi_alpha0.py | 0 bean/preprocessing/utils.py | 0 bean/qc/__init__.py | 0 bean/qc/guide_qc.py | 0 bean/qc/parser.py | 0 bean/qc/sample_qc.py | 0 bean/qc/utils.py | 0 bean/utils/__init__.py | 0 bean/utils/arithmetric.py | 0 bean/utils/plot.py | 0 docs/ReporterScreen_api.ipynb | 0 docs/ReporterScreen_api.rst | 0 docs/cds.rst | 0 docs/commands/count.md | 0 docs/commands/create-screen.md | 0 docs/commands/filter.md | 0 docs/commands/input.md | 0 docs/commands/profile.md | 0 docs/commands/qc.md | 0 docs/commands/run.md | 0 docs/conf.py | 0 docs/count.rst | 0 docs/count_samples.rst | 0 docs/editing_pattern_analysis.ipynb | 0 docs/exon_fa_format.md | 0 docs/filter.rst | 0 docs/gwas.rst | 0 docs/index.md | 0 docs/index_.rst | 0 docs/input.rst | 0 docs/profile.rst | 0 docs/qc.rst | 0 docs/qc_report_LDLvar.html | 0 docs/qc_report_LDLvar.ipynb | 0 docs/requirements.txt | 0 docs/run.rst | 0 docs/sample_quality_report.ipynb | 0 docs/subcommands.rst | 0 docs/tutorials/ldl_cds.md | 0 docs/tutorials/ldl_var.md | 0 environment_bean.yml | 0 imgs/anbe.svg | 0 imgs/bean.gif | Bin imgs/bean_title.svg | 0 imgs/dag_bean_v2.png | Bin imgs/data_structure_v2.png | Bin imgs/model_output.png | Bin imgs/output_20_2.png | Bin imgs/output_34_1.png | Bin imgs/profile_output.png | Bin imgs/qc_output.png | Bin imgs/reporter.jpg | Bin imgs/reporter_construct.png | Bin imgs/reporter_construct.svg | 0 imgs/reporter_screen.svg | 0 imgs/screendata.svg | 0 imgs/sequence_struct.png | Bin imgs/sorting_bins@8x.png | Bin imgs/tiling.png | Bin imgs/translation.png | Bin imgs/variant.png | Bin notebooks/profile_editing_preference.ipynb | 0 notebooks/sample_quality_report.ipynb | 0 pyproject.toml | 0 requirements.txt | 0 setup.py | 0 .../CRISPRessoCount_RUNNING_LOG.txt | 0 tests/data/accessibility_signal.bw | Bin tests/data/accessibility_signal_chr6.bw | Bin tests/data/bean_count_test_screen.h5ad | Bin tests/data/fastq/rep1_bot_R1.fastq | 0 tests/data/fastq/rep1_bot_R2.fastq | 0 tests/data/fastq/rep1_bulk_R1.fastq | 0 tests/data/fastq/rep1_bulk_R2.fastq | 0 tests/data/fastq/rep1_top_R1.fastq | 0 tests/data/fastq/rep1_top_R2.fastq | 0 tests/data/fastq/rep2_bot_R1.fastq | 0 tests/data/fastq/rep2_bot_R2.fastq | 0 tests/data/fastq/rep2_bulk_R1.fastq | 0 tests/data/fastq/rep2_bulk_R2.fastq | 0 tests/data/fastq/rep2_top_R1.fastq | 0 tests/data/fastq/rep2_top_R2.fastq | 0 tests/data/fastq/rep3_bot_R1.fastq | 0 tests/data/fastq/rep3_bot_R2.fastq | 0 tests/data/fastq/rep3_bulk_R1.fastq | 0 tests/data/fastq/rep3_bulk_R2.fastq | 0 tests/data/fastq/rep3_top_R1.fastq | 0 tests/data/fastq/rep3_top_R2.fastq | 0 tests/data/fastq/rep4_bot_R1.fastq | 0 tests/data/fastq/rep4_bot_R2.fastq | 0 tests/data/fastq/rep4_bulk_R1.fastq | 0 tests/data/fastq/rep4_bulk_R2.fastq | 0 tests/data/fastq/rep4_top_R1.fastq | 0 tests/data/fastq/rep4_top_R2.fastq | 0 tests/data/gene_symbols.txt | 0 tests/data/ldlr_exons.fa | 0 tests/data/sample_list.csv | 0 tests/data/sample_list_tiling.csv | 0 tests/data/test_R1.fastq | 0 tests/data/test_R2.fastq | 0 tests/data/test_guide_info_tiling.csv | 0 tests/data/test_guide_info_tiling_chrom.csv | 0 tests/data/test_tiling_R1.fastq | 0 tests/data/test_tiling_R2.fastq | 0 tests/data/tiling_mini_screen.h5ad | Bin tests/data/tiling_mini_screen_missing.h5ad | Bin tests/data/var_mini_counts.csv | 0 tests/data/var_mini_guides.csv | 0 tests/data/var_mini_samples.csv | 0 tests/data/var_mini_screen.h5ad | Bin ..._screen_annotated.translation_ref_mismatches.csv | 0 tests/data/var_mini_screen_missing.h5ad | Bin tests/test_Edit.py | 0 tests/test_count.py | 0 tests/test_filter.py | 0 tests/test_qc.py | 0 tests/test_res/.gitkeep | 0 tests/test_run.py | 0 163 files changed, 3 insertions(+), 4 deletions(-) mode change 100644 => 100755 LICENSE mode change 100644 => 100755 MANIFEST.in mode change 100644 => 100755 README.md mode change 100644 => 100755 bean/__init__.py mode change 100644 => 100755 bean/annotate/__init__.py mode change 100644 => 100755 bean/annotate/_supporting_fn.py mode change 100644 => 100755 bean/annotate/filter_alleles.py mode change 100644 => 100755 bean/annotate/ldlr_exons.fa mode change 100644 => 100755 bean/annotate/translate_allele.py mode change 100644 => 100755 bean/annotate/utils.py mode change 100644 => 100755 bean/cli/count.py mode change 100644 => 100755 bean/cli/count_samples.py mode change 100644 => 100755 bean/cli/create_screen.py mode change 100644 => 100755 bean/cli/filter.py mode change 100644 => 100755 bean/cli/profile.py mode change 100644 => 100755 bean/cli/qc.py mode change 100644 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tests/test_Edit.py mode change 100644 => 100755 tests/test_count.py mode change 100644 => 100755 tests/test_filter.py mode change 100644 => 100755 tests/test_qc.py mode change 100644 => 100755 tests/test_res/.gitkeep mode change 100644 => 100755 tests/test_run.py diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index bd348e0..b003783 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -19,8 +19,7 @@ jobs: - name: Build Documentation run: cd "$GITHUB_WORKSPACE/docs" && sphinx-build . _build - - name: Deploy - uses: peaceiris/actions-gh-pages@v3 + - uses: actions/upload-artifact@v3 with: - github_token: ${{ secrets.GITHUB_TOKEN }} - publish_dir: _build/html + name: Documentation + path: "$GITHUB_WORKSPACE/docs/_build/" diff --git a/LICENSE b/LICENSE old mode 100644 new mode 100755 diff --git a/MANIFEST.in b/MANIFEST.in old mode 100644 new mode 100755 diff --git a/README.md b/README.md old mode 100644 new mode 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a/tests/test_res/.gitkeep b/tests/test_res/.gitkeep old mode 100644 new mode 100755 diff --git a/tests/test_run.py b/tests/test_run.py old mode 100644 new mode 100755 From 5d722a11079d3a1abeda1bdd5ec4f8a495c9e64b Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 00:08:02 -0400 Subject: [PATCH 049/107] require pyro version that supports Torch2 --- .gitignore | 1 + 1 file changed, 1 insertion(+) diff --git a/.gitignore b/.gitignore index 0d20b64..00cb0e6 100644 --- a/.gitignore +++ b/.gitignore @@ -1 +1,2 @@ *.pyc +*.fastq From b5a7fb4aeaabc61a9ae28ace5a6c4c04c5ccb38d Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 00:10:09 -0400 Subject: [PATCH 050/107] try different working directory --- .github/workflows/documentation.yml | 2 +- requirements.txt | 2 +- 2 files changed, 2 insertions(+), 2 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index b003783..cb692a6 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -22,4 +22,4 @@ jobs: - uses: actions/upload-artifact@v3 with: name: Documentation - path: "$GITHUB_WORKSPACE/docs/_build/" + path: "$GITHUB_WORKSPACE/_build/" diff --git a/requirements.txt b/requirements.txt index 04bdbca..7c02022 100755 --- a/requirements.txt +++ b/requirements.txt @@ -11,7 +11,7 @@ liftover openpyxl>=3.0.10 papermill>=2.4.0 pyBigWig>=0.3.18 -pyro-ppl +pyro-ppl>=1.8.5 scikit-learn statsmodels>=0.12.1 ipykernel From 0abcb2fd5e8c83b16045c3e623183d5dca2af32c Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 00:12:21 -0400 Subject: [PATCH 051/107] try different working directory --- .github/workflows/documentation.yml | 5 +++-- 1 file changed, 3 insertions(+), 2 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index cb692a6..ea13754 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -17,9 +17,10 @@ jobs: pip install sphinx sphinx_markdown_builder sphinx_rtd_theme sphinx-argparse m2r pandas bio sudo apt-get install python3-distutils - name: Build Documentation - run: cd "$GITHUB_WORKSPACE/docs" && sphinx-build . _build + working-directory: $GITHUB_WORKSPACE/docs + run: sphinx-build . _build - uses: actions/upload-artifact@v3 with: name: Documentation - path: "$GITHUB_WORKSPACE/_build/" + path: $GITHUB_WORKSPACE/docs/_build/ From 98b748fa1a3c261083cec7dc2716f953ebb925e7 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 00:13:44 -0400 Subject: [PATCH 052/107] try different working directory --- .github/workflows/documentation.yml | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index ea13754..46a9ad6 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -17,10 +17,10 @@ jobs: pip install sphinx sphinx_markdown_builder sphinx_rtd_theme sphinx-argparse m2r pandas bio sudo apt-get install python3-distutils - name: Build Documentation - working-directory: $GITHUB_WORKSPACE/docs + working-directory: docs run: sphinx-build . _build - uses: actions/upload-artifact@v3 with: name: Documentation - path: $GITHUB_WORKSPACE/docs/_build/ + path: docs/_build/ From 3a292d763c0ba835146bab5f3193d4a6f332dfa4 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 00:21:28 -0400 Subject: [PATCH 053/107] try push --- .github/workflows/documentation.yml | 9 +++++++-- 1 file changed, 7 insertions(+), 2 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 46a9ad6..fc12121 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -20,7 +20,12 @@ jobs: working-directory: docs run: sphinx-build . _build - - uses: actions/upload-artifact@v3 + - uses: actions/checkout@v3 with: name: Documentation - path: docs/_build/ + run: | + git config --global user.name 'Your Name' + git config --global user.email 'your-username@users.noreply.github.com' + git add --all docs/_build/ + git commit -m "Documentation generated" + git push From e8a6df187d393a9c3cb76c83eaba75a827c458a3 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 00:22:56 -0400 Subject: [PATCH 054/107] Require Pyro-ppl >= 1.8.5 --- setup.py | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/setup.py b/setup.py index 04a28ce..5ffbc6a 100755 --- a/setup.py +++ b/setup.py @@ -46,7 +46,7 @@ "openpyxl>=3.0.10", "papermill>=2.4.0", "pyBigWig>=0.3.18", - "pyro-ppl==1.8.1", + "pyro-ppl>=1.8.5", "scikit-learn", "statsmodels>=0.12.1", "ipykernel", From 494924684c6e11e319fb51cd1958643cf50360c4 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 00:27:29 -0400 Subject: [PATCH 055/107] fix push --- .github/workflows/documentation.yml | 15 +++++++-------- 1 file changed, 7 insertions(+), 8 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index fc12121..ec4f2f9 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -21,11 +21,10 @@ jobs: run: sphinx-build . _build - uses: actions/checkout@v3 - with: - name: Documentation - run: | - git config --global user.name 'Your Name' - git config --global user.email 'your-username@users.noreply.github.com' - git add --all docs/_build/ - git commit -m "Documentation generated" - git push + - name: Push documentation + run: | + git config --global user.name 'Your Name' + git config --global user.email 'your-username@users.noreply.github.com' + git add --all docs/_build/ + git commit -m "Documentation generated" + git push From 3c88eb19232c2b13585aee17ffb404e2cfa0dd83 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 00:28:58 -0400 Subject: [PATCH 056/107] change scope of checkout --- .github/workflows/documentation.yml | 3 +-- 1 file changed, 1 insertion(+), 2 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index ec4f2f9..60c645e 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -16,11 +16,10 @@ jobs: run: | pip install sphinx sphinx_markdown_builder sphinx_rtd_theme sphinx-argparse m2r pandas bio sudo apt-get install python3-distutils + - uses: actions/checkout@v3 - name: Build Documentation working-directory: docs run: sphinx-build . _build - - - uses: actions/checkout@v3 - name: Push documentation run: | git config --global user.name 'Your Name' From 86aa6716cb997b411e15e97ec3c637f63858ae19 Mon Sep 17 00:00:00 2001 From: Your Name Date: Sat, 30 Mar 2024 04:30:11 +0000 Subject: [PATCH 057/107] Documentation generated --- docs/_build/.buildinfo | 4 + .../.doctrees/ReporterScreen_api.doctree | Bin 0 -> 97987 bytes docs/_build/.doctrees/cds.doctree | Bin 0 -> 31925 bytes docs/_build/.doctrees/commands/count.doctree | Bin 0 -> 12303 bytes .../.doctrees/commands/create-screen.doctree | Bin 0 -> 5140 bytes 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z@5Q8tBcyc0o2);TdSDIG6ErD&$rZyvQWa6sD7ACDI6jTROt~8Ay0yhVWTx9d1ws_B z0tLsm>744rO|)7xY#eL#3|vM>iWd0YMvw>n=%h}n6GKE6aBkI=`%u&rV|Q&tuuRvPmxm8t%xDCsKi**FcYv4$ov}jCV>Wa@RYTZY;dI z@CxY?%&mXzuI3bx2U#a1lsjwkgPE03L>0W + + + + + + + ReporterScreen API tutorial — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + + +

    +
    +
    + + +
    + +
    +

    ReporterScreen API tutorial¶

    +

    Load the required packages. (Anndata import isn’t required to use the package).

    +
    import numpy as np
+import pandas as pd
+import anndata as ad
+import seaborn as sns
+import matplotlib.pyplot as plt
+import bean as br
+
    +
    +

    bean ReporterScreen object and perturb-seq Screen object are both anndata compatible.

    +
    adata = ad.read_h5ad("bean_count_07+1021_LDLvar.h5ad")
+
    +
    +
    adata
+
    +
    +
    AnnData object with n_obs × n_vars = 3455 × 12
+    obs: 'name', 'Unnamed: 0', 'Target gene/variant', 'Target descriptor', 'Arbitrary number', 'gRNA position category', 'Target base position in gRNA', 'Target base position in reporter', 'BE', 'Group', 'sequence', 'Reporter', 'barcode', '5-nt PAM', 'offset', 'target', 'target_pos', 'Group2', 'masked_sequence', 'masked_barcode', 'edit_rate'
+    var: 'index', 'sort', 'replicate'
+    uns: 'allele_counts', 'edit_counts'
+    layers: 'X_bcmatch', 'edits'
+
    +
    +
    cdata = br.read_h5ad("bean_count_07+1021_LDLvar.h5ad")
+
    +
    +
    cdata
+
    +
    +
    Genome Editing Screen comprised of n_guides x n_conditions = 3455 x 12
+   guides:    'name', 'Unnamed: 0', 'Target gene/variant', 'Target descriptor', 'Arbitrary number', 'gRNA position category', 'Target base position in gRNA', 'Target base position in reporter', 'BE', 'Group', 'sequence', 'Reporter', 'barcode', '5-nt PAM', 'offset', 'target', 'target_pos', 'Group2', 'masked_sequence', 'masked_barcode', 'edit_rate'
+   samples:    'index', 'sort', 'replicate'
+   condit_m:
+   condit_p:
+   layers:    'X_bcmatch', 'edits'
+   uns:       'allele_counts', 'edit_counts'
+
    +
    +
      +

    • cdata.X: guide count

      • +

    • cdata.guides: guide metadata

      • +

    • cdata.samples: sample/condition metadata

      • +

    • cdata.layers["X_bcmatch"]: barcode-matched guide counts

      • +

    • cdata.layers["edits"]: edit counts

      • +

    • cdata.uns["allele_counts"]: allele counts per guide and condition

      • +

    • cdata.uns["edit_counts"]: edit counts per guide and condition

      • +
    +

    guides attribute contains the information about each guide.

    +
    cdata.guides
+
    +
    +
    + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
    nameUnnamed: 0Target gene/variantTarget descriptorArbitrary numbergRNA position categoryTarget base position in gRNATarget base position in reporterBEGroup...Reporterbarcode5-nt PAMoffsettargettarget_posGroup2masked_sequencemasked_barcodeedit_rate
    0CONTROL_1_g10CONTROLNaN1g1410ABENegCtrl...CCAAGCCCTACGCGGTAGGGAACTTTGGGAGCGTTTGGGAG-10CONTROL_19NegCtrlCCTGCGCGGTGGGGGGCTTTGTTT0.531163
    1CONTROL_1_g21CONTROLNaN1g2511ABENegCtrl...TCCAAGCCCTACGCGGTAGGGAACTTTGGGAGAACATGGGA-11CONTROL_110NegCtrlCCCTGCGCGGTGGGGGGCTTGGCG0.640765
    2CONTROL_1_g32CONTROLNaN1g3512ABENegCtrl...GTCCAAGCCCTACGCGGTAGGGAACTTTGGGACGCTTTGGG-12CONTROL_111NegCtrlCCCTGCGCGGTGGGGGGCTCGCT0.417709
    3CONTROL_1_g43CONTROLNaN1g4713ABENegCtrl...CGTCCAAGCCCTACGCGGTAGGGAACTTTGGGTGAGTTTGG-13CONTROL_112NegCtrlGGCCCTGCGCGGTGGGGGGCTGGG0.126400
    4CONTROL_1_g54CONTROLNaN1g5814ABENegCtrl...ACGTCCAAGCCCTACGCGGTAGGGAACTTTGGGTATCTTTG-14CONTROL_113NegCtrlGGGCCCTGCGCGGTGGGGGGGTGT0.201104
    ..................................................................
    3450rs9987289_Maj_ABE_347_g13450rs9987289Maj347g1310ABEVariant...TGCTTGGGCATCAATATCACGTGGAACCAGCCCAGTCCAGC-10rs9987289_Maj_ABE_3479VariantGCGTCGGTGTCGCGTGGGGCGGT0.087379
    3451rs9987289_Maj_ABE_347_g23451rs9987289Maj347g2411ABEVariant...ATGCTTGGGCATCAATATCACGTGGAACCAGCTCGCACCAG-11rs9987289_Maj_ABE_34710VariantGGCGTCGGTGTCGCGTGGGTCGC0.299923
    3452rs9987289_Maj_ABE_347_g33452rs9987289Maj347g3612ABEVariant...GATGCTTGGGCATCAATATCACGTGGAACCAGGCACAACCA-12rs9987289_Maj_ABE_34711VariantTGGGCGTCGGTGTCGCGTGGGCGC0.224973
    3453rs9987289_Maj_ABE_347_g43453rs9987289Maj347g4713ABEVariant...AGATGCTTGGGCATCAATATCACGTGGAACCATTGCGAACC-13rs9987289_Maj_ABE_34712VariantTTGGGCGTCGGTGTCGCGTGTTGC0.265378
    3454rs9987289_Maj_ABE_347_g53454rs9987289Maj347g5814ABEVariant...TAGATGCTTGGGCATCAATATCACGTGGAACCGCGAGGAAC-14rs9987289_Maj_ABE_34713VariantCTTGGGCGTCGGTGTCGCGTGCGG0.266573
    +

    3455 rows × 21 columns

    +

    samples attribute contains the sample and condition specific information.

    +
    cdata.samples
+
    +
    +
    + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
    indexsortreplicate
    0rep1_botbotrep1
    1rep2_botbotrep2
    2rep3_VPA_botbotrep3_VPA
    3rep4_VPA_botbotrep4_VPA
    4rep1_bulkbulkrep1
    5rep2_bulkbulkrep2
    6rep3_VPA_bulkbulkrep3_VPA
    7rep4_VPA_bulkbulkrep4_VPA
    8rep1_toptoprep1
    9rep2_toptoprep2
    10rep3_VPA_toptoprep3_VPA
    11rep4_VPA_toptoprep4_VPA
    +

    Allele_counts information is stored in .uns["allele_counts"].

    +
    cdata.uns["allele_counts"]
+
    +
    +
    + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
    guideallelerep1_botrep2_botrep3_VPA_botrep4_VPA_botrep1_bulkrep2_bulkrep3_VPA_bulkrep4_VPA_bulkrep1_toprep2_toprep3_VPA_toprep4_VPA_top
    012:51779544AGA_Maj_ABE_2_g10:9:+:A>G,5:14:+:A>G14201306152172214343
    112:51779544AGA_Maj_ABE_2_g1-4:5:+:A>G,-2:7:+:A>G,5:14:+:A>G,10:19:+:A>G100000000000
    212:51779544AGA_Maj_ABE_2_g1-7:2:+:A>G,0:9:+:A>G,5:14:+:A>G342010520010
    312:51779544AGA_Maj_ABE_2_g1-9:0:+:G>A,-8:1:+:G>A,-7:2:+:A>C,-6:3:+:C>A,-4...100102100010
    412:51779544AGA_Maj_ABE_2_g1-7:2:+:A>G,10:19:+:A>G110000000000
    .............................................
    438407rs9987289_Maj_ABE_347_g54:17:+:A>G,6:19:+:A>G,9:22:+:A>G000000000020
    438408rs9987289_Maj_ABE_347_g5-12:1:+:A>G,6:19:+:A>G,9:22:+:A>G,11:24:+:G>A000000000010
    438409rs9987289_Maj_ABE_347_g5-12:1:+:A>G,6:19:+:A>G,9:22:+:A>G,16:29:+:A>G000000000001
    438410rs9987289_Maj_ABE_347_g5-12:1:+:A>G,0:13:+:A>G,6:19:+:A>G,9:22:+:A>G,1...000000000100
    438411rs9987289_Maj_ABE_347_g5-12:1:+:A>G,6:19:+:A>G,9:22:+:A>G,12:25:+:T>G000000000001
    +

    438412 rows × 14 columns

    +

    Base-level edit counts can be saved at .uns[“edit_countsâ€].

    +
    cdata.uns["edit_counts"]
+
    +
    +
    + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
    guideeditrep1_botrep2_botrep3_VPA_botrep4_VPA_botrep1_bulkrep2_bulkrep3_VPA_bulkrep4_VPA_bulkrep1_toprep2_toprep3_VPA_toprep4_VPA_topref_basealt_base
    012:51779544AGA_Maj_ABE_2_g1-1:8:+:G>A000010000000GA
    112:51779544AGA_Maj_ABE_2_g1-1:8:+:G>C000000001010GC
    212:51779544AGA_Maj_ABE_2_g1-1:8:+:G>T000010000000GT
    312:51779544AGA_Maj_ABE_2_g1-2:7:+:A>C000000002010AC
    412:51779544AGA_Maj_ABE_2_g1-2:7:+:A>G1934404592566768481492AG
    ...................................................
    217563rs9987289_Maj_ABE_347_g58:21:+:C>A070001101000CA
    217564rs9987289_Maj_ABE_347_g58:21:+:C>G002008000180CG
    217565rs9987289_Maj_ABE_347_g58:21:+:C>T007000700000CT
    217566rs9987289_Maj_ABE_347_g59:22:+:A>G92130513746122058235947AG
    217567rs9987289_Maj_ABE_347_g59:22:+:A>T000000070000AT
    +

    217568 rows × 16 columns

    +
    +

    Subsetting & addition¶

    +

    Works as anndata, supports allele & edit count operations.

    +
    +

    Subsetting & selection¶

    +
    cdata_subset = cdata[:10,cdata.samples.sort == "bulk"]
+
    +
    +
    ['rep1_bulk', 'rep2_bulk', 'rep3_VPA_bulk', 'rep4_VPA_bulk']
+
    +
    +
    cdata_subset.uns["allele_counts"]
+
    +
    +
    + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
    guideallelerep1_bulkrep2_bulkrep3_VPA_bulkrep4_VPA_bulk
    14979CONTROL_10_g1-4:5:+:A>G,0:9:+:A>G8130
    14980CONTROL_10_g1-7:2:+:C>T00010
    14981CONTROL_10_g1-4:5:+:A>G2922925
    14982CONTROL_10_g11:10:+:A>G0641
    14983CONTROL_10_g1-4:5:+:A>G,1:10:+:A>G111512
    .....................
    22837CONTROL_1_g5-13:0:+:A>-,-12:1:+:C>T,-9:4:+:C>G,-8:5:+:C>T,...0000
    22838CONTROL_1_g5-6:7:+:A>C,7:20:+:A>G0000
    22839CONTROL_1_g5-13:0:+:A>G,-10:3:+:T>G,0:13:+:A>G,7:20:+:A>G0000
    22840CONTROL_1_g50:13:+:A>T0000
    22841CONTROL_1_g50:13:+:A>G,18:31:+:G>A0000
    +

    1080 rows × 6 columns

    +
    +
    +

    LFC calculation & Addition¶

    +
    cdata1 = br.read_h5ad("/data/pinello/PROJECTS/2021_08_ANBE/data/072121_ABE_topbot/bean_counts/LDLvar/032422_crispresso/bean_count_072121_ABE_topbot_LDLvar.h5ad")
+cdata2 = br.read_h5ad("/data/pinello/PROJECTS/2021_08_ANBE/data/102121_ABE_topbot/bean_counts/LDLvar/032422_crispresso/bean_count_102121_ABE_topbot_LDLvar.h5ad")
+
    +
    +
    cdata1.samples["sort"] = cdata1.samples["index"].map(lambda s: s.rsplit("_", 1)[-1])
+cdata1.samples["replicate"] = cdata1.samples["index"].map(lambda s: s.rsplit("_", 1)[0])
+cdata2.samples["sort"] = cdata2.samples["index"].map(lambda s: s.rsplit("_", 1)[-1])
+cdata2.samples["replicate"] = cdata2.samples["index"].map(lambda s: s.rsplit("_", 1)[0])
+
    +
    +
    cdata1.log_norm()
+lfc1 = cdata1.log_fold_change_reps("bot", "top")
+cdata2.log_norm()
+lfc2 = cdata2.log_fold_change_reps("bot", "top")
+lfcs = lfc1.join(lfc2, lsuffix = "_1", rsuffix = "_2")
+sns.pairplot(lfcs)
+
    +
    +_images/output_20_2.png +

    LFC can be aggregated for biological replicates.

    +
    cdata1.log_fold_change_aggregate("bot", "top", aggregate_condit = "replicate")
+
    +
    +
    cdata1.guides
+
    +
    +
    + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
    nameUnnamed: 0Target gene/variantTarget descriptorArbitrary numbergRNA position categoryTarget base position in gRNATarget base position in reporterBEGroup...Reporterbarcode5-nt PAMoffsettargettarget_posGroup2masked_sequencemasked_barcodebot_top.lfc.median
    0CONTROL_1_g10CONTROLNaN1g1410ABENegCtrl...CCAAGCCCTACGCGGTAGGGAACTTTGGGAGCGTTTGGGAG-10CONTROL_19NegCtrlCCTGCGCGGTGGGGGGCTTTGTTT-0.158787
    1CONTROL_1_g21CONTROLNaN1g2511ABENegCtrl...TCCAAGCCCTACGCGGTAGGGAACTTTGGGAGAACATGGGA-11CONTROL_110NegCtrlCCCTGCGCGGTGGGGGGCTTGGCG-0.212254
    2CONTROL_1_g32CONTROLNaN1g3512ABENegCtrl...GTCCAAGCCCTACGCGGTAGGGAACTTTGGGACGCTTTGGG-12CONTROL_111NegCtrlCCCTGCGCGGTGGGGGGCTCGCT0.186679
    3CONTROL_1_g43CONTROLNaN1g4713ABENegCtrl...CGTCCAAGCCCTACGCGGTAGGGAACTTTGGGTGAGTTTGG-13CONTROL_112NegCtrlGGCCCTGCGCGGTGGGGGGCTGGG-0.022441
    4CONTROL_1_g54CONTROLNaN1g5814ABENegCtrl...ACGTCCAAGCCCTACGCGGTAGGGAACTTTGGGTATCTTTG-14CONTROL_113NegCtrlGGGCCCTGCGCGGTGGGGGGGTGT0.457033
    ..................................................................
    3450rs9987289_Maj_ABE_347_g13450rs9987289Maj347g1310ABEVariant...TGCTTGGGCATCAATATCACGTGGAACCAGCCCAGTCCAGC-10rs9987289_Maj_ABE_3479VariantGCGTCGGTGTCGCGTGGGGCGGT-0.418312
    3451rs9987289_Maj_ABE_347_g23451rs9987289Maj347g2411ABEVariant...ATGCTTGGGCATCAATATCACGTGGAACCAGCTCGCACCAG-11rs9987289_Maj_ABE_34710VariantGGCGTCGGTGTCGCGTGGGTCGC-0.084936
    3452rs9987289_Maj_ABE_347_g33452rs9987289Maj347g3612ABEVariant...GATGCTTGGGCATCAATATCACGTGGAACCAGGCACAACCA-12rs9987289_Maj_ABE_34711VariantTGGGCGTCGGTGTCGCGTGGGCGC-0.339419
    3453rs9987289_Maj_ABE_347_g43453rs9987289Maj347g4713ABEVariant...AGATGCTTGGGCATCAATATCACGTGGAACCATTGCGAACC-13rs9987289_Maj_ABE_34712VariantTTGGGCGTCGGTGTCGCGTGTTGC-0.517138
    3454rs9987289_Maj_ABE_347_g53454rs9987289Maj347g5814ABEVariant...TAGATGCTTGGGCATCAATATCACGTGGAACCGCGAGGAAC-14rs9987289_Maj_ABE_34713VariantCTTGGGCGTCGGTGTCGCGTGCGG0.002245
    +

    3455 rows × 21 columns

    +

    Technical replicates show decent LFC correlation.

    +
    cdata = cdata1 + cdata2
+
    +
    +
    cdata
+
    +
    +
    Genome Editing Screen comprised of n_guides x n_conditions = 3455 x 12
+   guides:    'name', 'Unnamed: 0', 'Target gene/variant', 'Target descriptor', 'Arbitrary number', 'gRNA position category', 'Target base position in gRNA', 'Target base position in reporter', 'BE', 'Group', 'sequence', 'Reporter', 'barcode', '5-nt PAM', 'offset', 'target', 'target_pos', 'Group2', 'masked_sequence', 'masked_barcode', 'bot_top.lfc.median'
+   samples:    'index', 'sort', 'replicate'
+   condit_m:
+   condit_p:
+   layers:    'edits', 'X_bcmatch'
+   uns:       'allele_counts'
+
    +
    +

    You can concatenate different samples with shared guides.

    +
    br.concat((cdata1, cdata2))
+
    +
    +
    Genome Editing Screen comprised of n_guides x n_conditions = 3455 x 24
+   guides:    'name', 'Unnamed: 0', 'Target gene/variant', 'Target descriptor', 'Arbitrary number', 'gRNA position category', 'Target base position in gRNA', 'Target base position in reporter', 'BE', 'Group', 'sequence', 'Reporter', 'barcode', '5-nt PAM', 'offset', 'target', 'target_pos', 'Group2', 'masked_sequence', 'masked_barcode', 'bot_top.lfc.median'
+   samples:    'index', 'sort', 'replicate'
+   condit_m:
+   condit_p:
+   layers:    'X', 'X_bcmatch', 'edits', 'lognorm_counts', 'lognorm_edits'
+   uns:       'allele_counts'
+
    +
    +
    +
    +
    +

    Getting edit rates from allele counts¶

    +
    cdata.get_edit_rate(normalize_by_editable_base = False,
+                   edited_base = "A",
+                   editable_base_start = 3,
+                   editable_base_end = 8,
+                   bcmatch_thres = 10,
+                   prior_weight = 1)
+
    +
    +
    cdata.uns["edit_counts"] = cdata.get_edit_from_allele()
+
    +
    +
    cdata.get_edit_mat_from_uns("A", "G", match_target_position = True)
+cdata.get_edit_rate(edited_base = "A", bcmatch_thres = 10)
+plt.hist(cdata.guides.edit_rate, bins=30)
+plt.show()
+
    +
    +_images/output_34_1.png +
    +

    Calculating LFC¶

    +
    cdata.log_norm()
+cdata.log_fold_change_aggregate("bot", "top", aggregate_condit = "replicate")
+
    +
    +
    cdata.guides
+
    +
    +
    + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
    nameUnnamed: 0Target gene/variantTarget descriptorArbitrary numbergRNA position categoryTarget base position in gRNATarget base position in reporterBEGroup...barcode5-nt PAMoffsettargettarget_posGroup2masked_sequencemasked_barcodebot_top.lfc.medianedit_rate
    0CONTROL_1_g10CONTROLNaN1g1410ABENegCtrl...GTTTGGGAG-10CONTROL_19NegCtrlCCTGCGCGGTGGGGGGCTTTGTTT-0.1355500.531163
    1CONTROL_1_g21CONTROLNaN1g2511ABENegCtrl...AACATGGGA-11CONTROL_110NegCtrlCCCTGCGCGGTGGGGGGCTTGGCG-0.0593910.640765
    2CONTROL_1_g32CONTROLNaN1g3512ABENegCtrl...CGCTTTGGG-12CONTROL_111NegCtrlCCCTGCGCGGTGGGGGGCTCGCT0.1412900.417709
    3CONTROL_1_g43CONTROLNaN1g4713ABENegCtrl...TGAGTTTGG-13CONTROL_112NegCtrlGGCCCTGCGCGGTGGGGGGCTGGG-0.0723580.126400
    4CONTROL_1_g54CONTROLNaN1g5814ABENegCtrl...GTATCTTTG-14CONTROL_113NegCtrlGGGCCCTGCGCGGTGGGGGGGTGT0.2696500.201104
    ..................................................................
    3450rs9987289_Maj_ABE_347_g13450rs9987289Maj347g1310ABEVariant...CAGTCCAGC-10rs9987289_Maj_ABE_3479VariantGCGTCGGTGTCGCGTGGGGCGGT-0.2302640.087379
    3451rs9987289_Maj_ABE_347_g23451rs9987289Maj347g2411ABEVariant...TCGCACCAG-11rs9987289_Maj_ABE_34710VariantGGCGTCGGTGTCGCGTGGGTCGC-0.1821510.299923
    3452rs9987289_Maj_ABE_347_g33452rs9987289Maj347g3612ABEVariant...GCACAACCA-12rs9987289_Maj_ABE_34711VariantTGGGCGTCGGTGTCGCGTGGGCGC-0.1657780.224973
    3453rs9987289_Maj_ABE_347_g43453rs9987289Maj347g4713ABEVariant...TTGCGAACC-13rs9987289_Maj_ABE_34712VariantTTGGGCGTCGGTGTCGCGTGTTGC-0.3405900.265378
    3454rs9987289_Maj_ABE_347_g53454rs9987289Maj347g5814ABEVariant...GCGAGGAAC-14rs9987289_Maj_ABE_34713VariantCTTGGGCGTCGGTGTCGCGTGCGG0.0343650.266573
    +

    3455 rows × 22 columns

    +
    +
    +

    Allele translation¶

    +
    cdata_tiling = br.read_h5ad("../../072121_ABE_topbot/bean_counts/LDLRCDS/032422_crispresso/bean_count_072121_ABE_topbot_LDLRCDS.h5ad")
+
    +
    +
    cdata_tiling.uns["allele_counts"].allele
+
    +
    +
    0                                         11224415:14:+:A>G
+1                        11224401:0:+:A>G,11224415:14:+:A>G
+2                        11224410:9:+:A>G,11224415:14:+:A>G
+3         11224401:0:+:A>G,11224402:1:+:A>G,11224410:9:+...
+4                                          11224401:0:+:A>G
+                                ...
+438001    11203000:4:+:A>G,11203002:6:+:A>G,11203006:10:...
+438002    11224074:0:+:A>G,11224086:12:+:A>G,11224092:18...
+438003    0:0:+:A>G,3:3:+:A>G,11:11:+:A>G,13:13:+:A>G,17...
+438004                  11217409:23:+:G>-,11217417:31:+:->C
+438005    11226735:30:-:A>G,11226742:23:-:A>G,11226747:1...
+Name: allele, Length: 438006, dtype: object
+
    +
    +
    +
    +

    Writing¶

    +
    cdata.to_Excel("tmp.xlsx")
+
    +
    +
    Writing to: tmp.xlsx
+
+    Sheet 1:        X
+    Sheet 2:        edits
+    Sheet 3:        X_bcmatch
+    Sheet 4:        lognorm_counts
+    Sheet 5:        lognorm_edits
+    Sheet 6:        guides
+    Sheet 7:        samples
+    Sheet 8:        screen.uns.allele_counts
+    Sheet 9:        screen.uns.edit_counts
+
    +
    +
    cdata.to_mageck_input("mageck_input.txt", target_column='target')
+
    +
    +
    %%bash
+head mageck_input.txt
+
    +
    +
    sgRNA       gene    0       1       2       3       4       5       6       7       8       9       10      11
+CONTROL_1_g1        CONTROL_1       171     451     251     422     573     389     456     420     835     435     794     439
+CONTROL_1_g2        CONTROL_1       145     278     257     206     364     273     389     254     527     498     768     195
+CONTROL_1_g3        CONTROL_1       333     835     488     632     898     899     780     713     1189    626     1146    603
+CONTROL_1_g4        CONTROL_1       246     663     387     448     823     595     705     600     921     595     1143    506
+CONTROL_1_g5        CONTROL_1       243     647     434     529     776     451     700     676     1062    611     928     379
+CONTROL_10_g1       CONTROL_10      138     329     229     213     422     292     432     352     409     243     390     274
+CONTROL_10_g2       CONTROL_10      187     468     402     479     643     369     428     469     796     422     787     404
+CONTROL_10_g3       CONTROL_10      57      126     83      131     281     114     184     115     300     106     299     106
+CONTROL_10_g4       CONTROL_10      66      112     120     136     182     128     169     181     256     144     258     179
+
    +
    +
    +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
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    nameUnnamed: 0Target gene/variantTarget descriptorArbitrary numbergRNA position categoryTarget base position in gRNATarget base position in reporterBEGroup...Reporterbarcode5-nt PAMoffsettargettarget_posGroup2masked_sequencemasked_barcodeedit_rate
    0CONTROL_1_g10CONTROLNaN1g1410ABENegCtrl...CCAAGCCCTACGCGGTAGGGAACTTTGGGAGCGTTTGGGAG-10CONTROL_19NegCtrlCCTGCGCGGTGGGGGGCTTTGTTT0.531163
    1CONTROL_1_g21CONTROLNaN1g2511ABENegCtrl...TCCAAGCCCTACGCGGTAGGGAACTTTGGGAGAACATGGGA-11CONTROL_110NegCtrlCCCTGCGCGGTGGGGGGCTTGGCG0.640765
    2CONTROL_1_g32CONTROLNaN1g3512ABENegCtrl...GTCCAAGCCCTACGCGGTAGGGAACTTTGGGACGCTTTGGG-12CONTROL_111NegCtrlCCCTGCGCGGTGGGGGGCTCGCT0.417709
    3CONTROL_1_g43CONTROLNaN1g4713ABENegCtrl...CGTCCAAGCCCTACGCGGTAGGGAACTTTGGGTGAGTTTGG-13CONTROL_112NegCtrlGGCCCTGCGCGGTGGGGGGCTGGG0.126400
    4CONTROL_1_g54CONTROLNaN1g5814ABENegCtrl...ACGTCCAAGCCCTACGCGGTAGGGAACTTTGGGTATCTTTG-14CONTROL_113NegCtrlGGGCCCTGCGCGGTGGGGGGGTGT0.201104
    ..................................................................
    3450rs9987289_Maj_ABE_347_g13450rs9987289Maj347g1310ABEVariant...TGCTTGGGCATCAATATCACGTGGAACCAGCCCAGTCCAGC-10rs9987289_Maj_ABE_3479VariantGCGTCGGTGTCGCGTGGGGCGGT0.087379
    3451rs9987289_Maj_ABE_347_g23451rs9987289Maj347g2411ABEVariant...ATGCTTGGGCATCAATATCACGTGGAACCAGCTCGCACCAG-11rs9987289_Maj_ABE_34710VariantGGCGTCGGTGTCGCGTGGGTCGC0.299923
    3452rs9987289_Maj_ABE_347_g33452rs9987289Maj347g3612ABEVariant...GATGCTTGGGCATCAATATCACGTGGAACCAGGCACAACCA-12rs9987289_Maj_ABE_34711VariantTGGGCGTCGGTGTCGCGTGGGCGC0.224973
    3453rs9987289_Maj_ABE_347_g43453rs9987289Maj347g4713ABEVariant...AGATGCTTGGGCATCAATATCACGTGGAACCATTGCGAACC-13rs9987289_Maj_ABE_34712VariantTTGGGCGTCGGTGTCGCGTGTTGC0.265378
    3454rs9987289_Maj_ABE_347_g53454rs9987289Maj347g5814ABEVariant...TAGATGCTTGGGCATCAATATCACGTGGAACCGCGAGGAAC-14rs9987289_Maj_ABE_34713VariantCTTGGGCGTCGGTGTCGCGTGCGG0.266573
    +

    3455 rows × 21 columns

    + + + +:bash:`samples` attribute contains the sample and condition specific information. + +.. code:: ipython3 + + cdata.samples + + + + + +.. raw:: html + +
    + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
    indexsortreplicate
    0rep1_botbotrep1
    1rep2_botbotrep2
    2rep3_VPA_botbotrep3_VPA
    3rep4_VPA_botbotrep4_VPA
    4rep1_bulkbulkrep1
    5rep2_bulkbulkrep2
    6rep3_VPA_bulkbulkrep3_VPA
    7rep4_VPA_bulkbulkrep4_VPA
    8rep1_toptoprep1
    9rep2_toptoprep2
    10rep3_VPA_toptoprep3_VPA
    11rep4_VPA_toptoprep4_VPA
    +
    + + +Allele_counts information is stored in :bash:`.uns["allele_counts"]`. + +.. code:: ipython3 + + cdata.uns["allele_counts"] + + + + + +.. raw:: html + +
    + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
    guideallelerep1_botrep2_botrep3_VPA_botrep4_VPA_botrep1_bulkrep2_bulkrep3_VPA_bulkrep4_VPA_bulkrep1_toprep2_toprep3_VPA_toprep4_VPA_top
    012:51779544AGA_Maj_ABE_2_g10:9:+:A>G,5:14:+:A>G14201306152172214343
    112:51779544AGA_Maj_ABE_2_g1-4:5:+:A>G,-2:7:+:A>G,5:14:+:A>G,10:19:+:A>G100000000000
    212:51779544AGA_Maj_ABE_2_g1-7:2:+:A>G,0:9:+:A>G,5:14:+:A>G342010520010
    312:51779544AGA_Maj_ABE_2_g1-9:0:+:G>A,-8:1:+:G>A,-7:2:+:A>C,-6:3:+:C>A,-4...100102100010
    412:51779544AGA_Maj_ABE_2_g1-7:2:+:A>G,10:19:+:A>G110000000000
    .............................................
    438407rs9987289_Maj_ABE_347_g54:17:+:A>G,6:19:+:A>G,9:22:+:A>G000000000020
    438408rs9987289_Maj_ABE_347_g5-12:1:+:A>G,6:19:+:A>G,9:22:+:A>G,11:24:+:G>A000000000010
    438409rs9987289_Maj_ABE_347_g5-12:1:+:A>G,6:19:+:A>G,9:22:+:A>G,16:29:+:A>G000000000001
    438410rs9987289_Maj_ABE_347_g5-12:1:+:A>G,0:13:+:A>G,6:19:+:A>G,9:22:+:A>G,1...000000000100
    438411rs9987289_Maj_ABE_347_g5-12:1:+:A>G,6:19:+:A>G,9:22:+:A>G,12:25:+:T>G000000000001
    +

    438412 rows × 14 columns

    +
    + + +Base-level edit counts can be saved at `.uns["edit_counts"]`. + +.. code:: ipython3 + + cdata.uns["edit_counts"] + + + + + +.. raw:: html + +
    + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
    guideeditrep1_botrep2_botrep3_VPA_botrep4_VPA_botrep1_bulkrep2_bulkrep3_VPA_bulkrep4_VPA_bulkrep1_toprep2_toprep3_VPA_toprep4_VPA_topref_basealt_base
    012:51779544AGA_Maj_ABE_2_g1-1:8:+:G>A000010000000GA
    112:51779544AGA_Maj_ABE_2_g1-1:8:+:G>C000000001010GC
    212:51779544AGA_Maj_ABE_2_g1-1:8:+:G>T000010000000GT
    312:51779544AGA_Maj_ABE_2_g1-2:7:+:A>C000000002010AC
    412:51779544AGA_Maj_ABE_2_g1-2:7:+:A>G1934404592566768481492AG
    ...................................................
    217563rs9987289_Maj_ABE_347_g58:21:+:C>A070001101000CA
    217564rs9987289_Maj_ABE_347_g58:21:+:C>G002008000180CG
    217565rs9987289_Maj_ABE_347_g58:21:+:C>T007000700000CT
    217566rs9987289_Maj_ABE_347_g59:22:+:A>G92130513746122058235947AG
    217567rs9987289_Maj_ABE_347_g59:22:+:A>T000000070000AT
    +

    217568 rows × 16 columns

    +
    + + + + + +Subsetting & addition +--------------------- + +Works as anndata, supports allele & edit count operations. + +Subsetting & selection +~~~~~~~~~~~~~~~~~~~~~~ + +.. code:: ipython3 + + cdata_subset = cdata[:10,cdata.samples.sort == "bulk"] + + +.. parsed-literal:: + + ['rep1_bulk', 'rep2_bulk', 'rep3_VPA_bulk', 'rep4_VPA_bulk'] + + +.. code:: ipython3 + + cdata_subset.uns["allele_counts"] + + + + +.. raw:: html + +
    + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
    guideallelerep1_bulkrep2_bulkrep3_VPA_bulkrep4_VPA_bulk
    14979CONTROL_10_g1-4:5:+:A>G,0:9:+:A>G8130
    14980CONTROL_10_g1-7:2:+:C>T00010
    14981CONTROL_10_g1-4:5:+:A>G2922925
    14982CONTROL_10_g11:10:+:A>G0641
    14983CONTROL_10_g1-4:5:+:A>G,1:10:+:A>G111512
    .....................
    22837CONTROL_1_g5-13:0:+:A>-,-12:1:+:C>T,-9:4:+:C>G,-8:5:+:C>T,...0000
    22838CONTROL_1_g5-6:7:+:A>C,7:20:+:A>G0000
    22839CONTROL_1_g5-13:0:+:A>G,-10:3:+:T>G,0:13:+:A>G,7:20:+:A>G0000
    22840CONTROL_1_g50:13:+:A>T0000
    22841CONTROL_1_g50:13:+:A>G,18:31:+:G>A0000
    +

    1080 rows × 6 columns

    +
    + + + +LFC calculation & Addition +~~~~~~~~~~~~~~~~~~~~~~~~~~ + +.. code:: ipython3 + + cdata1 = br.read_h5ad("/data/pinello/PROJECTS/2021_08_ANBE/data/072121_ABE_topbot/bean_counts/LDLvar/032422_crispresso/bean_count_072121_ABE_topbot_LDLvar.h5ad") + cdata2 = br.read_h5ad("/data/pinello/PROJECTS/2021_08_ANBE/data/102121_ABE_topbot/bean_counts/LDLvar/032422_crispresso/bean_count_102121_ABE_topbot_LDLvar.h5ad") + + +.. code:: ipython3 + + cdata1.samples["sort"] = cdata1.samples["index"].map(lambda s: s.rsplit("_", 1)[-1]) + cdata1.samples["replicate"] = cdata1.samples["index"].map(lambda s: s.rsplit("_", 1)[0]) + cdata2.samples["sort"] = cdata2.samples["index"].map(lambda s: s.rsplit("_", 1)[-1]) + cdata2.samples["replicate"] = cdata2.samples["index"].map(lambda s: s.rsplit("_", 1)[0]) + +.. code:: ipython3 + + cdata1.log_norm() + lfc1 = cdata1.log_fold_change_reps("bot", "top") + cdata2.log_norm() + lfc2 = cdata2.log_fold_change_reps("bot", "top") + lfcs = lfc1.join(lfc2, lsuffix = "_1", rsuffix = "_2") + sns.pairplot(lfcs) + + +.. image:: ../imgs/output_20_2.png + + +LFC can be aggregated for biological replicates. + +.. code:: ipython3 + + cdata1.log_fold_change_aggregate("bot", "top", aggregate_condit = "replicate") + +.. code:: ipython3 + + cdata1.guides + + + + +.. raw:: html + +
    + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
    nameUnnamed: 0Target gene/variantTarget descriptorArbitrary numbergRNA position categoryTarget base position in gRNATarget base position in reporterBEGroup...Reporterbarcode5-nt PAMoffsettargettarget_posGroup2masked_sequencemasked_barcodebot_top.lfc.median
    0CONTROL_1_g10CONTROLNaN1g1410ABENegCtrl...CCAAGCCCTACGCGGTAGGGAACTTTGGGAGCGTTTGGGAG-10CONTROL_19NegCtrlCCTGCGCGGTGGGGGGCTTTGTTT-0.158787
    1CONTROL_1_g21CONTROLNaN1g2511ABENegCtrl...TCCAAGCCCTACGCGGTAGGGAACTTTGGGAGAACATGGGA-11CONTROL_110NegCtrlCCCTGCGCGGTGGGGGGCTTGGCG-0.212254
    2CONTROL_1_g32CONTROLNaN1g3512ABENegCtrl...GTCCAAGCCCTACGCGGTAGGGAACTTTGGGACGCTTTGGG-12CONTROL_111NegCtrlCCCTGCGCGGTGGGGGGCTCGCT0.186679
    3CONTROL_1_g43CONTROLNaN1g4713ABENegCtrl...CGTCCAAGCCCTACGCGGTAGGGAACTTTGGGTGAGTTTGG-13CONTROL_112NegCtrlGGCCCTGCGCGGTGGGGGGCTGGG-0.022441
    4CONTROL_1_g54CONTROLNaN1g5814ABENegCtrl...ACGTCCAAGCCCTACGCGGTAGGGAACTTTGGGTATCTTTG-14CONTROL_113NegCtrlGGGCCCTGCGCGGTGGGGGGGTGT0.457033
    ..................................................................
    3450rs9987289_Maj_ABE_347_g13450rs9987289Maj347g1310ABEVariant...TGCTTGGGCATCAATATCACGTGGAACCAGCCCAGTCCAGC-10rs9987289_Maj_ABE_3479VariantGCGTCGGTGTCGCGTGGGGCGGT-0.418312
    3451rs9987289_Maj_ABE_347_g23451rs9987289Maj347g2411ABEVariant...ATGCTTGGGCATCAATATCACGTGGAACCAGCTCGCACCAG-11rs9987289_Maj_ABE_34710VariantGGCGTCGGTGTCGCGTGGGTCGC-0.084936
    3452rs9987289_Maj_ABE_347_g33452rs9987289Maj347g3612ABEVariant...GATGCTTGGGCATCAATATCACGTGGAACCAGGCACAACCA-12rs9987289_Maj_ABE_34711VariantTGGGCGTCGGTGTCGCGTGGGCGC-0.339419
    3453rs9987289_Maj_ABE_347_g43453rs9987289Maj347g4713ABEVariant...AGATGCTTGGGCATCAATATCACGTGGAACCATTGCGAACC-13rs9987289_Maj_ABE_34712VariantTTGGGCGTCGGTGTCGCGTGTTGC-0.517138
    3454rs9987289_Maj_ABE_347_g53454rs9987289Maj347g5814ABEVariant...TAGATGCTTGGGCATCAATATCACGTGGAACCGCGAGGAAC-14rs9987289_Maj_ABE_34713VariantCTTGGGCGTCGGTGTCGCGTGCGG0.002245
    +

    3455 rows × 21 columns

    +
    + + + +Technical replicates show decent LFC correlation. + +.. code:: ipython3 + + cdata = cdata1 + cdata2 + + +.. code:: ipython3 + + cdata + + + + + +.. parsed-literal:: + + Genome Editing Screen comprised of n_guides x n_conditions = 3455 x 12 + guides: 'name', 'Unnamed: 0', 'Target gene/variant', 'Target descriptor', 'Arbitrary number', 'gRNA position category', 'Target base position in gRNA', 'Target base position in reporter', 'BE', 'Group', 'sequence', 'Reporter', 'barcode', '5-nt PAM', 'offset', 'target', 'target_pos', 'Group2', 'masked_sequence', 'masked_barcode', 'bot_top.lfc.median' + samples: 'index', 'sort', 'replicate' + condit_m: + condit_p: + layers: 'edits', 'X_bcmatch' + uns: 'allele_counts' + + + +You can concatenate different samples with shared guides. + +.. code:: ipython3 + + br.concat((cdata1, cdata2)) + + +.. parsed-literal:: + + Genome Editing Screen comprised of n_guides x n_conditions = 3455 x 24 + guides: 'name', 'Unnamed: 0', 'Target gene/variant', 'Target descriptor', 'Arbitrary number', 'gRNA position category', 'Target base position in gRNA', 'Target base position in reporter', 'BE', 'Group', 'sequence', 'Reporter', 'barcode', '5-nt PAM', 'offset', 'target', 'target_pos', 'Group2', 'masked_sequence', 'masked_barcode', 'bot_top.lfc.median' + samples: 'index', 'sort', 'replicate' + condit_m: + condit_p: + layers: 'X', 'X_bcmatch', 'edits', 'lognorm_counts', 'lognorm_edits' + uns: 'allele_counts' + + + +Getting edit rates from allele counts +------------------------------------- + +.. code:: ipython3 + + cdata.get_edit_rate(normalize_by_editable_base = False, + edited_base = "A", + editable_base_start = 3, + editable_base_end = 8, + bcmatch_thres = 10, + prior_weight = 1) + + +.. code:: ipython3 + + cdata.uns["edit_counts"] = cdata.get_edit_from_allele() + + + +.. code:: ipython3 + + cdata.get_edit_mat_from_uns("A", "G", match_target_position = True) + cdata.get_edit_rate(edited_base = "A", bcmatch_thres = 10) + plt.hist(cdata.guides.edit_rate, bins=30) + plt.show() + + +.. image:: ../imgs/output_34_1.png + + + +Calculating LFC +~~~~~~~~~~~~~~~ + +.. code:: ipython3 + + cdata.log_norm() + cdata.log_fold_change_aggregate("bot", "top", aggregate_condit = "replicate") + +.. code:: ipython3 + + cdata.guides + + + + +.. raw:: html + +
    + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
    nameUnnamed: 0Target gene/variantTarget descriptorArbitrary numbergRNA position categoryTarget base position in gRNATarget base position in reporterBEGroup...barcode5-nt PAMoffsettargettarget_posGroup2masked_sequencemasked_barcodebot_top.lfc.medianedit_rate
    0CONTROL_1_g10CONTROLNaN1g1410ABENegCtrl...GTTTGGGAG-10CONTROL_19NegCtrlCCTGCGCGGTGGGGGGCTTTGTTT-0.1355500.531163
    1CONTROL_1_g21CONTROLNaN1g2511ABENegCtrl...AACATGGGA-11CONTROL_110NegCtrlCCCTGCGCGGTGGGGGGCTTGGCG-0.0593910.640765
    2CONTROL_1_g32CONTROLNaN1g3512ABENegCtrl...CGCTTTGGG-12CONTROL_111NegCtrlCCCTGCGCGGTGGGGGGCTCGCT0.1412900.417709
    3CONTROL_1_g43CONTROLNaN1g4713ABENegCtrl...TGAGTTTGG-13CONTROL_112NegCtrlGGCCCTGCGCGGTGGGGGGCTGGG-0.0723580.126400
    4CONTROL_1_g54CONTROLNaN1g5814ABENegCtrl...GTATCTTTG-14CONTROL_113NegCtrlGGGCCCTGCGCGGTGGGGGGGTGT0.2696500.201104
    ..................................................................
    3450rs9987289_Maj_ABE_347_g13450rs9987289Maj347g1310ABEVariant...CAGTCCAGC-10rs9987289_Maj_ABE_3479VariantGCGTCGGTGTCGCGTGGGGCGGT-0.2302640.087379
    3451rs9987289_Maj_ABE_347_g23451rs9987289Maj347g2411ABEVariant...TCGCACCAG-11rs9987289_Maj_ABE_34710VariantGGCGTCGGTGTCGCGTGGGTCGC-0.1821510.299923
    3452rs9987289_Maj_ABE_347_g33452rs9987289Maj347g3612ABEVariant...GCACAACCA-12rs9987289_Maj_ABE_34711VariantTGGGCGTCGGTGTCGCGTGGGCGC-0.1657780.224973
    3453rs9987289_Maj_ABE_347_g43453rs9987289Maj347g4713ABEVariant...TTGCGAACC-13rs9987289_Maj_ABE_34712VariantTTGGGCGTCGGTGTCGCGTGTTGC-0.3405900.265378
    3454rs9987289_Maj_ABE_347_g53454rs9987289Maj347g5814ABEVariant...GCGAGGAAC-14rs9987289_Maj_ABE_34713VariantCTTGGGCGTCGGTGTCGCGTGCGG0.0343650.266573
    +

    3455 rows × 22 columns

    +
    + + + +Allele translation +~~~~~~~~~~~~~~~~~~ + +.. code:: ipython3 + + cdata_tiling = br.read_h5ad("../../072121_ABE_topbot/bean_counts/LDLRCDS/032422_crispresso/bean_count_072121_ABE_topbot_LDLRCDS.h5ad") + + +.. code:: ipython3 + + cdata_tiling.uns["allele_counts"].allele + + + + + + +.. parsed-literal:: + + 0 11224415:14:+:A>G + 1 11224401:0:+:A>G,11224415:14:+:A>G + 2 11224410:9:+:A>G,11224415:14:+:A>G + 3 11224401:0:+:A>G,11224402:1:+:A>G,11224410:9:+... + 4 11224401:0:+:A>G + ... + 438001 11203000:4:+:A>G,11203002:6:+:A>G,11203006:10:... + 438002 11224074:0:+:A>G,11224086:12:+:A>G,11224092:18... + 438003 0:0:+:A>G,3:3:+:A>G,11:11:+:A>G,13:13:+:A>G,17... + 438004 11217409:23:+:G>-,11217417:31:+:->C + 438005 11226735:30:-:A>G,11226742:23:-:A>G,11226747:1... + Name: allele, Length: 438006, dtype: object + + + +Writing +~~~~~~~ + +.. code:: ipython3 + + cdata.to_Excel("tmp.xlsx") + + +.. parsed-literal:: + + Writing to: tmp.xlsx + + Sheet 1: X + Sheet 2: edits + Sheet 3: X_bcmatch + Sheet 4: lognorm_counts + Sheet 5: lognorm_edits + Sheet 6: guides + Sheet 7: samples + Sheet 8: screen.uns.allele_counts + Sheet 9: screen.uns.edit_counts + + +.. code:: ipython3 + + cdata.to_mageck_input("mageck_input.txt", target_column='target') + +.. code:: bash + + %%bash + head mageck_input.txt + + +.. parsed-literal:: + + sgRNA gene 0 1 2 3 4 5 6 7 8 9 10 11 + CONTROL_1_g1 CONTROL_1 171 451 251 422 573 389 456 420 835 435 794 439 + CONTROL_1_g2 CONTROL_1 145 278 257 206 364 273 389 254 527 498 768 195 + CONTROL_1_g3 CONTROL_1 333 835 488 632 898 899 780 713 1189 626 1146 603 + CONTROL_1_g4 CONTROL_1 246 663 387 448 823 595 705 600 921 595 1143 506 + CONTROL_1_g5 CONTROL_1 243 647 434 529 776 451 700 676 1062 611 928 379 + CONTROL_10_g1 CONTROL_10 138 329 229 213 422 292 432 352 409 243 390 274 + CONTROL_10_g2 CONTROL_10 187 468 402 479 643 369 428 469 796 422 787 404 + CONTROL_10_g3 CONTROL_10 57 126 83 131 281 114 184 115 300 106 299 106 + CONTROL_10_g4 CONTROL_10 66 112 120 136 182 128 169 181 256 144 258 179 + diff --git a/docs/_build/_sources/cds.rst.txt b/docs/_build/_sources/cds.rst.txt new file mode 100644 index 0000000..5dabfde --- /dev/null +++ b/docs/_build/_sources/cds.rst.txt @@ -0,0 +1,5 @@ +Coding sequence tiling library +*********************** +.. mdinclude:: tutorials/ldl_cds.md + +See :ref:`subcommands` for the full details. diff --git a/docs/_build/_sources/commands/count.md.txt b/docs/_build/_sources/commands/count.md.txt new file mode 100644 index 0000000..5f04b4c --- /dev/null +++ b/docs/_build/_sources/commands/count.md.txt @@ -0,0 +1,31 @@ +# `bean count[-samples]`: Count (reporter) screen data +`bean count-samples` (or `bean count` for a single sample) maps guide into guide counts, **allowing for base transition in spacer sequence**. When the matched reporter information is provided, it can count the **target site edits** and **alleles produced by each guide**. Mapping is efficiently done based on [CRISPResso2](https://github.com/pinellolab/CRISPResso2) modified for base-edit-aware mapping. + + + +```python +bean count-samples \ + --input sample_list.csv `# sample with lines 'R1_filepath,R2_filepath,sample_name\n'` \ + -b A `# base that is being edited (A/G)` \ + -f sgRNA_info_table.csv `# sgRNA information` \ + -o . `# output directory` \ + -r `# read edit/allele information from reporter` \ + -t 12 `# number of threads` \ + --name my_sorting_screen `# name of this sample run` \ +``` +```python +bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r +``` +By default, `bean count[-samples]` assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure. + + Read structuren + +See full detail [below](#full-parameters). + +# Input file format +See :ref:`input` for input file formats. + +# Output file format +`count` or `count-samples` produces `.h5ad` and `.xlsx` file with guide and per-guide allele counts. +* `.h5ad`: This output file follows annotated matrix format compatible with `AnnData` and is based on `Screen` object in [purturb_tools](https://github.com/pinellolab/perturb-tools). See [Data Structure](#data-structure) section for more information. +* `.xlsx`: This output file contains `.guides`, `.samples`, `.X[_bcmatch,_edits]`. (`allele_tables` are often too large to write into an Excel!) diff --git a/docs/_build/_sources/commands/create-screen.md.txt b/docs/_build/_sources/commands/create-screen.md.txt new file mode 100644 index 0000000..d9200b7 --- /dev/null +++ b/docs/_build/_sources/commands/create-screen.md.txt @@ -0,0 +1,9 @@ +# `bean create-screen`: Create ReporterScreen object from flat files +```bash +bean create-screen gRNA_library.csv sample_list.csv gRNA_counts_table.csv +``` +## Input + * gRNA_library.csv + * sample_list.csv + * gRNA_counts_table.csv: Table with gRNA ID in the first column and sample IDs as the column names (first row) +`gRNA_library.csv` and `sample_list.csv` should be formatted as :ref:`input`. \ No newline at end of file diff --git a/docs/_build/_sources/commands/filter.md.txt b/docs/_build/_sources/commands/filter.md.txt new file mode 100644 index 0000000..53d6ec1 --- /dev/null +++ b/docs/_build/_sources/commands/filter.md.txt @@ -0,0 +1,37 @@ +# `filter`: Filtering (and optionally translating) alleles +As `tiling` mode of `bean run` accounts for any robustly observed alleles, `bean filter` filters for such alleles. +```bash +bean filter my_sorting_screen_masked.h5ad \ +-o my_sorting_screen_filtered.h5ad `# Output file path` \ +``` + +# Output +Above command produces +* `my_sorting_screen_filtered.h5ad` with filtered alleles stored in `.uns`, +* `my_sorting_screen_filtered.filtered_allele_stats.pdf`, and `my_sorting_screen_filtered.filter_log.txt` that report allele count stats in each filtering step. + +You may want to adjust the flitering parameters to obtain optimal balance between # guides per variant & # variants that are scored. See example outputs of filtering step [here](docs/example_filtering_output/). + + +# Translating alleles +If you want to obtain **amino acid level variant** for coding sequence tiling screens, provide coding sequence positions which variants occuring within the coding sequence will be translated. *This is optional, but **highly recommended** to increase per-(coding)variant support.* + +Allele translation + + +```bash +bean filter my_sorting_screen.h5ad \ +-o my_sorting_screen_masked.h5ad \ +--translate `# Translate coding variants` \ +[ --translate-gene-name GENE_SYMBOL OR + --translate-genes-list path_to_gene_names_file.txt OR + --translate-fasta gene_exon.fa, OR + --translate-fastas-csv gene_exon_fas.csv] +``` +* When library covers a single gene, do either of the following: + 1. Feed `--translate-gene-name GENE_SYMBOL` if your `genomic_pos` column of `sgRNA_info_tbl` is compatible with [MANE transcript](https://useast.ensembl.org/info/genome/genebuild/mane.html)'s reference genome. (Per 10/23/2023, GRCh38). This will automatically load the exon positions based on MANE transcript annotation. + 2. To use your custom coding sequence and exon positions, feed `--translate-fasta gene_exon.fa` argument where `gene_exon.fa` is the FASTA file with entries of exons. [See full details here](docs/exon_fa_format.md). +* When library covers multiple genes, do either of the following: + 1. Feed `--translate-genes-list path_to_gene_names_file.txt` where `path_to_gene_names_file.txt` is file with one gene symbol per line. + 2. Feed `--translate-fastas-csv gene_exon_fas.csv` where `gene_exon_fas.csv` is the csv file with lines `gene_id,gene_exon_fasta_path` without header. Each FASTA file in `gene_exon_fasta_path` is formatted [as the single-gene FASTA file](docs/exon_fa_format.md). +* Translation will keep the variants outside the coding sequence as nucleotide-level variants, while aggregating variants leading to the same coding sequence variants. diff --git a/docs/_build/_sources/commands/input.md.txt b/docs/_build/_sources/commands/input.md.txt new file mode 100644 index 0000000..9fa1de9 --- /dev/null +++ b/docs/_build/_sources/commands/input.md.txt @@ -0,0 +1,37 @@ +This document describes the input files of :ref:`count_samples`. +## sgRNA_info_table.csv +File should contain following columns. +* `name`: gRNA ID column +* `sequence`: gRNA sequence +* `barcode`: R2 barcode to help match reporter to gRNA, written in the sense direction (as in R1) +* In order to use accessibility in the [variant effect quantification](#bean-run-quantify-variant-effects), provide accessibility information in one of two options. (For non-targeting guides, provide NA values (empty cell).) + * Option 1: `chrom` & `genomic_pos`: Chromosome (ex. `chr19`) and genomic position of guide sequence. You will have to provide the path to the bigwig file with matching reference version in `bean run`. + * Option 2: `accessibility_signal`: ATAC-seq signal value of the target loci of each guide. +* For variant library (gRNAs are designed to target specific variants and ignores bystander edits) + * `target`: This column denotes which target variant/element of each gRNA. This is not used in `bean count[-samples]` but required to run `bean run` in later steps. + * `target_group`: If negative/positive control gRNA will be considered in `bean qc` and/or `bean run`, specify as "NegCtrl"/"PosCtrl" in this column. + * `target_pos`: If `--match_target_pos` flag is used, input file needs `target_pos` which specifies 0-based relative position of targeted base within Reporter sequence. +* For tiling library (gRNAs tile coding / noncoding sequences) + * `strand`: Specifies gRNA strand information relative to the reference genome. + * `chrom`: Chromosome of gRNA targeted locus. + * `start_pos`: gRNA starting position in the genome. Required when you provide `strand` column. Should specify the smaller coordinate value among start and end position regardless of gRNA strandedness. + +Also see examples for [variant library](tests/data/test_guide_info.csv) and [tiling library](tests/data/test_guide_info_tiling.csv). + +## sample_list.csv +File should contain following columns with header. +* `R1_filepath`: Path to read 1 `.fastq[.gz]` file +* `R2_filepath`: Path to read 1 `.fastq[.gz]` file +* `sample_id`: ID of sequencing sample +* `replicate`: Replicate # of this sample (Should NOT contain `.`) +* `condition`: Name of the sorting bin (ex. `top`, `bot`), or label of timepoint (ex. `D5`, `D18`) + +For FACS sorting screens: +* `upper_quantile`: FACS sorting upper quantile +* `lower_quantile`: FACS sorting lower quantile + +For proliferation / survival screens: +* `time`: Numeric time following the base editing of each sample. + + +Also see examples for [FACS sorting screen](tests/data/sample_list.csv). \ No newline at end of file diff --git a/docs/_build/_sources/commands/profile.md.txt b/docs/_build/_sources/commands/profile.md.txt new file mode 100644 index 0000000..bbe8a8c --- /dev/null +++ b/docs/_build/_sources/commands/profile.md.txt @@ -0,0 +1,8 @@ +# `bean profile`: Profile editing patterns +```bash +bean profile my_sorting_screen.h5ad -o output_prefix `# Prefix for editing profile report` +``` +# Output +Above command produces `prefix_editing_preference.[html,ipynb]` as editing preferences ([see example](../../notebooks/profile_editing_preference.ipynb)). + +Allele translation \ No newline at end of file diff --git a/docs/_build/_sources/commands/qc.md.txt b/docs/_build/_sources/commands/qc.md.txt new file mode 100644 index 0000000..82b719e --- /dev/null +++ b/docs/_build/_sources/commands/qc.md.txt @@ -0,0 +1,94 @@ +# `bean qc`: QC of reporter screen data +```bash +bean qc \ + my_sorting_screen.h5ad `# Input ReporterScreen .h5ad file path` \ + -o my_sorting_screen_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r qc_report_my_sorting_screen `# Prefix for QC report` \ + --ctrl-cond presort `# "condition" column in the control sample before selection. Mean gRNA editing rates in these samples are reported. ` \ +# Inspect the output qc_report_my_sorting_screen.html to tweak QC threshold + +bean qc \ + my_sorting_screen.h5ad \ + -o my_sorting_screen_masked.h5ad \ + -r qc_report_my_sorting_screen \ + #[--count-correlation-thres 0.7 ...]\ + -b +``` + +`bean qc` supports following quality control and masks samples with low quality. Specifically: + +Allele translation + +* Plots guide coverage and the uniformity of coverage +* Guide count correlation between samples +* Log fold change correlation when positive controls are provided +* Plots editing rate distribution +* Identify samples with low guide coverage/guide count correlation/editing rate and mask the sample in `bdata.samples.mask` +* Identify outlier guides to filter out + +# Output +Above command produces +* `my_sorting_screen_masked.h5ad` without problematic replicate and guides and with sample masks, and +* `qc_report_my_sorting_screen.[html,ipynb]` as QC report. +##### Optional arguments: +* `-o OUT_SCREEN_PATH`, `--out-screen-path OUT_SCREEN_PATH` + Path where quality-filtered ReporterScreen object to be written to +* `-r OUT_REPORT_PREFIX`, `--out-report-prefix OUT_REPORT_PREFIX` + Output prefix of qc report (prefix.html, prefix.ipynb) + +##### QC thresholds: +* `--count-correlation-thres COUNT_CORRELATION_THRES` + Correlation threshold to mask out. +* `--edit-rate-thres EDIT_RATE_THRES` + Mean editing rate threshold per sample to mask out. +* `--lfc-thres LFC_THRES` + Positive guides' correlation threshold to filter out. + +##### Run options: +* `-b`, `--remove-bad-replicates` + Remove replicates with at least two of its samples meet the QC threshold (bean run does not support having only one sorting bin sample for a replicate). +* `-i`, `--ignore-missing-samples` + If the flag is not provided, if the ReporterScreen object does not contain all condiitons for + each replicate, make fake empty samples. If the flag is provided, don't add dummy samples. +* `--no-editing` Ignore QC about editing. Can be used for QC of other editing modalities. +* `--dont-recalculate-edits` + When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from + ReporterScreen.uns['allele_count']. + +##### Input `.h5ad` formatting: +Note that these arguements will change the way the QC metrics are calculated for guides, samples, or replicates. +* `--tiling TILING` Specify that the guide library is tiling library without 'n guides per target' design +* `--replicate-label REPLICATE_LABEL` + Label of column in `bdata.samples` that describes replicate ID. +* `--sample-covariates SAMPLE_COVARIATES` + Comma-separated list of column names in `bdata.samples` that describes non-selective + experimental condition. (drug treatment, etc.) +* `--condition-label CONDITION_LABEL` + Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time, + etc.) +###### Editing rate calculation + * `--control-condition CTRL_COND` + Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate + to be calculated. Default is `None`, which considers all samples. + * `--rel-pos-is-reporter` + Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If + `False`, those are relative to spacer position. + Editing rate is calculated with following parameters in + * Variant screens: + * `--target-pos-col TARGET_POS_COL` + Target position column in `bdata.guides` specifying target edit position in reporter + * tiling screens: + * `--edit-start-pos EDIT_START_POS` + Edit start position to quantify editing rate on, 0-based inclusive. + * `--edit-end-pos EDIT_END_POS` + Edit end position to quantify editing rate on, 0-based exclusive. +###### LFC of positive controls + * `--posctrl-col POSCTRL_COL` + Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all + gRNAs, feed empty string ''. + * `--posctrl-val POSCTRL_VAL` + Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the + positive control in calculating log fold change. + * `--lfc-conds LFC_CONDS` + Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated + between, delimited by comma \ No newline at end of file diff --git a/docs/_build/_sources/commands/run.md.txt b/docs/_build/_sources/commands/run.md.txt new file mode 100644 index 0000000..4c45259 --- /dev/null +++ b/docs/_build/_sources/commands/run.md.txt @@ -0,0 +1,64 @@ +# `bean run`: Quantify variant effects +BEAN uses Bayesian network to incorporate gRNA editing outcome to provide posterior estimate of variant phenotype. The Bayesian network reflects data generation process. Briefly, +1. Cellular phenotype (either for cells are sorted upon for sorting screen, or log(proliferation rate)) is modeled as the Gaussian mixture distribution of wild-type phenotype and variant phenotype. +2. The weight of the mixture components are inferred from the reporter editing outcome and the chromatin accessibility of the loci. +3. Cells with each gRNA, formulated as the mixture distribution, is sorted by the phenotypic quantile to produce the gRNA counts. + +For the full detail, see the method section of the [BEAN manuscript](https://www.medrxiv.org/content/10.1101/2023.09.08.23295253v1). + +model + +

    + +# Usage example +```bash +bean run sorting[survival] variant[tiling] my_sorting_screen_filtered.h5ad \ +[--uniform-edit, --scale-by-acc [--acc-bw-path accessibility_signal.bw, --acc-col accessibility]] \ +-o output_prefix/ \ +--fit-negctrl +``` +See full list of parameters [below](#full-parameters). + + +# Input +`my_sorting_screen_filtered.h5ad` can be produced by one of the following: +1. [`bean count-samples`]((#bean-count-samples-count-reporter-screen-data)) when you have raw `.fastq` file +2. (Limited to `bean run variant` mode) `bean create-screen` when you have flat `.csv` tables of gRNA metadata table, sample metadata table, gRNA counts table (# guides x # samples), and optionally # edits table. + ```bash + bean create-screen gRNA_info_table.csv sample_info_table.csv gRNA_counts_table.csv \ + [--edits edit_counts_table.csv -o output.h5ad] + ``` + * `gRNA_info_table.csv` should have following columns. + * `name`: gRNA ID column + * `target`: This column denotes which target variant/element of each gRNA. + * `target_group [Optional]`: If negative control gRNA will be used, specify as "NegCtrl" in this column. + * `sample_info_table.csv` should have following columns. + * `sample_id`: ID of sequencing sample + * `replicate`: Replicate # of this sample + * `bin`: Name of the sorting bin + * `upper_quantile`: FACS sorting upper quantile + * `lower_quantile`: FACS sorting lower quantile + * `gRNA_counts_table.csv` should be formatted as follows. + * Columns include one of `sample_id` columns in `sample_info_table.csv` file. + * 1st row (row index) follows `name` (gRNA ID) in `gRNA_info_table.csv` file. +3. You can manually create the `AnnData` object with more annotations including allele counts: see [API tutorial](#using-bean-as-python-module) for full detail. + + +# Output +model + +Above command produces +* `output_prefix/bean_element_result.[model_type].csv` with following columns: + * Estimated variant effect sizes + * `mu` (Effect size): Mean of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. + * `mu_sd`: Mean of variant phenotype `mu` is modeled as normal distribution. The column shows fitted standard deviation of `mu` that quantify the uncertainty of the variant effect. + * `mu_z`: z-score of `mu` + * `sd`: Standard deviation of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. + * `CI[0.025`, `0.975]`: Credible interval of `mu` + * When negative control is provided, above columns with `_adj` suffix are provided, which are the corresponding values adjusted for negative control. + * Metrics on per-variant evidence provided in input (provided in `tiling` mode) + * `effective_edit_rate`: Sum of per-variant editing rates over all alleles observed in the input. Allele-level editing rate is divided by the number of variants observed in the allele prior to summing up. + * `n_guides`: # of guides covering the variant. + * `n_coocc`: # of cooccurring variants with a given variant in any alleles observed in the input. +* `output_prefix/bean_sgRNA_result.[model_type].csv`: + * `edit_rate`: Estimated editing rate at the target loci. diff --git a/docs/_build/_sources/count.rst.txt b/docs/_build/_sources/count.rst.txt new file mode 100644 index 0000000..206dbd2 --- /dev/null +++ b/docs/_build/_sources/count.rst.txt @@ -0,0 +1,10 @@ +`bean count` +*********************** +.. mdinclude:: commands/count.md + +Full parameters +================== +.. argparse:: + :filename: ../bean/mapping/utils.py + :func: get_input_parser_count + :prog: bean count \ No newline at end of file diff --git a/docs/_build/_sources/count_samples.rst.txt b/docs/_build/_sources/count_samples.rst.txt new file mode 100644 index 0000000..fbbf8f1 --- /dev/null +++ b/docs/_build/_sources/count_samples.rst.txt @@ -0,0 +1,11 @@ +.. _count_samples: +`bean count-samples` +*********************** +.. mdinclude:: commands/count.md + +Full parameters +================== +.. argparse:: + :filename: ../bean/mapping/utils.py + :func: get_input_parser + :prog: bean count-samples \ No newline at end of file diff --git a/docs/_build/_sources/exon_fa_format.md.txt b/docs/_build/_sources/exon_fa_format.md.txt new file mode 100644 index 0000000..2498b39 --- /dev/null +++ b/docs/_build/_sources/exon_fa_format.md.txt @@ -0,0 +1,8 @@ +# Input .fa file format for `bean-filter` +You can provide custom FASTA file with exon sequence entries. Currently only supports positive strand genes. + +* Exon FASTA files can be downloaded from UCSC Genomic sequences / Table Browser: [see the instruction video](https://www.youtube.com/watch?v=T4E0Ez5Vjz8) +* You can manually format as: + * Header line has ` range=chrom:start-end ` and `strand=+/-` tag that is parsed. + * fasta entry has the sequence of exons, where the first (includes 5'-UTR) and last (includes 3'-UTR) exon sequence has lower-case sequence denoting noncoding sequences. +* See the example .fa [here](../tests/data/ldlr_exons.fa). \ No newline at end of file diff --git a/docs/_build/_sources/filter.rst.txt b/docs/_build/_sources/filter.rst.txt new file mode 100644 index 0000000..ce485ab --- /dev/null +++ b/docs/_build/_sources/filter.rst.txt @@ -0,0 +1,11 @@ +.. _filter: +`bean filter` +*********************** +.. mdinclude:: commands/filter.md + +Full parameters +================== +.. argparse:: + :filename: ../bean/annotate/utils.py + :func: parse_args + :prog: bean filter \ No newline at end of file diff --git a/docs/_build/_sources/gwas.rst.txt b/docs/_build/_sources/gwas.rst.txt new file mode 100644 index 0000000..e407ad9 --- /dev/null +++ b/docs/_build/_sources/gwas.rst.txt @@ -0,0 +1,5 @@ +GWAS variant library +*********************** +.. mdinclude:: tutorials/ldl_var.md + +See :ref:`subcommands` for the full details. diff --git a/docs/_build/_sources/index.md.txt b/docs/_build/_sources/index.md.txt new file mode 100644 index 0000000..45e2414 --- /dev/null +++ b/docs/_build/_sources/index.md.txt @@ -0,0 +1,4 @@ +--- +layout: default +title: CRISPR-BEAN +--- diff --git a/docs/_build/_sources/index_.rst.txt b/docs/_build/_sources/index_.rst.txt new file mode 100644 index 0000000..d78bc9a --- /dev/null +++ b/docs/_build/_sources/index_.rst.txt @@ -0,0 +1,38 @@ +.. bean documentation master file, created by + sphinx-quickstart on Fri Mar 29 19:10:46 2024. + You can adapt this file completely to your liking, but it should at least + contain the root `toctree` directive. + +Welcome to `bean`'s documentation! +================================ +=================== +Workflows +=================== +.. toctree:: + :maxdepth: 2 + + gwas + cds + input + +=================== +`bean` subcommands +=================== +.. toctree:: + :maxdepth: 3 + + subcommands + +=================== +Screen data structure +=================== +.. toctree:: + ReporterScreen_api + +================== +Indices and tables +================== + +* :ref:`genindex` +* :ref:`modindex` +* :ref:`search` diff --git a/docs/_build/_sources/input.rst.txt b/docs/_build/_sources/input.rst.txt new file mode 100644 index 0000000..d63417e --- /dev/null +++ b/docs/_build/_sources/input.rst.txt @@ -0,0 +1,4 @@ +.. _input: +Input file format +*********************** +.. mdinclude:: commands/input.md \ No newline at end of file diff --git a/docs/_build/_sources/profile.rst.txt b/docs/_build/_sources/profile.rst.txt new file mode 100644 index 0000000..3ee525f --- /dev/null +++ b/docs/_build/_sources/profile.rst.txt @@ -0,0 +1,10 @@ +`bean profile` +*********************** +.. mdinclude:: commands/profile.md + +Full parameters +================== +.. argparse:: + :filename: ../bean/plotting/utils.py + :func: parse_args + :prog: bean profile \ No newline at end of file diff --git a/docs/_build/_sources/qc.rst.txt b/docs/_build/_sources/qc.rst.txt new file mode 100644 index 0000000..604e77a --- /dev/null +++ b/docs/_build/_sources/qc.rst.txt @@ -0,0 +1,11 @@ +.. _qc: +`bean qc` +*********************** +.. mdinclude:: commands/qc.md + +Full parameters +================== +.. argparse:: + :filename: ../bean/qc/parser.py + :func: parse_args + :prog: bean qc \ No newline at end of file diff --git a/docs/_build/_sources/run.rst.txt b/docs/_build/_sources/run.rst.txt new file mode 100644 index 0000000..a6dc1fc --- /dev/null +++ b/docs/_build/_sources/run.rst.txt @@ -0,0 +1,11 @@ +.. _run: +`bean run` +*********************** +.. mdinclude:: commands/run.md + +Full parameters +================== +.. argparse:: + :filename: ../bean/model/parser.py + :func: parse_args + :prog: bean run \ No newline at end of file diff --git a/docs/_build/_sources/subcommands.rst.txt b/docs/_build/_sources/subcommands.rst.txt new file mode 100644 index 0000000..56319ca --- /dev/null +++ b/docs/_build/_sources/subcommands.rst.txt @@ -0,0 +1,14 @@ +.. _subcommands: +=================== +Subcommands +=================== +.. toctree:: + :maxdepth: 2 + + count + count_samples + profile + qc + filter + run + create_screen \ No newline at end of file diff --git a/docs/_build/_sources/tutorials/ldl_cds.md.txt b/docs/_build/_sources/tutorials/ldl_cds.md.txt new file mode 100644 index 0000000..ba78a00 --- /dev/null +++ b/docs/_build/_sources/tutorials/ldl_cds.md.txt @@ -0,0 +1,148 @@ +# Tiling sorting screen tutorial +Tiling screen that tiles gRNA densely across locus or multiple loci, selected based on FACS signal quantiles. + + + + + + + + + + +
    Library designTiling (gRNAs tile each locus densely)
    tiling library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design
    + +

    + +## Example workflow +```bash +screen_id=my_sorting_tiling_screen + +# 1. Count gRNA & reporter +bean-count-samples \ +--input tests/data/sample_list_tiling.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f tests/data/test_guide_info_tiling_chrom.csv `# Contains gRNA metadata; see test file for example.`\ +-o ./ `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen` \ +--tiling + +# 2. QC samples & guides +bean-qc \ + bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r qc_report_${screen_id} `# Prefix for QC report` \ + +# 3. Filter & translate alleles +bean-filter ./bean_count_${screen_id}_masked.h5ad \ +-o ./bean_count_${screen_id}_alleleFiltered \ +--filter-target-basechange `# Filter based on intended base changes. If -b A was provided in bean-count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\ +--filter-window --edit-start-pos 0 --edit-end-pos 19 `# Filter based on editing window in spacer position within reporter.`\ +--filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \ +--translate --translate-genes-list tests/data/gene_symbols.txt + +# 4. Quantify variant effect +bean-run sorting tiling \ + ./bean_count_${screen_id}_alleleFiltered.h5ad \ + -o tests/test_res/var/ \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility +``` +See more details below. + +## 1. Count gRNA & reporter (:ref:`count_samples`) +``` +screen_id=my_sorting_tiling_screen + +bean-count-samples \ +--input tests/data/sample_list_tiling.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f tests/data/test_guide_info_tiling_chrom.csv `# Contains gRNA metadata; see test file for example.`\ +-o ./ `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen` \ +--tiling +``` +Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. + +## 2. QC (:ref:`qc`) +Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean-qc`](../../README#bean-qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) +``` +bean-qc \ + bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r qc_report_${screen_id} `# Prefix for QC report` \ + [--tiling] `# Not required if you have passed --tiling in counting step` +``` + + + +If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. + +## 3. Filter alleles (:ref:`filter`) +As tiling library doesn't have designated per-gRNA target variant, any base edit observed in reporter may be the candidate variant, while having too many variants with very low editing rate significantly decreases the power. Variants are filtered based on multiple criteria in `bean-fitler`. + +If the screen targets coding sequence, it's beneficial to translate edits into coding varaints whenever possible for better power. For translation, provide `--translate` and one of the following: +``` +[ --translate-gene-name GENE_SYMBOL OR + --translate-genes-list path_to_gene_names_file.txt OR + --translate-fasta gene_exon.fa, OR + --translate-fastas-csv gene_exon_fas.csv] +``` +where `path_to_gene_names_file.txt` has one gene symbol per line, and gene symbol uses its MANE transcript (hg38) coordinates of exons. In order to use other reference versions or transcript ID, you'll need to feed in fasta file. See detailed formatting of fasta file [here](../../README#translating-alleles). + +Example allele filtering given we're translating based on MANE transcript exons of multiple gene symbols: + +```bash +bean-filter ./bean_count_${screen_id}_masked.h5ad \ +-o ./bean_count_${screen_id}_alleleFiltered \ +--filter-target-basechange `# Filter based on intended base changes. If -b A was provided in bean-count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\ +--filter-window --edit-start-pos 0 --edit-end-pos 19 `# Filter based on editing window in spacer position within reporter.`\ +--filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \ +--translate --translate-genes-list tests/data/gene_symbols.txt +``` + +Ouptut file `` shows number of alleles per guide and number of guides per variant, where we want high enough values for the latter. See the typical output for dataset with good editing coverage & filtering result [here](../example_filtering_ouptut/). + +## 4. Quantify variant effect (:ref:`run`) +By default, `bean-run [sorting,survival] tiling` uses most filtered allele counts table for variant identification and quantification of their effects. **Check [allele filtering output](../example_filtering_ouptut/)** and choose alternative filtered allele counts table if necessary. + +`bean-run` can take 3 run options to quantify editing rate: +1. From **reporter + accessibility** + 1-1. If your gRNA metadata table (`tests/data/test_guide_info.csv` above) included per-gRNA accessibility score, + ``` + bean-run sorting tiling \ + ./bean_count_${screen_id}_alleleFiltered.h5ad \ + -o tests/test_res/var/ \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility + ``` + 1-2. If your gRNA metadata table (`tests/data/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, + ``` + bean-run sorting tiling \ + ./bean_count_${screen_id}_alleleFiltered.h5ad \ + -o tests/test_res/var/ \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-bw accessibility.bw + ``` + +2. From **reporter** + ``` + bean-run sorting tiling \ + ./bean_count_${screen_id}_alleleFiltered.h5ad \ + -o tests/test_res/var/ \ + --fit-negctrl + ``` +3. No reporter information, assume the same editing efficiency of all gRNAs. + Use this option if your data don't have editing rate information. + ``` + bean-run sorting tiling \ + ./bean_count_${screen_id}_alleleFiltered.h5ad \ + -o tests/test_res/var/ \ + --fit-negctrl \ + --uniform-edit + ``` \ No newline at end of file diff --git a/docs/_build/_sources/tutorials/ldl_var.md.txt b/docs/_build/_sources/tutorials/ldl_var.md.txt new file mode 100644 index 0000000..1542536 --- /dev/null +++ b/docs/_build/_sources/tutorials/ldl_var.md.txt @@ -0,0 +1,116 @@ +# Variant sorting screen tutorial +GWAS variant screen with per-variant gRNA tiling design, selected based on FACS signal quantiles. + + + + + + + + + + +
    Library designVariant (gRNAs tile each target variant)
    variant library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design
    + +

    + +## Example workflow +```bash +screen_id=my_sorting_tiling_screen + +# 1. Count gRNA & reporter +bean-count-samples \ +--input tests/data/sample_list.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f tests/data/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ +-o ./ `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen to be counted` + +# 2. QC samples & guides +bean-qc \ + bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r qc_report_${screen_id} `# Prefix for QC report` \ + -b ` # Remove replicates with no good samples. + +# 3. Quantify variant effect +bean-run sorting variant \ + tests/data/bean_count_${screen_id}_masked.h5ad \ + -o tests/test_res/var/ \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility +``` +See more details below. + +## 1. Count gRNA & reporter (:ref:`count_samples`) +```bash +screen_id=my_sorting_tiling_screen + +# 1. Count gRNA & reporter +bean-count-samples \ +--input tests/data/sample_list.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f tests/data/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ +-o ./ `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen to be counted` +``` +Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. + +## 2. QC samples & guides (:ref:`qc`) +Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean-qc`](../../README#bean-qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) +``` +bean-qc \ + bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r qc_report_${screen_id} `# Prefix for QC report` +``` + + + +If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. + + +## 3. Quantify variant effect (:ref:`run`) + +`bean-run` can take 3 run options to quantify editing rate: +1. From **reporter + accessibility** + If your gRNA metadata table (`tests/data/test_guide_info.csv` above) included per-gRNA accessibility score, + ``` + bean-run sorting variant \ + tests/data/bean_count_${screen_id}_masked.h5ad \ + -o tests/test_res/var/ \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility + ``` + If your gRNA metadata table (`tests/data/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, + ``` + bean-run sorting variant \ + tests/data/bean_count_${screen_id}_masked.h5ad \ + -o tests/test_res/var/ \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-bw accessibility.bw + ``` + +2. From **reporter**, without accessibility + + This assumes the all target sites have the uniform chromatin accessibility. + ``` + bean-run sorting variant \ + tests/data/bean_count_${screen_id}_masked.h5ad \ + -o tests/test_res/var/ \ + --fit-negctrl + ``` +3. No reporter information, assume the same editing efficiency of all gRNAs. + Use this option if your data don't have editing outcome information. + ``` + bean-run sorting variant \ + tests/data/bean_count_${screen_id}_masked.h5ad \ + -o tests/test_res/var/ \ + --fit-negctrl \ + --uniform-edit + ``` \ No newline at end of file diff --git a/docs/_build/_static/alabaster.css b/docs/_build/_static/alabaster.css new file mode 100644 index 0000000..e3174bf --- /dev/null +++ b/docs/_build/_static/alabaster.css @@ -0,0 +1,708 @@ +@import url("basic.css"); + +/* -- page layout ----------------------------------------------------------- */ + +body { + font-family: Georgia, serif; + font-size: 17px; + background-color: #fff; + color: #000; + margin: 0; + padding: 0; +} + + +div.document { + width: 940px; + margin: 30px auto 0 auto; +} + +div.documentwrapper { + float: left; + width: 100%; +} + +div.bodywrapper { + margin: 0 0 0 220px; +} + +div.sphinxsidebar { + width: 220px; + font-size: 14px; + line-height: 1.5; 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+ } + + .document { + width: auto; + } + + .footer { + width: auto; + } + + .footer { + width: auto; + } + + .github { + display: none; + } +} + + +/* misc. */ + +.revsys-inline { + display: none!important; +} + +/* Hide ugly table cell borders in ..bibliography:: directive output */ +table.docutils.citation, table.docutils.citation td, table.docutils.citation th { + border: none; + /* Below needed in some edge cases; if not applied, bottom shadows appear */ + -moz-box-shadow: none; + -webkit-box-shadow: none; + box-shadow: none; +} + + +/* relbar */ + +.related { + line-height: 30px; + width: 100%; + font-size: 0.9rem; +} + +.related.top { + border-bottom: 1px solid #EEE; + margin-bottom: 20px; +} + +.related.bottom { + border-top: 1px solid #EEE; +} + +.related ul { + padding: 0; + margin: 0; + list-style: none; +} + +.related li { + display: inline; +} + +nav#rellinks { + float: right; +} + +nav#rellinks li+li:before { + content: "|"; +} + +nav#breadcrumbs li+li:before { + content: "\00BB"; +} + +/* Hide certain items when printing */ +@media print { + div.related { + display: none; + } +} \ No newline at end of file diff --git a/docs/_build/_static/basic.css b/docs/_build/_static/basic.css new file mode 100644 index 0000000..4157edf --- /dev/null +++ b/docs/_build/_static/basic.css @@ -0,0 +1,925 @@ +/* + * basic.css + * ~~~~~~~~~ + * + * Sphinx stylesheet -- basic theme. + * + * :copyright: Copyright 2007-2023 by the Sphinx team, see AUTHORS. + * :license: BSD, see LICENSE for details. + * + */ + +/* -- main layout ----------------------------------------------------------- */ + +div.clearer { + clear: both; +} + +div.section::after { + display: block; + content: ''; + clear: left; +} + +/* -- relbar ---------------------------------------------------------------- */ + +div.related { + width: 100%; + font-size: 90%; +} + +div.related h3 { + display: none; +} + +div.related ul { + margin: 0; + padding: 0 0 0 10px; + list-style: none; +} + +div.related li { + display: inline; +} + +div.related li.right { + float: right; + margin-right: 5px; +} + +/* -- sidebar --------------------------------------------------------------- */ + +div.sphinxsidebarwrapper { + padding: 10px 5px 0 10px; +} + +div.sphinxsidebar { + float: left; + width: 230px; + margin-left: -100%; + font-size: 90%; + word-wrap: break-word; + overflow-wrap : break-word; +} + +div.sphinxsidebar ul { + list-style: none; +} + +div.sphinxsidebar ul ul, +div.sphinxsidebar ul.want-points { + margin-left: 20px; + list-style: square; +} + +div.sphinxsidebar ul ul { + margin-top: 0; + margin-bottom: 0; +} + +div.sphinxsidebar form { + margin-top: 10px; +} + +div.sphinxsidebar input { + border: 1px solid #98dbcc; + font-family: sans-serif; + font-size: 1em; +} + +div.sphinxsidebar #searchbox form.search { + overflow: hidden; +} + +div.sphinxsidebar #searchbox input[type="text"] { + float: left; + width: 80%; + padding: 0.25em; + box-sizing: border-box; +} + +div.sphinxsidebar #searchbox input[type="submit"] { + float: left; + width: 20%; + border-left: none; + padding: 0.25em; + box-sizing: border-box; +} + + +img { + border: 0; + max-width: 100%; +} + +/* -- search page ----------------------------------------------------------- */ + +ul.search { + margin: 10px 0 0 20px; + padding: 0; +} + +ul.search li { + padding: 5px 0 5px 20px; + background-image: url(file.png); + background-repeat: no-repeat; + background-position: 0 7px; +} + +ul.search li a { + font-weight: bold; +} + +ul.search li p.context { + color: #888; + margin: 2px 0 0 30px; + text-align: left; +} + +ul.keywordmatches li.goodmatch a { + font-weight: bold; +} + +/* -- index page ------------------------------------------------------------ */ + +table.contentstable { + width: 90%; + margin-left: auto; + margin-right: auto; +} + +table.contentstable p.biglink { + line-height: 150%; +} + +a.biglink { + font-size: 1.3em; +} + +span.linkdescr { + font-style: italic; + padding-top: 5px; + font-size: 90%; +} + +/* -- general index --------------------------------------------------------- */ + +table.indextable { + width: 100%; +} + +table.indextable td { + text-align: left; + vertical-align: top; +} + +table.indextable ul { + margin-top: 0; + margin-bottom: 0; + list-style-type: none; +} + +table.indextable > tbody > tr > td > ul { + padding-left: 0em; +} + +table.indextable tr.pcap { + height: 10px; +} + +table.indextable tr.cap { + margin-top: 10px; + background-color: #f2f2f2; +} + +img.toggler { + margin-right: 3px; + margin-top: 3px; + cursor: pointer; +} + +div.modindex-jumpbox { + border-top: 1px solid #ddd; + border-bottom: 1px solid #ddd; + margin: 1em 0 1em 0; + padding: 0.4em; +} + +div.genindex-jumpbox { + border-top: 1px solid #ddd; + border-bottom: 1px solid #ddd; + margin: 1em 0 1em 0; + padding: 0.4em; +} + +/* -- domain module index --------------------------------------------------- */ + +table.modindextable td { + padding: 2px; + border-collapse: collapse; +} + +/* -- general body styles --------------------------------------------------- */ + +div.body { + min-width: inherit; + max-width: 800px; +} + +div.body p, div.body dd, div.body li, div.body blockquote { + -moz-hyphens: auto; + -ms-hyphens: auto; + -webkit-hyphens: auto; + hyphens: auto; +} + +a.headerlink { + visibility: hidden; +} + +a:visited { + color: #551A8B; +} + +h1:hover > a.headerlink, +h2:hover > a.headerlink, +h3:hover > a.headerlink, +h4:hover > a.headerlink, +h5:hover > a.headerlink, +h6:hover > a.headerlink, +dt:hover > a.headerlink, +caption:hover > a.headerlink, +p.caption:hover > a.headerlink, +div.code-block-caption:hover > a.headerlink { + visibility: visible; +} + +div.body p.caption { + text-align: inherit; +} + +div.body td { + text-align: left; +} + +.first { + margin-top: 0 !important; +} + +p.rubric { + margin-top: 30px; + font-weight: bold; +} + +img.align-left, figure.align-left, .figure.align-left, object.align-left { + clear: left; + float: left; + margin-right: 1em; +} + +img.align-right, figure.align-right, .figure.align-right, object.align-right { + clear: right; + float: right; + margin-left: 1em; +} + +img.align-center, figure.align-center, .figure.align-center, object.align-center { + display: block; + margin-left: auto; + margin-right: auto; +} + +img.align-default, figure.align-default, .figure.align-default { + display: block; + margin-left: auto; + margin-right: auto; +} + +.align-left { + text-align: left; +} + +.align-center { + text-align: center; +} + +.align-default { + text-align: center; +} + +.align-right { + text-align: right; +} + +/* -- sidebars -------------------------------------------------------------- */ + +div.sidebar, +aside.sidebar { + margin: 0 0 0.5em 1em; + border: 1px solid #ddb; + padding: 7px; + background-color: #ffe; + width: 40%; + float: right; + clear: right; + overflow-x: auto; +} + +p.sidebar-title { + font-weight: bold; +} + +nav.contents, +aside.topic, +div.admonition, div.topic, blockquote { + clear: left; +} + +/* -- topics ---------------------------------------------------------------- */ + +nav.contents, +aside.topic, +div.topic { + border: 1px solid #ccc; + padding: 7px; + margin: 10px 0 10px 0; +} + +p.topic-title { + font-size: 1.1em; + font-weight: bold; + margin-top: 10px; +} + +/* -- admonitions ----------------------------------------------------------- */ + +div.admonition { + margin-top: 10px; + margin-bottom: 10px; + padding: 7px; +} + +div.admonition dt { + font-weight: bold; +} + +p.admonition-title { + margin: 0px 10px 5px 0px; + font-weight: bold; +} + +div.body p.centered { + text-align: center; + margin-top: 25px; +} + +/* -- content of sidebars/topics/admonitions -------------------------------- */ + +div.sidebar > :last-child, +aside.sidebar > :last-child, +nav.contents > :last-child, +aside.topic > :last-child, +div.topic > :last-child, +div.admonition > :last-child { + margin-bottom: 0; +} + +div.sidebar::after, +aside.sidebar::after, +nav.contents::after, +aside.topic::after, +div.topic::after, +div.admonition::after, +blockquote::after { + display: block; + content: ''; + clear: both; +} + +/* -- tables ---------------------------------------------------------------- */ + +table.docutils { + margin-top: 10px; + margin-bottom: 10px; + border: 0; + border-collapse: collapse; +} + +table.align-center { + margin-left: auto; + margin-right: auto; +} + +table.align-default { + margin-left: auto; + margin-right: auto; +} + +table caption span.caption-number { + font-style: italic; +} + +table caption span.caption-text { +} + +table.docutils td, table.docutils th { + padding: 1px 8px 1px 5px; + border-top: 0; + border-left: 0; + border-right: 0; + border-bottom: 1px solid #aaa; +} + +th { + text-align: left; + padding-right: 5px; +} + +table.citation { + border-left: solid 1px gray; + margin-left: 1px; +} + +table.citation td { + border-bottom: none; +} + +th > :first-child, +td > :first-child { + margin-top: 0px; +} + +th > :last-child, +td > :last-child { + margin-bottom: 0px; +} + +/* -- figures --------------------------------------------------------------- */ + +div.figure, figure { + margin: 0.5em; + padding: 0.5em; +} + +div.figure p.caption, figcaption { + padding: 0.3em; +} + +div.figure p.caption span.caption-number, +figcaption span.caption-number { + font-style: italic; +} + +div.figure p.caption span.caption-text, +figcaption span.caption-text { +} + +/* -- field list styles ----------------------------------------------------- */ + +table.field-list td, table.field-list th { + border: 0 !important; +} + +.field-list ul { + margin: 0; + padding-left: 1em; +} + +.field-list p { + margin: 0; +} + +.field-name { + -moz-hyphens: manual; + -ms-hyphens: manual; + -webkit-hyphens: manual; + hyphens: manual; +} + +/* -- hlist styles ---------------------------------------------------------- */ + +table.hlist { + margin: 1em 0; +} + +table.hlist td { + vertical-align: top; +} + +/* -- object description styles --------------------------------------------- */ + +.sig { + font-family: 'Consolas', 'Menlo', 'DejaVu Sans Mono', 'Bitstream Vera Sans Mono', monospace; +} + +.sig-name, code.descname { + background-color: transparent; + font-weight: bold; +} + +.sig-name { + font-size: 1.1em; +} + +code.descname { + font-size: 1.2em; +} + +.sig-prename, code.descclassname { + background-color: transparent; +} + +.optional { + font-size: 1.3em; +} + +.sig-paren { + font-size: larger; +} + +.sig-param.n { + font-style: italic; +} + +/* C++ specific styling */ + +.sig-inline.c-texpr, +.sig-inline.cpp-texpr { + font-family: unset; +} + +.sig.c .k, .sig.c .kt, +.sig.cpp .k, .sig.cpp .kt { + color: #0033B3; +} + +.sig.c .m, +.sig.cpp .m { + color: #1750EB; +} + +.sig.c .s, .sig.c .sc, +.sig.cpp .s, .sig.cpp .sc { + color: #067D17; +} + + +/* -- other body styles ----------------------------------------------------- */ + +ol.arabic { + list-style: decimal; +} + +ol.loweralpha { + list-style: lower-alpha; +} + +ol.upperalpha { + list-style: upper-alpha; +} + +ol.lowerroman { + list-style: lower-roman; +} + +ol.upperroman { + list-style: upper-roman; +} + +:not(li) > ol > li:first-child > :first-child, +:not(li) > ul > li:first-child > :first-child { + margin-top: 0px; +} + +:not(li) > ol > li:last-child > :last-child, +:not(li) > ul > li:last-child > :last-child { + margin-bottom: 0px; +} + +ol.simple ol p, +ol.simple ul p, +ul.simple ol p, +ul.simple ul p { + margin-top: 0; +} + +ol.simple > li:not(:first-child) > p, +ul.simple > li:not(:first-child) > p { + margin-top: 0; +} + +ol.simple p, +ul.simple p { + margin-bottom: 0; +} + +aside.footnote > span, +div.citation > span { + float: left; +} +aside.footnote > span:last-of-type, +div.citation > span:last-of-type { + padding-right: 0.5em; +} +aside.footnote > p { + margin-left: 2em; +} +div.citation > p { + margin-left: 4em; +} +aside.footnote > p:last-of-type, +div.citation > p:last-of-type { + margin-bottom: 0em; +} +aside.footnote > p:last-of-type:after, +div.citation > p:last-of-type:after { + content: ""; + clear: both; +} + +dl.field-list { + display: grid; + grid-template-columns: fit-content(30%) auto; +} + +dl.field-list > dt { + font-weight: bold; + word-break: break-word; + padding-left: 0.5em; + padding-right: 5px; +} + +dl.field-list > dd { + padding-left: 0.5em; + margin-top: 0em; + margin-left: 0em; + margin-bottom: 0em; +} + +dl { + margin-bottom: 15px; +} + +dd > :first-child { + margin-top: 0px; +} + +dd ul, dd table { + margin-bottom: 10px; +} + +dd { + margin-top: 3px; + margin-bottom: 10px; + margin-left: 30px; +} + +.sig dd { + margin-top: 0px; + margin-bottom: 0px; +} + +.sig dl { + margin-top: 0px; + margin-bottom: 0px; +} + +dl > dd:last-child, +dl > dd:last-child > :last-child { + margin-bottom: 0; +} + +dt:target, span.highlighted { + background-color: #fbe54e; +} + +rect.highlighted { + fill: #fbe54e; +} + +dl.glossary dt { + font-weight: bold; + font-size: 1.1em; +} + +.versionmodified { + font-style: italic; +} + +.system-message { + background-color: #fda; + padding: 5px; + border: 3px solid red; +} + +.footnote:target { + background-color: #ffa; +} + +.line-block { + display: block; + margin-top: 1em; + margin-bottom: 1em; +} + +.line-block .line-block { + margin-top: 0; + margin-bottom: 0; + margin-left: 1.5em; +} + +.guilabel, .menuselection { + font-family: sans-serif; +} + +.accelerator { + text-decoration: underline; +} + +.classifier { + font-style: oblique; +} + +.classifier:before { + font-style: normal; + margin: 0 0.5em; + content: ":"; + display: inline-block; +} + +abbr, acronym { + border-bottom: dotted 1px; + cursor: help; +} + +.translated { + background-color: rgba(207, 255, 207, 0.2) +} + +.untranslated { + background-color: rgba(255, 207, 207, 0.2) +} + +/* -- code displays --------------------------------------------------------- */ + +pre { + overflow: auto; + overflow-y: hidden; /* fixes display issues on Chrome browsers */ +} + +pre, div[class*="highlight-"] { + clear: both; +} + +span.pre { + -moz-hyphens: none; + -ms-hyphens: none; + -webkit-hyphens: none; + hyphens: none; + white-space: nowrap; +} + +div[class*="highlight-"] { + margin: 1em 0; +} + +td.linenos pre { + border: 0; + background-color: transparent; + color: #aaa; +} + +table.highlighttable { + display: block; +} + +table.highlighttable tbody { + display: block; +} + +table.highlighttable tr { + display: flex; +} + +table.highlighttable td { + margin: 0; + padding: 0; +} + +table.highlighttable td.linenos { + padding-right: 0.5em; +} + +table.highlighttable td.code { + flex: 1; + overflow: hidden; +} + +.highlight .hll { + display: block; +} + +div.highlight pre, +table.highlighttable pre { + margin: 0; +} + +div.code-block-caption + div { + margin-top: 0; +} + +div.code-block-caption { + margin-top: 1em; + padding: 2px 5px; + font-size: small; +} + +div.code-block-caption code { + background-color: transparent; +} + +table.highlighttable td.linenos, +span.linenos, +div.highlight span.gp { /* gp: Generic.Prompt */ + user-select: none; + -webkit-user-select: text; /* Safari fallback only */ + -webkit-user-select: none; /* Chrome/Safari */ + -moz-user-select: none; /* Firefox */ + -ms-user-select: none; /* IE10+ */ +} + +div.code-block-caption span.caption-number { + padding: 0.1em 0.3em; + font-style: italic; +} + +div.code-block-caption span.caption-text { +} + +div.literal-block-wrapper { + margin: 1em 0; +} + +code.xref, a code { + background-color: transparent; + font-weight: bold; +} + +h1 code, h2 code, h3 code, h4 code, h5 code, h6 code { + background-color: transparent; +} + +.viewcode-link { + float: right; +} + +.viewcode-back { + float: right; + font-family: sans-serif; +} + +div.viewcode-block:target { + margin: -1px -10px; + padding: 0 10px; +} + +/* -- math display ---------------------------------------------------------- */ + +img.math { + vertical-align: middle; +} + +div.body div.math p { + text-align: center; +} + +span.eqno { + float: right; +} + +span.eqno a.headerlink { + position: absolute; + z-index: 1; +} + +div.math:hover a.headerlink { + visibility: visible; +} + +/* -- printout stylesheet --------------------------------------------------- */ + +@media print { + div.document, + div.documentwrapper, + div.bodywrapper { + margin: 0 !important; + width: 100%; + } + + div.sphinxsidebar, + div.related, + div.footer, + #top-link { + display: none; + } +} \ No newline at end of file diff --git a/docs/_build/_static/custom.css b/docs/_build/_static/custom.css new file mode 100644 index 0000000..2a924f1 --- /dev/null +++ b/docs/_build/_static/custom.css @@ -0,0 +1 @@ +/* This file intentionally left blank. */ diff --git a/docs/_build/_static/doctools.js b/docs/_build/_static/doctools.js new file mode 100644 index 0000000..d06a71d --- /dev/null +++ b/docs/_build/_static/doctools.js @@ -0,0 +1,156 @@ +/* + * doctools.js + * ~~~~~~~~~~~ + * + * Base JavaScript utilities for all Sphinx HTML documentation. + * + * :copyright: Copyright 2007-2023 by the Sphinx team, see AUTHORS. + * :license: BSD, see LICENSE for details. + * + */ +"use strict"; + +const BLACKLISTED_KEY_CONTROL_ELEMENTS = new Set([ + "TEXTAREA", + "INPUT", + "SELECT", + "BUTTON", +]); + +const _ready = (callback) => { + if (document.readyState !== "loading") { + callback(); + } else { + document.addEventListener("DOMContentLoaded", callback); + } +}; + +/** + * Small JavaScript module for the documentation. + */ +const Documentation = { + init: () => { + Documentation.initDomainIndexTable(); + Documentation.initOnKeyListeners(); + }, + + /** + * i18n support + */ + TRANSLATIONS: {}, + PLURAL_EXPR: (n) => (n === 1 ? 0 : 1), + LOCALE: "unknown", + + // gettext and ngettext don't access this so that the functions + // can safely bound to a different name (_ = Documentation.gettext) + gettext: (string) => { + const translated = Documentation.TRANSLATIONS[string]; + switch (typeof translated) { + case "undefined": + return string; // no translation + case "string": + return translated; // translation exists + default: + return translated[0]; // (singular, plural) translation tuple exists + } + }, + + ngettext: (singular, plural, n) => { + const translated = Documentation.TRANSLATIONS[singular]; + if (typeof translated !== "undefined") + return translated[Documentation.PLURAL_EXPR(n)]; + return n === 1 ? singular : plural; + }, + + addTranslations: (catalog) => { + Object.assign(Documentation.TRANSLATIONS, catalog.messages); + Documentation.PLURAL_EXPR = new Function( + "n", + `return (${catalog.plural_expr})` + ); + Documentation.LOCALE = catalog.locale; + }, + + /** + * helper function to focus on search bar + */ + focusSearchBar: () => { + document.querySelectorAll("input[name=q]")[0]?.focus(); + }, + + /** + * Initialise the domain index toggle buttons + */ + initDomainIndexTable: () => { + const toggler = (el) => { + const idNumber = el.id.substr(7); + const toggledRows = document.querySelectorAll(`tr.cg-${idNumber}`); + if (el.src.substr(-9) === "minus.png") { + el.src = `${el.src.substr(0, el.src.length - 9)}plus.png`; + toggledRows.forEach((el) => (el.style.display = "none")); + } else { + el.src = `${el.src.substr(0, el.src.length - 8)}minus.png`; + toggledRows.forEach((el) => (el.style.display = "")); + } + }; + + const togglerElements = document.querySelectorAll("img.toggler"); + togglerElements.forEach((el) => + el.addEventListener("click", (event) => toggler(event.currentTarget)) + ); + togglerElements.forEach((el) => (el.style.display = "")); + if (DOCUMENTATION_OPTIONS.COLLAPSE_INDEX) togglerElements.forEach(toggler); + }, + + initOnKeyListeners: () => { + // only install a listener if it is really needed + if ( + !DOCUMENTATION_OPTIONS.NAVIGATION_WITH_KEYS && + !DOCUMENTATION_OPTIONS.ENABLE_SEARCH_SHORTCUTS + ) + return; + + document.addEventListener("keydown", (event) => { + // bail for input elements + if (BLACKLISTED_KEY_CONTROL_ELEMENTS.has(document.activeElement.tagName)) return; + // bail with special keys + if (event.altKey || event.ctrlKey || event.metaKey) return; + + if (!event.shiftKey) { + switch (event.key) { + case "ArrowLeft": + if (!DOCUMENTATION_OPTIONS.NAVIGATION_WITH_KEYS) break; + + const prevLink = document.querySelector('link[rel="prev"]'); + if (prevLink && prevLink.href) { + window.location.href = prevLink.href; + event.preventDefault(); + } + break; + case "ArrowRight": + if (!DOCUMENTATION_OPTIONS.NAVIGATION_WITH_KEYS) break; + + const nextLink = document.querySelector('link[rel="next"]'); + if (nextLink && nextLink.href) { + window.location.href = nextLink.href; + event.preventDefault(); + } + break; + } + } + + // some keyboard layouts may need Shift to get / + switch (event.key) { + case "/": + if (!DOCUMENTATION_OPTIONS.ENABLE_SEARCH_SHORTCUTS) break; + Documentation.focusSearchBar(); + event.preventDefault(); + } + }); + }, +}; + +// quick alias for translations +const _ = Documentation.gettext; + +_ready(Documentation.init); diff --git a/docs/_build/_static/documentation_options.js b/docs/_build/_static/documentation_options.js new file mode 100644 index 0000000..89435bb --- /dev/null +++ b/docs/_build/_static/documentation_options.js @@ -0,0 +1,13 @@ +const DOCUMENTATION_OPTIONS = { + VERSION: '1.0.0', + LANGUAGE: 'en', + COLLAPSE_INDEX: false, + BUILDER: 'html', + FILE_SUFFIX: '.html', + LINK_SUFFIX: '.html', + HAS_SOURCE: true, + SOURCELINK_SUFFIX: '.txt', + NAVIGATION_WITH_KEYS: false, + SHOW_SEARCH_SUMMARY: true, + ENABLE_SEARCH_SHORTCUTS: true, +}; \ No newline at end of file diff --git a/docs/_build/_static/file.png b/docs/_build/_static/file.png new file mode 100644 index 0000000000000000000000000000000000000000..a858a410e4faa62ce324d814e4b816fff83a6fb3 GIT binary patch literal 286 zcmV+(0pb3MP)s`hMrGg#P~ix$^RISR_I47Y|r1 z_CyJOe}D1){SET-^Amu_i71Lt6eYfZjRyw@I6OQAIXXHDfiX^GbOlHe=Ae4>0m)d(f|Me07*qoM6N<$f}vM^LjV8( literal 0 HcmV?d00001 diff --git a/docs/_build/_static/language_data.js b/docs/_build/_static/language_data.js new file mode 100644 index 0000000..250f566 --- /dev/null +++ b/docs/_build/_static/language_data.js @@ -0,0 +1,199 @@ +/* + * language_data.js + * ~~~~~~~~~~~~~~~~ + * + * This script contains the language-specific data used by searchtools.js, + * namely the list of stopwords, stemmer, scorer and splitter. + * + * :copyright: Copyright 2007-2023 by the Sphinx team, see AUTHORS. + * :license: BSD, see LICENSE for details. + * + */ + +var stopwords = ["a", "and", "are", "as", "at", "be", "but", "by", "for", "if", "in", "into", "is", "it", "near", "no", "not", "of", "on", "or", "such", "that", "the", "their", "then", "there", "these", "they", "this", "to", "was", "will", "with"]; + + +/* Non-minified version is copied as a separate JS file, is available */ + +/** + * Porter Stemmer + */ +var Stemmer = function() { + + var step2list = { + ational: 'ate', + tional: 'tion', + enci: 'ence', + anci: 'ance', + izer: 'ize', + bli: 'ble', + alli: 'al', + entli: 'ent', + eli: 'e', + ousli: 'ous', + ization: 'ize', + ation: 'ate', + ator: 'ate', + alism: 'al', + iveness: 'ive', + fulness: 'ful', + ousness: 'ous', + aliti: 'al', + iviti: 'ive', + biliti: 'ble', + logi: 'log' + }; + + var step3list = { + icate: 'ic', + ative: '', + alize: 'al', + iciti: 'ic', + ical: 'ic', + ful: '', + ness: '' + }; + + var c = "[^aeiou]"; // consonant + var v = "[aeiouy]"; // vowel + var C = c + "[^aeiouy]*"; // consonant sequence + var V = v + "[aeiou]*"; // vowel sequence + + var mgr0 = "^(" + C + ")?" + V + C; // [C]VC... is m>0 + var meq1 = "^(" + C + ")?" + V + C + "(" + V + ")?$"; // [C]VC[V] is m=1 + var mgr1 = "^(" + C + ")?" + V + C + V + C; // [C]VCVC... is m>1 + var s_v = "^(" + C + ")?" + v; // vowel in stem + + this.stemWord = function (w) { + var stem; + var suffix; + var firstch; + var origword = w; + + if (w.length < 3) + return w; + + var re; + var re2; + var re3; + var re4; + + firstch = w.substr(0,1); + if (firstch == "y") + w = firstch.toUpperCase() + w.substr(1); + + // Step 1a + re = /^(.+?)(ss|i)es$/; + re2 = /^(.+?)([^s])s$/; + + if (re.test(w)) + w = w.replace(re,"$1$2"); + else if (re2.test(w)) + w = w.replace(re2,"$1$2"); + + // Step 1b + re = /^(.+?)eed$/; + re2 = /^(.+?)(ed|ing)$/; + if (re.test(w)) { + var fp = re.exec(w); + re = new RegExp(mgr0); + if (re.test(fp[1])) { + re = /.$/; + w = w.replace(re,""); + } + } + else if (re2.test(w)) { + var fp = re2.exec(w); + stem = fp[1]; + re2 = new RegExp(s_v); + if (re2.test(stem)) { + w = stem; + re2 = /(at|bl|iz)$/; + re3 = new RegExp("([^aeiouylsz])\\1$"); + re4 = new RegExp("^" + C + v + "[^aeiouwxy]$"); + if (re2.test(w)) + w = w + "e"; + else if (re3.test(w)) { + re = /.$/; + w = w.replace(re,""); + } + else if (re4.test(w)) + w = w + "e"; + } + } + + // Step 1c + re = /^(.+?)y$/; + if (re.test(w)) { + var fp = re.exec(w); + stem = fp[1]; + re = new RegExp(s_v); + if (re.test(stem)) + w = stem + "i"; + } + + // Step 2 + re = /^(.+?)(ational|tional|enci|anci|izer|bli|alli|entli|eli|ousli|ization|ation|ator|alism|iveness|fulness|ousness|aliti|iviti|biliti|logi)$/; + if (re.test(w)) { + var fp = re.exec(w); + stem = fp[1]; + suffix = fp[2]; + re = new RegExp(mgr0); + if (re.test(stem)) + w = stem + step2list[suffix]; + } + + // Step 3 + re = /^(.+?)(icate|ative|alize|iciti|ical|ful|ness)$/; + if (re.test(w)) { + var fp = re.exec(w); + stem = fp[1]; + suffix = fp[2]; + re = new RegExp(mgr0); + if (re.test(stem)) + w = stem + step3list[suffix]; + } + + // Step 4 + re = /^(.+?)(al|ance|ence|er|ic|able|ible|ant|ement|ment|ent|ou|ism|ate|iti|ous|ive|ize)$/; + re2 = /^(.+?)(s|t)(ion)$/; + if (re.test(w)) { + var fp = re.exec(w); + stem = fp[1]; + re = new RegExp(mgr1); + if (re.test(stem)) + w = stem; + } + else if (re2.test(w)) { + var fp = re2.exec(w); + stem = fp[1] + fp[2]; + re2 = new RegExp(mgr1); + if (re2.test(stem)) + w = stem; + } + + // Step 5 + re = /^(.+?)e$/; + if (re.test(w)) { + var fp = re.exec(w); + stem = fp[1]; + re = new RegExp(mgr1); + re2 = new RegExp(meq1); + re3 = new RegExp("^" + C + v + "[^aeiouwxy]$"); + if (re.test(stem) || (re2.test(stem) && !(re3.test(stem)))) + w = stem; + } + re = /ll$/; + re2 = new RegExp(mgr1); + if (re.test(w) && re2.test(w)) { + re = /.$/; + w = w.replace(re,""); + } + + // and turn initial Y back to y + if (firstch == "y") + w = firstch.toLowerCase() + w.substr(1); + return w; + } +} + diff --git a/docs/_build/_static/minus.png b/docs/_build/_static/minus.png new file mode 100644 index 0000000000000000000000000000000000000000..d96755fdaf8bb2214971e0db9c1fd3077d7c419d GIT binary patch literal 90 zcmeAS@N?(olHy`uVBq!ia0vp^+#t*WBp7;*Yy1LIik>cxAr*|t7R?Mi>2?kWtu=nj kDsEF_5m^0CR;1wuP-*O&G^0G}KYk!hp00i_>zopr08q^qX#fBK literal 0 HcmV?d00001 diff --git a/docs/_build/_static/plus.png b/docs/_build/_static/plus.png new file mode 100644 index 0000000000000000000000000000000000000000..7107cec93a979b9a5f64843235a16651d563ce2d GIT binary patch literal 90 zcmeAS@N?(olHy`uVBq!ia0vp^+#t*WBp7;*Yy1LIik>cxAr*|t7R?Mi>2?kWtu>-2 m3q%Vub%g%s<8sJhVPMczOq}xhg9DJoz~JfX=d#Wzp$Pyb1r*Kz literal 0 HcmV?d00001 diff --git a/docs/_build/_static/pygments.css b/docs/_build/_static/pygments.css new file mode 100644 index 0000000..04a4174 --- /dev/null +++ b/docs/_build/_static/pygments.css @@ -0,0 +1,84 @@ +pre { line-height: 125%; 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+ +/** + * Simple result scoring code. + */ +if (typeof Scorer === "undefined") { + var Scorer = { + // Implement the following function to further tweak the score for each result + // The function takes a result array [docname, title, anchor, descr, score, filename] + // and returns the new score. + /* + score: result => { + const [docname, title, anchor, descr, score, filename] = result + return score + }, + */ + + // query matches the full name of an object + objNameMatch: 11, + // or matches in the last dotted part of the object name + objPartialMatch: 6, + // Additive scores depending on the priority of the object + objPrio: { + 0: 15, // used to be importantResults + 1: 5, // used to be objectResults + 2: -5, // used to be unimportantResults + }, + // Used when the priority is not in the mapping. + objPrioDefault: 0, + + // query found in title + title: 15, + partialTitle: 7, + // query found in terms + term: 5, + partialTerm: 2, + }; +} + +const _removeChildren = (element) => { + while (element && element.lastChild) element.removeChild(element.lastChild); +}; + +/** + * See https://developer.mozilla.org/en-US/docs/Web/JavaScript/Guide/Regular_Expressions#escaping + */ +const _escapeRegExp = (string) => + string.replace(/[.*+\-?^${}()|[\]\\]/g, "\\$&"); // $& means the whole matched string + +const _displayItem = (item, searchTerms, highlightTerms) => { + const docBuilder = DOCUMENTATION_OPTIONS.BUILDER; + const docFileSuffix = DOCUMENTATION_OPTIONS.FILE_SUFFIX; + const docLinkSuffix = DOCUMENTATION_OPTIONS.LINK_SUFFIX; + const showSearchSummary = DOCUMENTATION_OPTIONS.SHOW_SEARCH_SUMMARY; + const contentRoot = document.documentElement.dataset.content_root; + + const [docName, title, anchor, descr, score, _filename] = item; + + let listItem = document.createElement("li"); + let requestUrl; + let linkUrl; + if (docBuilder === "dirhtml") { + // dirhtml builder + let dirname = docName + "/"; + if (dirname.match(/\/index\/$/)) + dirname = dirname.substring(0, dirname.length - 6); + else if (dirname === "index/") dirname = ""; + requestUrl = contentRoot + dirname; + linkUrl = requestUrl; + } else { + // normal html builders + requestUrl = contentRoot + docName + docFileSuffix; + linkUrl = docName + docLinkSuffix; + } + let linkEl = listItem.appendChild(document.createElement("a")); + linkEl.href = linkUrl + anchor; + linkEl.dataset.score = score; + linkEl.innerHTML = title; + if (descr) { + listItem.appendChild(document.createElement("span")).innerHTML = + " (" + descr + ")"; + // highlight search terms in the description + if (SPHINX_HIGHLIGHT_ENABLED) // set in sphinx_highlight.js + highlightTerms.forEach((term) => _highlightText(listItem, term, "highlighted")); + } + else if (showSearchSummary) + fetch(requestUrl) + .then((responseData) => responseData.text()) + .then((data) => { + if (data) + listItem.appendChild( + Search.makeSearchSummary(data, searchTerms) + ); + // highlight search terms in the summary + if (SPHINX_HIGHLIGHT_ENABLED) // set in sphinx_highlight.js + highlightTerms.forEach((term) => _highlightText(listItem, term, "highlighted")); + }); + Search.output.appendChild(listItem); +}; +const _finishSearch = (resultCount) => { + Search.stopPulse(); + Search.title.innerText = _("Search Results"); + if (!resultCount) + Search.status.innerText = Documentation.gettext( + "Your search did not match any documents. Please make sure that all words are spelled correctly and that you've selected enough categories." + ); + else + Search.status.innerText = _( + `Search finished, found ${resultCount} page(s) matching the search query.` + ); +}; +const _displayNextItem = ( + results, + resultCount, + searchTerms, + highlightTerms, +) => { + // results left, load the summary and display it + // this is intended to be dynamic (don't sub resultsCount) + if (results.length) { + _displayItem(results.pop(), searchTerms, highlightTerms); + setTimeout( + () => _displayNextItem(results, resultCount, searchTerms, highlightTerms), + 5 + ); + } + // search finished, update title and status message + else _finishSearch(resultCount); +}; + +/** + * Default splitQuery function. Can be overridden in ``sphinx.search`` with a + * custom function per language. + * + * The regular expression works by splitting the string on consecutive characters + * that are not Unicode letters, numbers, underscores, or emoji characters. + * This is the same as ``\W+`` in Python, preserving the surrogate pair area. + */ +if (typeof splitQuery === "undefined") { + var splitQuery = (query) => query + .split(/[^\p{Letter}\p{Number}_\p{Emoji_Presentation}]+/gu) + .filter(term => term) // remove remaining empty strings +} + +/** + * Search Module + */ +const Search = { + _index: null, + _queued_query: null, + _pulse_status: -1, + + htmlToText: (htmlString) => { + const htmlElement = new DOMParser().parseFromString(htmlString, 'text/html'); + htmlElement.querySelectorAll(".headerlink").forEach((el) => { el.remove() }); + const docContent = htmlElement.querySelector('[role="main"]'); + if (docContent !== undefined) return docContent.textContent; + console.warn( + "Content block not found. Sphinx search tries to obtain it via '[role=main]'. Could you check your theme or template." + ); + return ""; + }, + + init: () => { + const query = new URLSearchParams(window.location.search).get("q"); + document + .querySelectorAll('input[name="q"]') + .forEach((el) => (el.value = query)); + if (query) Search.performSearch(query); + }, + + loadIndex: (url) => + (document.body.appendChild(document.createElement("script")).src = url), + + setIndex: (index) => { + Search._index = index; + if (Search._queued_query !== null) { + const query = Search._queued_query; + Search._queued_query = null; + Search.query(query); + } + }, + + hasIndex: () => Search._index !== null, + + deferQuery: (query) => (Search._queued_query = query), + + stopPulse: () => (Search._pulse_status = -1), + + startPulse: () => { + if (Search._pulse_status >= 0) return; + + const pulse = () => { + Search._pulse_status = (Search._pulse_status + 1) % 4; + Search.dots.innerText = ".".repeat(Search._pulse_status); + if (Search._pulse_status >= 0) window.setTimeout(pulse, 500); + }; + pulse(); + }, + + /** + * perform a search for something (or wait until index is loaded) + */ + performSearch: (query) => { + // create the required interface elements + const searchText = document.createElement("h2"); + searchText.textContent = _("Searching"); + const searchSummary = document.createElement("p"); + searchSummary.classList.add("search-summary"); + searchSummary.innerText = ""; + const searchList = document.createElement("ul"); + searchList.classList.add("search"); + + const out = document.getElementById("search-results"); + Search.title = out.appendChild(searchText); + Search.dots = Search.title.appendChild(document.createElement("span")); + Search.status = out.appendChild(searchSummary); + Search.output = out.appendChild(searchList); + + const searchProgress = document.getElementById("search-progress"); + // Some themes don't use the search progress node + if (searchProgress) { + searchProgress.innerText = _("Preparing search..."); + } + Search.startPulse(); + + // index already loaded, the browser was quick! + if (Search.hasIndex()) Search.query(query); + else Search.deferQuery(query); + }, + + /** + * execute search (requires search index to be loaded) + */ + query: (query) => { + const filenames = Search._index.filenames; + const docNames = Search._index.docnames; + const titles = Search._index.titles; + const allTitles = Search._index.alltitles; + const indexEntries = Search._index.indexentries; + + // stem the search terms and add them to the correct list + const stemmer = new Stemmer(); + const searchTerms = new Set(); + const excludedTerms = new Set(); + const highlightTerms = new Set(); + const objectTerms = new Set(splitQuery(query.toLowerCase().trim())); + splitQuery(query.trim()).forEach((queryTerm) => { + const queryTermLower = queryTerm.toLowerCase(); + + // maybe skip this "word" + // stopwords array is from language_data.js + if ( + stopwords.indexOf(queryTermLower) !== -1 || + queryTerm.match(/^\d+$/) + ) + return; + + // stem the word + let word = stemmer.stemWord(queryTermLower); + // select the correct list + if (word[0] === "-") excludedTerms.add(word.substr(1)); + else { + searchTerms.add(word); + highlightTerms.add(queryTermLower); + } + }); + + if (SPHINX_HIGHLIGHT_ENABLED) { // set in sphinx_highlight.js + localStorage.setItem("sphinx_highlight_terms", [...highlightTerms].join(" ")) + } + + // console.debug("SEARCH: searching for:"); + // console.info("required: ", [...searchTerms]); + // console.info("excluded: ", [...excludedTerms]); + + // array of [docname, title, anchor, descr, score, filename] + let results = []; + _removeChildren(document.getElementById("search-progress")); + + const queryLower = query.toLowerCase(); + for (const [title, foundTitles] of Object.entries(allTitles)) { + if (title.toLowerCase().includes(queryLower) && (queryLower.length >= title.length/2)) { + for (const [file, id] of foundTitles) { + let score = Math.round(100 * queryLower.length / title.length) + results.push([ + docNames[file], + titles[file] !== title ? `${titles[file]} > ${title}` : title, + id !== null ? "#" + id : "", + null, + score, + filenames[file], + ]); + } + } + } + + // search for explicit entries in index directives + for (const [entry, foundEntries] of Object.entries(indexEntries)) { + if (entry.includes(queryLower) && (queryLower.length >= entry.length/2)) { + for (const [file, id] of foundEntries) { + let score = Math.round(100 * queryLower.length / entry.length) + results.push([ + docNames[file], + titles[file], + id ? "#" + id : "", + null, + score, + filenames[file], + ]); + } + } + } + + // lookup as object + objectTerms.forEach((term) => + results.push(...Search.performObjectSearch(term, objectTerms)) + ); + + // lookup as search terms in fulltext + results.push(...Search.performTermsSearch(searchTerms, excludedTerms)); + + // let the scorer override scores with a custom scoring function + if (Scorer.score) results.forEach((item) => (item[4] = Scorer.score(item))); + + // now sort the results by score (in opposite order of appearance, since the + // display function below uses pop() to retrieve items) and then + // alphabetically + results.sort((a, b) => { + const leftScore = a[4]; + const rightScore = b[4]; + if (leftScore === rightScore) { + // same score: sort alphabetically + const leftTitle = a[1].toLowerCase(); + const rightTitle = b[1].toLowerCase(); + if (leftTitle === rightTitle) return 0; + return leftTitle > rightTitle ? -1 : 1; // inverted is intentional + } + return leftScore > rightScore ? 1 : -1; + }); + + // remove duplicate search results + // note the reversing of results, so that in the case of duplicates, the highest-scoring entry is kept + let seen = new Set(); + results = results.reverse().reduce((acc, result) => { + let resultStr = result.slice(0, 4).concat([result[5]]).map(v => String(v)).join(','); + if (!seen.has(resultStr)) { + acc.push(result); + seen.add(resultStr); + } + return acc; + }, []); + + results = results.reverse(); + + // for debugging + //Search.lastresults = results.slice(); // a copy + // console.info("search results:", Search.lastresults); + + // print the results + _displayNextItem(results, results.length, searchTerms, highlightTerms); + }, + + /** + * search for object names + */ + performObjectSearch: (object, objectTerms) => { + const filenames = Search._index.filenames; + const docNames = Search._index.docnames; + const objects = Search._index.objects; + const objNames = Search._index.objnames; + const titles = Search._index.titles; + + const results = []; + + const objectSearchCallback = (prefix, match) => { + const name = match[4] + const fullname = (prefix ? prefix + "." : "") + name; + const fullnameLower = fullname.toLowerCase(); + if (fullnameLower.indexOf(object) < 0) return; + + let score = 0; + const parts = fullnameLower.split("."); + + // check for different match types: exact matches of full name or + // "last name" (i.e. last dotted part) + if (fullnameLower === object || parts.slice(-1)[0] === object) + score += Scorer.objNameMatch; + else if (parts.slice(-1)[0].indexOf(object) > -1) + score += Scorer.objPartialMatch; // matches in last name + + const objName = objNames[match[1]][2]; + const title = titles[match[0]]; + + // If more than one term searched for, we require other words to be + // found in the name/title/description + const otherTerms = new Set(objectTerms); + otherTerms.delete(object); + if (otherTerms.size > 0) { + const haystack = `${prefix} ${name} ${objName} ${title}`.toLowerCase(); + if ( + [...otherTerms].some((otherTerm) => haystack.indexOf(otherTerm) < 0) + ) + return; + } + + let anchor = match[3]; + if (anchor === "") anchor = fullname; + else if (anchor === "-") anchor = objNames[match[1]][1] + "-" + fullname; + + const descr = objName + _(", in ") + title; + + // add custom score for some objects according to scorer + if (Scorer.objPrio.hasOwnProperty(match[2])) + score += Scorer.objPrio[match[2]]; + else score += Scorer.objPrioDefault; + + results.push([ + docNames[match[0]], + fullname, + "#" + anchor, + descr, + score, + filenames[match[0]], + ]); + }; + Object.keys(objects).forEach((prefix) => + objects[prefix].forEach((array) => + objectSearchCallback(prefix, array) + ) + ); + return results; + }, + + /** + * search for full-text terms in the index + */ + performTermsSearch: (searchTerms, excludedTerms) => { + // prepare search + const terms = Search._index.terms; + const titleTerms = Search._index.titleterms; + const filenames = Search._index.filenames; + const docNames = Search._index.docnames; + const titles = Search._index.titles; + + const scoreMap = new Map(); + const fileMap = new Map(); + + // perform the search on the required terms + searchTerms.forEach((word) => { + const files = []; + const arr = [ + { files: terms[word], score: Scorer.term }, + { files: titleTerms[word], score: Scorer.title }, + ]; + // add support for partial matches + if (word.length > 2) { + const escapedWord = _escapeRegExp(word); + Object.keys(terms).forEach((term) => { + if (term.match(escapedWord) && !terms[word]) + arr.push({ files: terms[term], score: Scorer.partialTerm }); + }); + Object.keys(titleTerms).forEach((term) => { + if (term.match(escapedWord) && !titleTerms[word]) + arr.push({ files: titleTerms[word], score: Scorer.partialTitle }); + }); + } + + // no match but word was a required one + if (arr.every((record) => record.files === undefined)) return; + + // found search word in contents + arr.forEach((record) => { + if (record.files === undefined) return; + + let recordFiles = record.files; + if (recordFiles.length === undefined) recordFiles = [recordFiles]; + files.push(...recordFiles); + + // set score for the word in each file + recordFiles.forEach((file) => { + if (!scoreMap.has(file)) scoreMap.set(file, {}); + scoreMap.get(file)[word] = record.score; + }); + }); + + // create the mapping + files.forEach((file) => { + if (fileMap.has(file) && fileMap.get(file).indexOf(word) === -1) + fileMap.get(file).push(word); + else fileMap.set(file, [word]); + }); + }); + + // now check if the files don't contain excluded terms + const results = []; + for (const [file, wordList] of fileMap) { + // check if all requirements are matched + + // as search terms with length < 3 are discarded + const filteredTermCount = [...searchTerms].filter( + (term) => term.length > 2 + ).length; + if ( + wordList.length !== searchTerms.size && + wordList.length !== filteredTermCount + ) + continue; + + // ensure that none of the excluded terms is in the search result + if ( + [...excludedTerms].some( + (term) => + terms[term] === file || + titleTerms[term] === file || + (terms[term] || []).includes(file) || + (titleTerms[term] || []).includes(file) + ) + ) + break; + + // select one (max) score for the file. + const score = Math.max(...wordList.map((w) => scoreMap.get(file)[w])); + // add result to the result list + results.push([ + docNames[file], + titles[file], + "", + null, + score, + filenames[file], + ]); + } + return results; + }, + + /** + * helper function to return a node containing the + * search summary for a given text. keywords is a list + * of stemmed words. + */ + makeSearchSummary: (htmlText, keywords) => { + const text = Search.htmlToText(htmlText); + if (text === "") return null; + + const textLower = text.toLowerCase(); + const actualStartPosition = [...keywords] + .map((k) => textLower.indexOf(k.toLowerCase())) + .filter((i) => i > -1) + .slice(-1)[0]; + const startWithContext = Math.max(actualStartPosition - 120, 0); + + const top = startWithContext === 0 ? "" : "..."; + const tail = startWithContext + 240 < text.length ? "..." : ""; + + let summary = document.createElement("p"); + summary.classList.add("context"); + summary.textContent = top + text.substr(startWithContext, 240).trim() + tail; + + return summary; + }, +}; + +_ready(Search.init); diff --git a/docs/_build/_static/sphinx_highlight.js b/docs/_build/_static/sphinx_highlight.js new file mode 100644 index 0000000..8a96c69 --- /dev/null +++ b/docs/_build/_static/sphinx_highlight.js @@ -0,0 +1,154 @@ +/* Highlighting utilities for Sphinx HTML documentation. */ +"use strict"; + +const SPHINX_HIGHLIGHT_ENABLED = true + +/** + * highlight a given string on a node by wrapping it in + * span elements with the given class name. + */ +const _highlight = (node, addItems, text, className) => { + if (node.nodeType === Node.TEXT_NODE) { + const val = node.nodeValue; + const parent = node.parentNode; + const pos = val.toLowerCase().indexOf(text); + if ( + pos >= 0 && + !parent.classList.contains(className) && + !parent.classList.contains("nohighlight") + ) { + let span; + + const closestNode = parent.closest("body, svg, foreignObject"); + const isInSVG = closestNode && closestNode.matches("svg"); + if (isInSVG) { + span = document.createElementNS("http://www.w3.org/2000/svg", "tspan"); + } else { + span = document.createElement("span"); + span.classList.add(className); + } + + span.appendChild(document.createTextNode(val.substr(pos, text.length))); + const rest = document.createTextNode(val.substr(pos + text.length)); + parent.insertBefore( + span, + parent.insertBefore( + rest, + node.nextSibling + ) + ); + node.nodeValue = val.substr(0, pos); + /* There may be more occurrences of search term in this node. So call this + * function recursively on the remaining fragment. + */ + _highlight(rest, addItems, text, className); + + if (isInSVG) { + const rect = document.createElementNS( + "http://www.w3.org/2000/svg", + "rect" + ); + const bbox = parent.getBBox(); + rect.x.baseVal.value = bbox.x; + rect.y.baseVal.value = bbox.y; + rect.width.baseVal.value = bbox.width; + rect.height.baseVal.value = bbox.height; + rect.setAttribute("class", className); + addItems.push({ parent: parent, target: rect }); + } + } + } else if (node.matches && !node.matches("button, select, textarea")) { + node.childNodes.forEach((el) => _highlight(el, addItems, text, className)); + } +}; +const _highlightText = (thisNode, text, className) => { + let addItems = []; + _highlight(thisNode, addItems, text, className); + addItems.forEach((obj) => + obj.parent.insertAdjacentElement("beforebegin", obj.target) + ); +}; + +/** + * Small JavaScript module for the documentation. + */ +const SphinxHighlight = { + + /** + * highlight the search words provided in localstorage in the text + */ + highlightSearchWords: () => { + if (!SPHINX_HIGHLIGHT_ENABLED) return; // bail if no highlight + + // get and clear terms from localstorage + const url = new URL(window.location); + const highlight = + localStorage.getItem("sphinx_highlight_terms") + || url.searchParams.get("highlight") + || ""; + localStorage.removeItem("sphinx_highlight_terms") + url.searchParams.delete("highlight"); + window.history.replaceState({}, "", url); + + // get individual terms from highlight string + const terms = highlight.toLowerCase().split(/\s+/).filter(x => x); + if (terms.length === 0) return; // nothing to do + + // There should never be more than one element matching "div.body" + const divBody = document.querySelectorAll("div.body"); + const body = divBody.length ? divBody[0] : document.querySelector("body"); + window.setTimeout(() => { + terms.forEach((term) => _highlightText(body, term, "highlighted")); + }, 10); + + const searchBox = document.getElementById("searchbox"); + if (searchBox === null) return; + searchBox.appendChild( + document + .createRange() + .createContextualFragment( + '

    ' + + '' + + _("Hide Search Matches") + + "

    " + ) + ); + }, + + /** + * helper function to hide the search marks again + */ + hideSearchWords: () => { + document + .querySelectorAll("#searchbox .highlight-link") + .forEach((el) => el.remove()); + document + .querySelectorAll("span.highlighted") + .forEach((el) => el.classList.remove("highlighted")); + localStorage.removeItem("sphinx_highlight_terms") + }, + + initEscapeListener: () => { + // only install a listener if it is really needed + if (!DOCUMENTATION_OPTIONS.ENABLE_SEARCH_SHORTCUTS) return; + + document.addEventListener("keydown", (event) => { + // bail for input elements + if (BLACKLISTED_KEY_CONTROL_ELEMENTS.has(document.activeElement.tagName)) return; + // bail with special keys + if (event.shiftKey || event.altKey || event.ctrlKey || event.metaKey) return; + if (DOCUMENTATION_OPTIONS.ENABLE_SEARCH_SHORTCUTS && (event.key === "Escape")) { + SphinxHighlight.hideSearchWords(); + event.preventDefault(); + } + }); + }, +}; + +_ready(() => { + /* Do not call highlightSearchWords() when we are on the search page. + * It will highlight words from the *previous* search query. + */ + if (typeof Search === "undefined") SphinxHighlight.highlightSearchWords(); + SphinxHighlight.initEscapeListener(); +}); diff --git a/docs/_build/cds.html b/docs/_build/cds.html new file mode 100644 index 0000000..8b93f22 --- /dev/null +++ b/docs/_build/cds.html @@ -0,0 +1,272 @@ + + + + + + + + Coding sequence tiling library — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    Coding sequence tiling library¶

    +
    +

    Tiling sorting screen tutorial¶

    +

    Tiling screen that tiles gRNA densely across locus or multiple loci, selected based on FACS signal quantiles.

    + + + + + + + + + +
    Library designTiling (gRNAs tile each locus densely)
    tiling library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design



    +
    +

    Example workflow¶

    +
    screen_id=my_sorting_tiling_screen
+
+# 1. Count gRNA & reporter
+bean-count-samples \
+--input tests/data/sample_list_tiling.csv          `# Contains fastq file path; see test file for example.`\
+-b A                                               `# Base A is edited (into G)` \
+-f tests/data/test_guide_info_tiling_chrom.csv     `# Contains gRNA metadata; see test file for example.`\
+-o ./                                              `# Output directory` \
+-r                                                 `# Quantify reporter edits` \
+-n ${screen_id}                                       `# ID of the screen` \
+--tiling
+
+# 2. QC samples & guides
+bean-qc \
+  bean_count_${screen_id}.h5ad           `# Input ReporterScreen .h5ad file path` \
+  -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \
+  -r qc_report_${screen_id}              `# Prefix for QC report` \
+
+# 3. Filter & translate alleles
+bean-filter ./bean_count_${screen_id}_masked.h5ad \
+-o ./bean_count_${screen_id}_alleleFiltered \
+--filter-target-basechange                             `# Filter based on intended base changes. If -b A was provided in bean-count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\
+--filter-window --edit-start-pos 0 --edit-end-pos 19   `# Filter based on editing window in spacer position within reporter.`\
+--filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \
+--translate --translate-genes-list tests/data/gene_symbols.txt
+
+# 4. Quantify variant effect
+bean-run sorting tiling \
+    ./bean_count_${screen_id}_alleleFiltered.h5ad \
+    -o tests/test_res/var/ \
+    --fit-negctrl \
+    --scale-by-acc \
+    --accessibility-col accessibility
+
    +
    +

    See more details below.

    +
    +
    +

    1. Count gRNA & reporter (bean count-samples)¶

    +
    screen_id=my_sorting_tiling_screen
+
+bean-count-samples \
+--input tests/data/sample_list_tiling.csv          `# Contains fastq file path; see test file for example.`\
+-b A                                               `# Base A is edited (into G)` \
+-f tests/data/test_guide_info_tiling_chrom.csv     `# Contains gRNA metadata; see test file for example.`\
+-o ./                                              `# Output directory` \
+-r                                                 `# Quantify reporter edits` \
+-n ${screen_id}                                       `# ID of the screen` \
+--tiling
+
    +
    +

    Make sure you follow the input file format for seamless downstream steps. This will produce ./bean_count_${screen_id}.h5ad.

    +
    +
    +

    2. QC (bean qc)¶

    +

    Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the input file guideline, but you can change the parameters with the full argument list of ``bean-qc` <../../README#bean-qc-qc-of-reporter-screen-data>`_. (Common factors you may want to tweak is --ctrl-cond=bulk and --lfc-conds=top,bot if you have different sample condition labels.)

    +
    bean-qc \
+  bean_count_${screen_id}.h5ad           `# Input ReporterScreen .h5ad file path` \
+  -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \
+  -r qc_report_${screen_id}              `# Prefix for QC report` \
+  [--tiling]                          `# Not required if you have passed --tiling in counting step`
+
    +
    +

    If the data does not include reporter editing data, you can provide --no-editing flag to omit the editing rate QC.

    +
    +
    +

    3. Filter alleles (bean filter)¶

    +

    As tiling library doesn’t have designated per-gRNA target variant, any base edit observed in reporter may be the candidate variant, while having too many variants with very low editing rate significantly decreases the power. Variants are filtered based on multiple criteria in bean-fitler.

    +

    If the screen targets coding sequence, it’s beneficial to translate edits into coding varaints whenever possible for better power. For translation, provide --translate and one of the following:

    +
    [ --translate-gene-name GENE_SYMBOL OR
+  --translate-genes-list path_to_gene_names_file.txt OR
+  --translate-fasta gene_exon.fa, OR
+  --translate-fastas-csv gene_exon_fas.csv]
+
    +
    +

    where path_to_gene_names_file.txt has one gene symbol per line, and gene symbol uses its MANE transcript (hg38) coordinates of exons. In order to use other reference versions or transcript ID, you’ll need to feed in fasta file. See detailed formatting of fasta file here.

    +

    Example allele filtering given we’re translating based on MANE transcript exons of multiple gene symbols:

    +
    bean-filter ./bean_count_${screen_id}_masked.h5ad \
+-o ./bean_count_${screen_id}_alleleFiltered \
+--filter-target-basechange                             `# Filter based on intended base changes. If -b A was provided in bean-count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\
+--filter-window --edit-start-pos 0 --edit-end-pos 19   `# Filter based on editing window in spacer position within reporter.`\
+--filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \
+--translate --translate-genes-list tests/data/gene_symbols.txt
+
    +
    +

    Ouptut file `` shows number of alleles per guide and number of guides per variant, where we want high enough values for the latter. See the typical output for dataset with good editing coverage & filtering result here.

    +
    +
    +

    4. Quantify variant effect (bean run)¶

    +

    By default, bean-run [sorting,survival] tiling uses most filtered allele counts table for variant identification and quantification of their effects. **Check allele filtering output** and choose alternative filtered allele counts table if necessary.

    +

    bean-run can take 3 run options to quantify editing rate:

    +
      +
    1. +
      From reporter + accessibility

      1-1. If your gRNA metadata table (tests/data/test_guide_info.csv above) included per-gRNA accessibility score,

      +
      +
      +
         bean-run sorting tiling \
+   ./bean_count_${screen_id}_alleleFiltered.h5ad \
+   -o tests/test_res/var/ \
+   --fit-negctrl \
+   --scale-by-acc \
+   --accessibility-col accessibility
+
+1-2. If your gRNA metadata table (\ ``tests/data/test_guide_info.csv`` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal,
+
      +
      +
      bean-run sorting tiling \
+./bean_count_${screen_id}_alleleFiltered.h5ad \
+-o tests/test_res/var/ \
+--fit-negctrl \
+--scale-by-acc \
+--accessibility-bw accessibility.bw
+
      +
      +
      2. +

    2. From reporter

      +
      bean-run sorting tiling \
+./bean_count_${screen_id}_alleleFiltered.h5ad \
+-o tests/test_res/var/ \
+--fit-negctrl
+
      +
      +
      3. +
    3. +
      No reporter information, assume the same editing efficiency of all gRNAs.

      Use this option if your data don’t have editing rate information.

      +
      +
      +
      bean-run sorting tiling \
+./bean_count_${screen_id}_alleleFiltered.h5ad \
+-o tests/test_res/var/ \
+--fit-negctrl \
+--uniform-edit
+
      +
      +
      4. +
    +

    See Subcommands for the full details.

    +
    +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/commands/count.html b/docs/_build/commands/count.html new file mode 100644 index 0000000..4b364ed --- /dev/null +++ b/docs/_build/commands/count.html @@ -0,0 +1,142 @@ + + + + + + + + bean count[-samples]: Count (reporter) screen data — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    bean count[-samples]: Count (reporter) screen data¶

    +

    bean count-samples (or bean count for a single sample) maps guide into guide counts, allowing for base transition in spacer sequence. When the matched reporter information is provided, it can count the target site edits and alleles produced by each guide. Mapping is efficiently done based on CRISPResso2 modified for base-edit-aware mapping.

    +
    bean count-samples \
+  --input sample_list.csv   `# sample with lines 'R1_filepath,R2_filepath,sample_name\n'` \
+  -b A                      `# base that is being edited (A/G)` \
+  -f sgRNA_info_table.csv   `# sgRNA information` \
+  -o .                      `# output directory` \
+  -r                        `# read edit/allele information from reporter` \
+  -t 12                     `# number of threads` \
+  --name my_sorting_screen  `# name of this sample run` \
+
    +
    +
    bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r
+
    +
    +

    By default, bean count[-samples] assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure.

    +
    +

    Read structuren

    +
    +

    See full detail below.

    +
    +
    +

    Input file format¶

    +

    See Input file format for input file formats.

    +
    +
    +

    Output file format¶

    +

    count or count-samples produces .h5ad and .xlsx file with guide and per-guide allele counts.

    +
      +

    • .h5ad: This output file follows annotated matrix format compatible with AnnData and is based on Screen object in [purturb_tools](https://github.com/pinellolab/perturb-tools). See Data Structure section for more information.

      • +

    • .xlsx: This output file contains .guides, .samples, .X[_bcmatch,_edits]. (allele_tables are often too large to write into an Excel!)

      • +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/commands/create-screen.html b/docs/_build/commands/create-screen.html new file mode 100644 index 0000000..8ea5daf --- /dev/null +++ b/docs/_build/commands/create-screen.html @@ -0,0 +1,123 @@ + + + + + + + + bean create-screen: Create ReporterScreen object from flat files — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    bean create-screen: Create ReporterScreen object from flat files¶

    +
    bean create-screen gRNA_library.csv sample_list.csv gRNA_counts_table.csv
+
    +
    +
    +

    Input¶

    +
      +

    • gRNA_library.csv

      • +

    • sample_list.csv

      • +

    • gRNA_counts_table.csv: Table with gRNA ID in the first column and sample IDs as the column names (first row) +gRNA_library.csv and sample_list.csv should be formatted as Input file format.

      • +
    +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/commands/filter.html b/docs/_build/commands/filter.html new file mode 100644 index 0000000..22d1c8e --- /dev/null +++ b/docs/_build/commands/filter.html @@ -0,0 +1,154 @@ + + + + + + + + filter: Filtering (and optionally translating) alleles — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    filter: Filtering (and optionally translating) alleles¶

    +

    As tiling mode of bean run accounts for any robustly observed alleles, bean filter filters for such alleles.

    +
    bean filter my_sorting_screen_masked.h5ad \
+-o my_sorting_screen_filtered.h5ad  `# Output file path` \
+
    +
    +
    +
    +

    Output¶

    +

    Above command produces

    +
      +

    • my_sorting_screen_filtered.h5ad with filtered alleles stored in .uns,

      • +

    • my_sorting_screen_filtered.filtered_allele_stats.pdf, and my_sorting_screen_filtered.filter_log.txt that report allele count stats in each filtering step.

      • +
    +

    You may want to adjust the flitering parameters to obtain optimal balance between # guides per variant & # variants that are scored. See example outputs of filtering step here.

    +
    +
    +

    Translating alleles¶

    +

    If you want to obtain amino acid level variant for coding sequence tiling screens, provide coding sequence positions which variants occuring within the coding sequence will be translated. This is optional, but **highly recommended* to increase per-(coding)variant support.*

    +

    Allele translation

    +
    bean filter my_sorting_screen.h5ad \
+-o my_sorting_screen_masked.h5ad \
+--translate   `# Translate coding variants` \
+[ --translate-gene-name GENE_SYMBOL OR
+  --translate-genes-list path_to_gene_names_file.txt OR
+  --translate-fasta gene_exon.fa, OR
+  --translate-fastas-csv gene_exon_fas.csv]
+
    +
    +
      +

    • When library covers a single gene, do either of the following:

      +
        +

      1. Feed --translate-gene-name GENE_SYMBOL if your genomic_pos column of sgRNA_info_tbl is compatible with MANE transcript‘s reference genome. (Per 10/23/2023, GRCh38). This will automatically load the exon positions based on MANE transcript annotation.

        2. +

      2. To use your custom coding sequence and exon positions, feed --translate-fasta gene_exon.fa argument where gene_exon.fa is the FASTA file with entries of exons. See full details here.

        3. +
      +
      • +

    • When library covers multiple genes, do either of the following:

      +
        +

      1. Feed --translate-genes-list path_to_gene_names_file.txt where path_to_gene_names_file.txt is file with one gene symbol per line.

        2. +

      2. Feed --translate-fastas-csv gene_exon_fas.csv where gene_exon_fas.csv is the csv file with lines gene_id,gene_exon_fasta_path without header. Each FASTA file in gene_exon_fasta_path is formatted as the single-gene FASTA file.

        3. +
      +
      • +

    • Translation will keep the variants outside the coding sequence as nucleotide-level variants, while aggregating variants leading to the same coding sequence variants.

      • +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/commands/input.html b/docs/_build/commands/input.html new file mode 100644 index 0000000..f0b9c24 --- /dev/null +++ b/docs/_build/commands/input.html @@ -0,0 +1,160 @@ + + + + + + + + sgRNA_info_table.csv — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +

    This document describes the input files of bean count-samples.

    +
    +

    sgRNA_info_table.csv¶

    +

    File should contain following columns.

    +
      +

    • name: gRNA ID column

      • +

    • sequence: gRNA sequence

      • +

    • barcode: R2 barcode to help match reporter to gRNA, written in the sense direction (as in R1)

      • +

    • In order to use accessibility in the variant effect quantification, provide accessibility information in one of two options. (For non-targeting guides, provide NA values (empty cell).)

      +
        +

      • Option 1: chrom & genomic_pos: Chromosome (ex. chr19) and genomic position of guide sequence. You will have to provide the path to the bigwig file with matching reference version in bean run.

        • +

      • Option 2: accessibility_signal: ATAC-seq signal value of the target loci of each guide.

        • +
      +
      • +

    • For variant library (gRNAs are designed to target specific variants and ignores bystander edits)

      +
        +

      • target: This column denotes which target variant/element of each gRNA. This is not used in bean count[-samples] but required to run bean run in later steps.

        • +

      • target_group: If negative/positive control gRNA will be considered in bean qc and/or bean run, specify as “NegCtrlâ€/â€PosCtrl†in this column.

        • +

      • target_pos: If --match_target_pos flag is used, input file needs target_pos which specifies 0-based relative position of targeted base within Reporter sequence.

        • +
      +
      • +

    • For tiling library (gRNAs tile coding / noncoding sequences)

      +
        +

      • strand: Specifies gRNA strand information relative to the reference genome.

        • +

      • chrom: Chromosome of gRNA targeted locus.

        • +

      • start_pos: gRNA starting position in the genome. Required when you provide strand column. Should specify the smaller coordinate value among start and end position regardless of gRNA strandedness.

        • +
      +
      • +
    +

    Also see examples for variant library and tiling library.

    +
    +
    +

    sample_list.csv¶

    +

    File should contain following columns with header.

    +
      +

    • R1_filepath: Path to read 1 .fastq[.gz] file

      • +

    • R2_filepath: Path to read 1 .fastq[.gz] file

      • +

    • sample_id: ID of sequencing sample

      • +

    • replicate: Replicate # of this sample (Should NOT contain .)

      • +

    • condition: Name of the sorting bin (ex. top, bot), or label of timepoint (ex. D5, D18)

      • +
    +

    For FACS sorting screens:

    +
      +

    • upper_quantile: FACS sorting upper quantile

      • +

    • lower_quantile: FACS sorting lower quantile

      • +
    +

    For proliferation / survival screens:

    +
      +

    • time: Numeric time following the base editing of each sample.

      • +
    +

    Also see examples for FACS sorting screen.

    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/commands/profile.html b/docs/_build/commands/profile.html new file mode 100644 index 0000000..32f78d4 --- /dev/null +++ b/docs/_build/commands/profile.html @@ -0,0 +1,119 @@ + + + + + + + + bean profile: Profile editing patterns — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    bean profile: Profile editing patterns¶

    +
    bean profile my_sorting_screen.h5ad -o output_prefix `# Prefix for editing profile report`
+
    +
    +
    +
    +

    Output¶

    +

    Above command produces prefix_editing_preference.[html,ipynb] as editing preferences (see example).

    +

    Allele translation

    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/commands/qc.html b/docs/_build/commands/qc.html new file mode 100644 index 0000000..5526162 --- /dev/null +++ b/docs/_build/commands/qc.html @@ -0,0 +1,282 @@ + + + + + + + + bean qc: QC of reporter screen data — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    bean qc: QC of reporter screen data¶

    +
    bean qc \
+  my_sorting_screen.h5ad             `# Input ReporterScreen .h5ad file path` \
+  -o my_sorting_screen_masked.h5ad   `# Output ReporterScreen .h5ad file path` \
+  -r qc_report_my_sorting_screen     `# Prefix for QC report` \
+  --ctrl-cond presort                `# "condition" column in the control sample before selection. Mean gRNA editing rates in these samples are reported. ` \
+# Inspect the output qc_report_my_sorting_screen.html to tweak QC threshold
+
+bean qc \
+  my_sorting_screen.h5ad              \
+  -o my_sorting_screen_masked.h5ad    \
+  -r qc_report_my_sorting_screen      \
+  #[--count-correlation-thres 0.7 ...]\
+  -b
+
    +
    +

    bean qc supports following quality control and masks samples with low quality. Specifically:

    +

    Allele translation

    +
      +

    • Plots guide coverage and the uniformity of coverage

      • +

    • Guide count correlation between samples

      • +

    • Log fold change correlation when positive controls are provided

      • +

    • Plots editing rate distribution

      • +

    • Identify samples with low guide coverage/guide count correlation/editing rate and mask the sample in bdata.samples.mask

      • +

    • Identify outlier guides to filter out

      • +
    +
    +
    +

    Output¶

    +

    Above command produces

    +
      +

    • my_sorting_screen_masked.h5ad without problematic replicate and guides and with sample masks, and

      • +

    • qc_report_my_sorting_screen.[html,ipynb] as QC report.
       +##### Optional arguments:

      • +

    • -o OUT_SCREEN_PATH, --out-screen-path OUT_SCREEN_PATH

      +
      Path where quality-filtered ReporterScreen object to be written to
+
      +
      +
      • +

    • -r OUT_REPORT_PREFIX, --out-report-prefix OUT_REPORT_PREFIX

      +
      Output prefix of qc report (prefix.html, prefix.ipynb)
+
      +
      +
      • +
    +
    +

    QC thresholds:¶

    +
      +

    • --count-correlation-thres COUNT_CORRELATION_THRES

      +
      Correlation threshold to mask out.
+
      +
      +
      • +

    • --edit-rate-thres EDIT_RATE_THRES

      +
      Mean editing rate threshold per sample to mask out.
+
      +
      +
      • +

    • --lfc-thres LFC_THRES

      +
      Positive guides' correlation threshold to filter out.
+
      +
      +
      • +
    +
    +
    +

    Run options:¶

    +
      +

    • -b, --remove-bad-replicates

      +
      Remove replicates with at least two of its samples meet the QC threshold (bean run does not support having only one sorting bin sample for a replicate).
+
      +
      +
      • +

    • -i, --ignore-missing-samples

      +
      If the flag is not provided, if the ReporterScreen object does not contain all condiitons for
+each replicate, make fake empty samples. If the flag is provided, don't add dummy samples.
+
      +
      +
      • +

    • --no-editing Ignore QC about editing. Can be used for QC of other editing modalities.

      • +

    • --dont-recalculate-edits

      +
      When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from
+ReporterScreen.uns['allele_count'].
+
      +
      +
      • +
    +
    +
    +

    Input .h5ad formatting:¶

    +

    Note that these arguements will change the way the QC metrics are calculated for guides, samples, or replicates.

    +
      +

    • --tiling TILING Specify that the guide library is tiling library without ‘n guides per target’ design

      • +

    • --replicate-label REPLICATE_LABEL

      +
      Label of column in `bdata.samples` that describes replicate ID.
+
      +
      +
      • +

    • --sample-covariates SAMPLE_COVARIATES

      +
      Comma-separated list of column names in `bdata.samples` that describes non-selective
+experimental condition. (drug treatment, etc.)
+
      +
      +
      • +

    • --condition-label CONDITION_LABEL

      +
      Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time,
+etc.)
+
      +
      +

      ###### Editing rate calculation

      +
        +

      • --control-condition CTRL_COND

        +
        Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate
+to be calculated. Default is `None`, which considers all samples.
+
        +
        +
        • +

      • --rel-pos-is-reporter

        +
        Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If
+`False`, those are relative to spacer position.
+
        +
        +

        Editing rate is calculated with following parameters in

        +
          +

        • Variant screens:

          +
            +

          • --target-pos-col TARGET_POS_COL

            +
            Target position column in `bdata.guides` specifying target edit position in reporter
+
            +
            +
            • +
          +
          • +

        • tiling screens:

          +
            +

          • --edit-start-pos EDIT_START_POS

            +
            Edit start position to quantify editing rate on, 0-based inclusive.
+
            +
            +
            • +

          • --edit-end-pos EDIT_END_POS

            +
            Edit end position to quantify editing rate on, 0-based exclusive.
+
            +
            +

            ###### LFC of positive controls

            +
            • +
          +
          • +
        +
        • +

      • --posctrl-col POSCTRL_COL

        +
        Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all
+gRNAs, feed empty string ''.
+
        +
        +
        • +

      • --posctrl-val POSCTRL_VAL

        +
        Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the
+positive control in calculating log fold change.
+
        +
        +
        • +

      • --lfc-conds LFC_CONDS

        +
        Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated
+between, delimited by comma
+
        +
        +
        • +
      +
      • +
    +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/commands/run.html b/docs/_build/commands/run.html new file mode 100644 index 0000000..4711982 --- /dev/null +++ b/docs/_build/commands/run.html @@ -0,0 +1,201 @@ + + + + + + + + bean run: Quantify variant effects — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    bean run: Quantify variant effects¶

    +

    BEAN uses Bayesian network to incorporate gRNA editing outcome to provide posterior estimate of variant phenotype. The Bayesian network reflects data generation process. Briefly,

    +
      +

    1. Cellular phenotype (either for cells are sorted upon for sorting screen, or log(proliferation rate)) is modeled as the Gaussian mixture distribution of wild-type phenotype and variant phenotype.

      2. +

    2. The weight of the mixture components are inferred from the reporter editing outcome and the chromatin accessibility of the loci.

      3. +

    3. Cells with each gRNA, formulated as the mixture distribution, is sorted by the phenotypic quantile to produce the gRNA counts.

      4. +
    +

    For the full detail, see the method section of the BEAN manuscript.

    +

    model

    +



    +
    +
    +

    Usage example¶

    +
    bean run sorting[survival] variant[tiling] my_sorting_screen_filtered.h5ad \
+[--uniform-edit, --scale-by-acc [--acc-bw-path accessibility_signal.bw, --acc-col accessibility]] \
+-o output_prefix/ \
+--fit-negctrl
+
    +
    +

    See full list of parameters below.

    +
    +
    +

    Input¶

    +

    my_sorting_screen_filtered.h5ad can be produced by one of the following:

    +
      +

    1. ``bean count-samples` <(#bean-count-samples-count-reporter-screen-data>`_) when you have raw .fastq file

      2. +

    2. (Limited to bean run variant mode) bean create-screen when you have flat .csv tables of gRNA metadata table, sample metadata table, gRNA counts table (# guides x # samples), and optionally # edits table. +.. code-block:: bash

      +
      +

      bean create-screen gRNA_info_table.csv sample_info_table.csv gRNA_counts_table.csv [–edits edit_counts_table.csv -o output.h5ad]

      +
      +
        +

      • gRNA_info_table.csv should have following columns.

        +
          +

        • name: gRNA ID column

          • +

        • target: This column denotes which target variant/element of each gRNA.

          • +

        • target_group [Optional]: If negative control gRNA will be used, specify as “NegCtrl†in this column.

          • +
        +
        • +

      • sample_info_table.csv should have following columns.

        +
          +

        • sample_id: ID of sequencing sample

          • +

        • replicate: Replicate # of this sample

          • +

        • bin: Name of the sorting bin

          • +

        • upper_quantile: FACS sorting upper quantile

          • +

        • lower_quantile: FACS sorting lower quantile

          • +
        +
        • +

      • gRNA_counts_table.csv should be formatted as follows.

        +
          +

        • Columns include one of sample_id columns in sample_info_table.csv file.

          • +

        • 1st row (row index) follows name (gRNA ID) in gRNA_info_table.csv file.

          • +
        +
        • +
      +
      3. +

    3. You can manually create the AnnData object with more annotations including allele counts: see API tutorial for full detail.

      4. +
    +
    +
    +

    Output¶

    +

    model

    +

    Above command produces

    +
      +

    • output_prefix/bean_element_result.[model_type].csv with following columns:

      +
        +

      • Estimated variant effect sizes

        +
          +

        • mu (Effect size): Mean of variant phenotype, given the wild type has standard normal phenotype distribution of mu = 0, sd = 1.

          • +

        • mu_sd: Mean of variant phenotype mu is modeled as normal distribution. The column shows fitted standard deviation of mu that quantify the uncertainty of the variant effect.

          • +

        • mu_z: z-score of mu

          • +

        • sd: Standard deviation of variant phenotype, given the wild type has standard normal phenotype distribution of mu = 0, sd = 1.

          • +

        • CI[0.025, 0.975]: Credible interval of mu

          • +

        • When negative control is provided, above columns with _adj suffix are provided, which are the corresponding values adjusted for negative control.

          • +
        +
        • +

      • Metrics on per-variant evidence provided in input (provided in tiling mode)

        +
          +

        • effective_edit_rate: Sum of per-variant editing rates over all alleles observed in the input. Allele-level editing rate is divided by the number of variants observed in the allele prior to summing up.

          • +

        • n_guides: # of guides covering the variant.

          • +

        • n_coocc: # of cooccurring variants with a given variant in any alleles observed in the input.

          • +
        +
        • +
      +
      • +

    • output_prefix/bean_sgRNA_result.[model_type].csv:

      +
        +

      • edit_rate: Estimated editing rate at the target loci.

        • +
      +
      • +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/count.html b/docs/_build/count.html new file mode 100644 index 0000000..2aa8294 --- /dev/null +++ b/docs/_build/count.html @@ -0,0 +1,299 @@ + + + + + + + + bean count — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    bean count¶

    +
    +

    bean count[-samples]: Count (reporter) screen data¶

    +

    bean count-samples (or bean count for a single sample) maps guide into guide counts, allowing for base transition in spacer sequence. When the matched reporter information is provided, it can count the target site edits and alleles produced by each guide. Mapping is efficiently done based on CRISPResso2 modified for base-edit-aware mapping.

    +
    bean count-samples \
+  --input sample_list.csv   `# sample with lines 'R1_filepath,R2_filepath,sample_name\n'` \
+  -b A                      `# base that is being edited (A/G)` \
+  -f sgRNA_info_table.csv   `# sgRNA information` \
+  -o .                      `# output directory` \
+  -r                        `# read edit/allele information from reporter` \
+  -t 12                     `# number of threads` \
+  --name my_sorting_screen  `# name of this sample run` \
+
    +
    +
    bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r
+
    +
    +

    By default, bean count[-samples] assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure.

    +
    +

    Read structuren

    +
    +

    See full detail below.

    +
    +
    +

    Input file format¶

    +

    See Input file format for input file formats.

    +
    +
    +

    Output file format¶

    +

    count or count-samples produces .h5ad and .xlsx file with guide and per-guide allele counts.

    +
      +

    • .h5ad: This output file follows annotated matrix format compatible with AnnData and is based on Screen object in [purturb_tools](https://github.com/pinellolab/perturb-tools). See Data Structure section for more information.

      • +

    • .xlsx: This output file contains .guides, .samples, .X[_bcmatch,_edits]. (allele_tables are often too large to write into an Excel!)

      • +
    +
    +
    +

    Full parameters¶

    +
    usage: bean count [-h] --R1 R1 --R2 R2 -b EDITED_BASE -f SGRNA_FILENAME
+                  [--guide-start-seq GUIDE_START_SEQ]
+                  [--guide-end-seq GUIDE_END_SEQ]
+                  [--barcode-start-seq BARCODE_START_SEQ] [-r]
+                  [-q MIN_AVERAGE_READ_QUALITY] [-s MIN_SINGLE_BP_QUALITY]
+                  [-n NAME] [-o OUTPUT_FOLDER] [-l REPORTER_LENGTH]
+                  [--keep-intermediate] [--qstart-R1 QSTART_R1]
+                  [--qend-R1 QEND_R1] [--qstart-R2 QSTART_R2]
+                  [--qend-R2 QEND_R2] [--gstart-reporter GSTART_REPORTER]
+                  [--match-target-pos] [--target-pos-col TARGET_POS_COL]
+                  [--guide-bc GUIDE_BC] [--guide-bc-len GUIDE_BC_LEN]
+                  [--offset] [--align-fasta ALIGN_FASTA] [--string-allele]
+                  [-g] [-m] [--tiling]
+
    +
    +
    +

    Named Arguments¶

    +
    +
    --R1
    +

    FASTQ file path for read 1

    +
    +
    --R2
    +

    FASTQ file path for read 2.

    +
    +
    -b, --edited-base
    +

    For base editors, the base that should be ignored when matching the gRNA sequence

    +
    +
    -f, --sgRNA-filename
    +

    sgRNA description file. The format requires three columns: name, sequence, barcode [ reporter [,strand, target_pos], [start_pos, offset] ].

    +
    +
    --guide-start-seq
    +

    Guide starts after this sequence in R1

    +

    Default: “â€

    +
    +
    --guide-end-seq
    +

    Guide starts after this sequence in R1

    +

    Default: “â€

    +
    +
    --barcode-start-seq
    +

    Barcode + reporter starts after this sequence in R2, denoted as the sense direction (the same sequence direction as R1).

    +

    Default: “â€

    +
    +
    -r, --count-reporter
    +

    Count reporter edits.

    +

    Default: False

    +
    +
    -q, --min-average-read-quality
    +

    Minimum average quality score (phred33) to keep a read

    +

    Default: 30

    +
    +
    -s, --min-single-bp-quality
    +

    Minimum single bp score (phred33) to keep a read

    +

    Default: 0

    +
    +
    -n, --name
    +

    Output name

    +

    Default: “â€

    +
    +
    -o, --output-folder
    +

    Default: “â€

    +
    +
    -l, --reporter-length
    +

    length of the reporter

    +

    Default: 32

    +
    +
    --keep-intermediate
    +

    Keep all the intermediate files

    +

    Default: False

    +
    +
    --qstart-R1
    +

    Start position of the read when filtering for quality score of the read 1

    +

    Default: 0

    +
    +
    --qend-R1
    +

    End position of the read when filtering for quality score of the read 1

    +

    Default: 47

    +
    +
    --qstart-R2
    +

    Same as qstart_R1, for read 2 fastq file

    +

    Default: 0

    +
    +
    --qend-R2
    +

    Same as qstart_R2, for read 2 fastq file

    +

    Default: 36

    +
    +
    --gstart-reporter
    +

    Start position of the guide sequence in the reporter

    +

    Default: 6

    +
    +
    --match-target-pos
    +

    Count the edit in the exact target position.

    +

    Default: False

    +
    +
    --target-pos-col
    +

    Column name specifying the relative target position within reporter sequence.

    +

    Default: “target_posâ€

    +
    +
    --guide-bc
    +

    Construct has guide barcode

    +

    Default: True

    +
    +
    --guide-bc-len
    +

    Guide barcode sequence length at the beginning of the R2

    +

    Default: 4

    +
    +
    --offset
    +

    Guide file has offest column that will be added to the relative position of reporters.

    +

    Default: False

    +
    +
    --align-fasta
    +

    gRNA is aligned to this sequence to infer the offset. Can be used when the exact offset is not provided.

    +

    Default: “â€

    +
    +
    --string-allele
    +

    Store allele as quality filtered string instead of Allele object

    +

    Default: False

    +
    +
    -g, --count-guide-edits
    +

    count the self editing of guides

    +

    Default: False

    +
    +
    -m, --count-guide-reporter-alleles
    +

    count the matched allele of guide and reporter edit

    +

    Default: False

    +
    +
    --tiling
    +

    Specify that the guide library is tiling library without ‘n guides per target’ design

    +

    Default: False

    +
    +
    +
    +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/count_samples.html b/docs/_build/count_samples.html new file mode 100644 index 0000000..6f7d4af --- /dev/null +++ b/docs/_build/count_samples.html @@ -0,0 +1,320 @@ + + + + + + + + bean count-samples — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    bean count-samples¶

    +
    +

    bean count[-samples]: Count (reporter) screen data¶

    +

    bean count-samples (or bean count for a single sample) maps guide into guide counts, allowing for base transition in spacer sequence. When the matched reporter information is provided, it can count the target site edits and alleles produced by each guide. Mapping is efficiently done based on CRISPResso2 modified for base-edit-aware mapping.

    +
    bean count-samples \
+  --input sample_list.csv   `# sample with lines 'R1_filepath,R2_filepath,sample_name\n'` \
+  -b A                      `# base that is being edited (A/G)` \
+  -f sgRNA_info_table.csv   `# sgRNA information` \
+  -o .                      `# output directory` \
+  -r                        `# read edit/allele information from reporter` \
+  -t 12                     `# number of threads` \
+  --name my_sorting_screen  `# name of this sample run` \
+
    +
    +
    bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r
+
    +
    +

    By default, bean count[-samples] assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure.

    +
    +

    Read structuren

    +
    +

    See full detail below.

    +
    +
    +

    Input file format¶

    +

    See Input file format for input file formats.

    +
    +
    +

    Output file format¶

    +

    count or count-samples produces .h5ad and .xlsx file with guide and per-guide allele counts.

    +
      +

    • .h5ad: This output file follows annotated matrix format compatible with AnnData and is based on Screen object in [purturb_tools](https://github.com/pinellolab/perturb-tools). See Data Structure section for more information.

      • +

    • .xlsx: This output file contains .guides, .samples, .X[_bcmatch,_edits]. (allele_tables are often too large to write into an Excel!)

      • +
    +
    +
    +

    Full parameters¶

    +
    usage: bean count-samples [-h] -i SAMPLE_LIST -b EDITED_BASE -f SGRNA_FILENAME
+                          [--guide-start-seq GUIDE_START_SEQ]
+                          [--guide-end-seq GUIDE_END_SEQ]
+                          [--barcode-start-seq BARCODE_START_SEQ] [-r]
+                          [-q MIN_AVERAGE_READ_QUALITY]
+                          [-s MIN_SINGLE_BP_QUALITY] [-n NAME]
+                          [-o OUTPUT_FOLDER] [-l REPORTER_LENGTH]
+                          [--keep-intermediate] [--qstart-R1 QSTART_R1]
+                          [--qend-R1 QEND_R1] [--qstart-R2 QSTART_R2]
+                          [--qend-R2 QEND_R2]
+                          [--gstart-reporter GSTART_REPORTER]
+                          [--match-target-pos]
+                          [--target-pos-col TARGET_POS_COL]
+                          [--guide-bc GUIDE_BC] [--guide-bc-len GUIDE_BC_LEN]
+                          [--offset] [--align-fasta ALIGN_FASTA]
+                          [--string-allele] [-g] [-m] [--tiling] [-t THREADS]
+                          [--guide-start-seqs-file GUIDE_START_SEQS_FILE]
+                          [--guide-end-seqs-file GUIDE_END_SEQS_FILE]
+                          [--barcode-start-seqs-file BARCODE_START_SEQS_FILE]
+                          [--rerun]
+
    +
    +
    +

    Named Arguments¶

    +
    +
    -i, --sample-list
    +

    List of fastq and sample ids. Formatted as R1_filepath,R2_filepath,sample_id

    +
    +
    -b, --edited-base
    +

    For base editors, the base that should be ignored when matching the gRNA sequence

    +
    +
    -f, --sgRNA-filename
    +

    sgRNA description file. The format requires three columns: name, sequence, barcode [ reporter [,strand, target_pos], [start_pos, offset] ].

    +
    +
    --guide-start-seq
    +

    Guide starts after this sequence in R1

    +

    Default: “â€

    +
    +
    --guide-end-seq
    +

    Guide starts after this sequence in R1

    +

    Default: “â€

    +
    +
    --barcode-start-seq
    +

    Barcode + reporter starts after this sequence in R2, denoted as the sense direction (the same sequence direction as R1).

    +

    Default: “â€

    +
    +
    -r, --count-reporter
    +

    Count reporter edits.

    +

    Default: False

    +
    +
    -q, --min-average-read-quality
    +

    Minimum average quality score (phred33) to keep a read

    +

    Default: 30

    +
    +
    -s, --min-single-bp-quality
    +

    Minimum single bp score (phred33) to keep a read

    +

    Default: 0

    +
    +
    -n, --name
    +

    Output name

    +

    Default: “â€

    +
    +
    -o, --output-folder
    +

    Default: “â€

    +
    +
    -l, --reporter-length
    +

    length of the reporter

    +

    Default: 32

    +
    +
    --keep-intermediate
    +

    Keep all the intermediate files

    +

    Default: False

    +
    +
    --qstart-R1
    +

    Start position of the read when filtering for quality score of the read 1

    +

    Default: 0

    +
    +
    --qend-R1
    +

    End position of the read when filtering for quality score of the read 1

    +

    Default: 47

    +
    +
    --qstart-R2
    +

    Same as qstart_R1, for read 2 fastq file

    +

    Default: 0

    +
    +
    --qend-R2
    +

    Same as qstart_R2, for read 2 fastq file

    +

    Default: 36

    +
    +
    --gstart-reporter
    +

    Start position of the guide sequence in the reporter

    +

    Default: 6

    +
    +
    --match-target-pos
    +

    Count the edit in the exact target position.

    +

    Default: False

    +
    +
    --target-pos-col
    +

    Column name specifying the relative target position within reporter sequence.

    +

    Default: “target_posâ€

    +
    +
    --guide-bc
    +

    Construct has guide barcode

    +

    Default: True

    +
    +
    --guide-bc-len
    +

    Guide barcode sequence length at the beginning of the R2

    +

    Default: 4

    +
    +
    --offset
    +

    Guide file has offest column that will be added to the relative position of reporters.

    +

    Default: False

    +
    +
    --align-fasta
    +

    gRNA is aligned to this sequence to infer the offset. Can be used when the exact offset is not provided.

    +

    Default: “â€

    +
    +
    --string-allele
    +

    Store allele as quality filtered string instead of Allele object

    +

    Default: False

    +
    +
    -g, --count-guide-edits
    +

    count the self editing of guides

    +

    Default: False

    +
    +
    -m, --count-guide-reporter-alleles
    +

    count the matched allele of guide and reporter edit

    +

    Default: False

    +
    +
    --tiling
    +

    Specify that the guide library is tiling library without ‘n guides per target’ design

    +

    Default: False

    +
    +
    -t, --threads
    +

    Number of threads

    +

    Default: 10

    +
    +
    --guide-start-seqs-file
    +

    CSV file path with per-sample guide_start_seq to be used.Formatted as sample_id, guide_start_seq

    +
    +
    --guide-end-seqs-file
    +

    CSV file path with per-sample guide_end_seq to be used.Formatted as sample_id,guide_end_seq

    +
    +
    --barcode-start-seqs-file
    +

    CSV file path with per-sample barcode_start_seq to be used.Formatted as sample_id,guide_end_seq

    +
    +
    --rerun
    +

    Recount each sample

    +

    Default: False

    +
    +
    +
    +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/exon_fa_format.html b/docs/_build/exon_fa_format.html new file mode 100644 index 0000000..36916fe --- /dev/null +++ b/docs/_build/exon_fa_format.html @@ -0,0 +1,122 @@ + + + + + + + + Input .fa file format for bean-filter — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    Input .fa file format for bean-filter¶

    +

    You can provide custom FASTA file with exon sequence entries. Currently only supports positive strand genes.

    +
      +

    • Exon FASTA files can be downloaded from UCSC Genomic sequences / Table Browser: see the instruction video

      • +

    • You can manually format as:

      +
        +

      • Header line has range=chrom:start-end and strand=+/- tag that is parsed.

        • +

      • fasta entry has the sequence of exons, where the first (includes 5’-UTR) and last (includes 3’-UTR) exon sequence has lower-case sequence denoting noncoding sequences.

        • +
      +
      • +

    • See the example .fa here.

      • +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/filter.html b/docs/_build/filter.html new file mode 100644 index 0000000..688d41f --- /dev/null +++ b/docs/_build/filter.html @@ -0,0 +1,274 @@ + + + + + + + + bean filter — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    bean filter¶

    +
    +

    filter: Filtering (and optionally translating) alleles¶

    +

    As tiling mode of bean run accounts for any robustly observed alleles, bean filter filters for such alleles.

    +
    bean filter my_sorting_screen_masked.h5ad \
+-o my_sorting_screen_filtered.h5ad  `# Output file path` \
+
    +
    +
    +
    +

    Output¶

    +

    Above command produces

    +
      +

    • my_sorting_screen_filtered.h5ad with filtered alleles stored in .uns,

      • +

    • my_sorting_screen_filtered.filtered_allele_stats.pdf, and my_sorting_screen_filtered.filter_log.txt that report allele count stats in each filtering step.

      • +
    +

    You may want to adjust the flitering parameters to obtain optimal balance between # guides per variant & # variants that are scored. See example outputs of filtering step here.

    +
    +
    +

    Translating alleles¶

    +

    If you want to obtain amino acid level variant for coding sequence tiling screens, provide coding sequence positions which variants occuring within the coding sequence will be translated. This is optional, but **highly recommended* to increase per-(coding)variant support.*

    +

    Allele translation

    +
    bean filter my_sorting_screen.h5ad \
+-o my_sorting_screen_masked.h5ad \
+--translate   `# Translate coding variants` \
+[ --translate-gene-name GENE_SYMBOL OR
+  --translate-genes-list path_to_gene_names_file.txt OR
+  --translate-fasta gene_exon.fa, OR
+  --translate-fastas-csv gene_exon_fas.csv]
+
    +
    +
      +

    • When library covers a single gene, do either of the following:

      +
        +

      1. Feed --translate-gene-name GENE_SYMBOL if your genomic_pos column of sgRNA_info_tbl is compatible with MANE transcript‘s reference genome. (Per 10/23/2023, GRCh38). This will automatically load the exon positions based on MANE transcript annotation.

        2. +

      2. To use your custom coding sequence and exon positions, feed --translate-fasta gene_exon.fa argument where gene_exon.fa is the FASTA file with entries of exons. See full details here.

        3. +
      +
      • +

    • When library covers multiple genes, do either of the following:

      +
        +

      1. Feed --translate-genes-list path_to_gene_names_file.txt where path_to_gene_names_file.txt is file with one gene symbol per line.

        2. +

      2. Feed --translate-fastas-csv gene_exon_fas.csv where gene_exon_fas.csv is the csv file with lines gene_id,gene_exon_fasta_path without header. Each FASTA file in gene_exon_fasta_path is formatted as the single-gene FASTA file.

        3. +
      +
      • +

    • Translation will keep the variants outside the coding sequence as nucleotide-level variants, while aggregating variants leading to the same coding sequence variants.

      • +
    +
    +
    +

    Full parameters¶

    +

    Filter alleles based on edit position in spacer and frequency across samples.

    +

    +
    usage: bean filter [-h] [--output-prefix OUTPUT_PREFIX]
+                   [--plasmid-path PLASMID_PATH]
+                   [--edit-start-pos EDIT_START_POS]
+                   [--edit-end-pos EDIT_END_POS]
+                   [--jaccard-threshold JACCARD_THRESHOLD] [--filter-window]
+                   [--keep-indels] [--filter-target-basechange] [--translate]
+                   [--translate-fasta TRANSLATE_FASTA]
+                   [--translate-fastas-csv TRANSLATE_FASTAS_CSV]
+                   [--translate-gene TRANSLATE_GENE]
+                   [--translate-genes-list TRANSLATE_GENES_LIST]
+                   [--filter-allele-proportion FILTER_ALLELE_PROPORTION]
+                   [--filter-allele-count FILTER_ALLELE_COUNT]
+                   [--filter-sample-proportion FILTER_SAMPLE_PROPORTION]
+                   [--load-tmp]
+                   bdata_path
+
    +
    +
    +

    Positional Arguments¶

    +
    +
    bdata_path
    +

    Input ReporterScreen file of which allele will be filtered out.

    +
    +
    +
    +
    +

    Named Arguments¶

    +
    +
    --output-prefix, -o
    +

    Output prefix for log and ReporterScreen file with allele assignment

    +
    +
    --plasmid-path, -p
    +

    Plasmid ReporterScreen object path. If provided, alleles are filtered based on if a nucleotide edit is more significantly enriched in sample compared to the plasmid data. Negative control data where no edit is expected can be fed in instead of plasmid library.

    +
    +
    --edit-start-pos, -s
    +

    0-based start posiiton (inclusive) of edit relative to the start of guide spacer.

    +

    Default: 2

    +
    +
    --edit-end-pos, -e
    +

    0-based end position (exclusive) of edit relative to the start of guide spacer.

    +

    Default: 7

    +
    +
    --jaccard-threshold, -j
    +

    Jaccard Index threshold when the alleles are mapped to the most similar alleles. In each filtering step, allele counts of filtered out alleles will be mapped to the most similar allele only if they have Jaccard Index of shared edit higher than this threshold.

    +

    Default: 0.3

    +
    +
    --filter-window, -w
    +

    Only consider edit within window provided by (edit-start-pos, edit-end-pos). If this flag is not provided, –edit-start-pos and –edit-end-pos flags are ignored.

    +

    Default: False

    +
    +
    --keep-indels, -i
    +

    Include indels.

    +

    Default: False

    +
    +
    --filter-target-basechange, -b
    +

    Only consider target edit (stored in bdata.uns[‘target_base_change’])

    +

    Default: False

    +
    +
    --translate, -t
    +

    Translate alleles

    +

    Default: False

    +
    +
    --translate-fasta, -f
    +

    fasta file path with exon positions. If not provided, LDLR hg19 coordinates will be used.

    +
    +
    --translate-fastas-csv, -fs
    +

    .csv with two columns with gene IDs and FASTA file path corresponding to each gene.

    +
    +
    --translate-gene, -g
    +

    Gene symbol if a gene is tiled. If not provided, LDLR hg19 coordinates will be used.

    +
    +
    --translate-genes-list, -gs
    +

    File with gene symbols, one per line, if multiple genes are tiled.

    +
    +
    --filter-allele-proportion, -ap
    +

    If provided, alleles that exceed filter_allele_proportion in filter-sample-proportion will be retained.

    +

    Default: 0.05

    +
    +
    --filter-allele-count, -ac
    +

    If provided, alleles that exceed filter_allele_proportion AND filter_allele_count in filter-sample-proportion will be retained.

    +

    Default: 5

    +
    +
    --filter-sample-proportion, -sp
    +

    If filter_allele_proportion is provided, alleles that exceed filter_allele_proportion in filter-sample-proportion will be retained.

    +

    Default: 0.2

    +
    +
    --load-tmp
    +

    Load temporary file and work from there.

    +

    Default: False

    +
    +
    +
    +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/genindex.html b/docs/_build/genindex.html new file mode 100644 index 0000000..a301425 --- /dev/null +++ b/docs/_build/genindex.html @@ -0,0 +1,110 @@ + + + + + + + Index — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + + +

    Index

    + +
    + +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/gwas.html b/docs/_build/gwas.html new file mode 100644 index 0000000..7655e7f --- /dev/null +++ b/docs/_build/gwas.html @@ -0,0 +1,243 @@ + + + + + + + + GWAS variant library — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    GWAS variant library¶

    +
    +

    Variant sorting screen tutorial¶

    +

    GWAS variant screen with per-variant gRNA tiling design, selected based on FACS signal quantiles.

    + + + + + + + + + +
    Library designVariant (gRNAs tile each target variant)
    variant library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design



    +
    +

    Example workflow¶

    +
    screen_id=my_sorting_tiling_screen
+
+# 1. Count gRNA & reporter
+bean-count-samples \
+--input tests/data/sample_list.csv    `# Contains fastq file path; see test file for example.`\
+-b A                                  `# Base A is edited (into G)` \
+-f tests/data/test_guide_info.csv     `# Contains gRNA metadata; see test file for example.`\
+-o ./                                 `# Output directory` \
+-r                                    `# Quantify reporter edits` \
+-n ${screen_id}                          `# ID of the screen to be counted`
+
+# 2. QC samples & guides
+bean-qc \
+  bean_count_${screen_id}.h5ad             `# Input ReporterScreen .h5ad file path` \
+  -o bean_count_${screen_id}_masked.h5ad   `# Output ReporterScreen .h5ad file path` \
+  -r qc_report_${screen_id}                `# Prefix for QC report` \
+  -b                                       ` # Remove replicates with no good samples.
+
+# 3. Quantify variant effect
+bean-run sorting variant \
+    tests/data/bean_count_${screen_id}_masked.h5ad \
+    -o tests/test_res/var/ \
+    --fit-negctrl \
+    --scale-by-acc \
+    --accessibility-col accessibility
+
    +
    +

    See more details below.

    +
    +
    +

    1. Count gRNA & reporter (bean count-samples)¶

    +
    screen_id=my_sorting_tiling_screen
+
+# 1. Count gRNA & reporter
+bean-count-samples \
+--input tests/data/sample_list.csv    `# Contains fastq file path; see test file for example.`\
+-b A                                  `# Base A is edited (into G)` \
+-f tests/data/test_guide_info.csv     `# Contains gRNA metadata; see test file for example.`\
+-o ./                                 `# Output directory` \
+-r                                    `# Quantify reporter edits` \
+-n ${screen_id}                          `# ID of the screen to be counted`
+
    +
    +

    Make sure you follow the input file format for seamless downstream steps. This will produce ./bean_count_${screen_id}.h5ad.

    +
    +
    +

    2. QC samples & guides (bean qc)¶

    +

    Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the input file guideline, but you can change the parameters with the full argument list of ``bean-qc` <../../README#bean-qc-qc-of-reporter-screen-data>`_. (Common factors you may want to tweak is --ctrl-cond=bulk and --lfc-conds=top,bot if you have different sample condition labels.)

    +
    bean-qc \
+  bean_count_${screen_id}.h5ad    `# Input ReporterScreen .h5ad file path` \
+  -o bean_count_${screen_id}_masked.h5ad   `# Output ReporterScreen .h5ad file path` \
+  -r qc_report_${screen_id}   `# Prefix for QC report`
+
    +
    +

    If the data does not include reporter editing data, you can provide --no-editing flag to omit the editing rate QC.

    +
    +
    +

    3. Quantify variant effect (bean run)¶

    +

    bean-run can take 3 run options to quantify editing rate:

    +
      +
    1. +
      From reporter + accessibility

      If your gRNA metadata table (tests/data/test_guide_info.csv above) included per-gRNA accessibility score,

      +
      +
      +
         bean-run sorting variant \
+   tests/data/bean_count_${screen_id}_masked.h5ad \
+   -o tests/test_res/var/ \
+   --fit-negctrl \
+   --scale-by-acc \
+   --accessibility-col accessibility
+
+If your gRNA metadata table (\ ``tests/data/test_guide_info.csv`` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal,
+
      +
      +
      bean-run sorting variant \
+tests/data/bean_count_${screen_id}_masked.h5ad \
+-o tests/test_res/var/ \
+--fit-negctrl \
+--scale-by-acc \
+--accessibility-bw accessibility.bw
+
      +
      +
      2. +

    2. From reporter, without accessibility

      +
      +

      This assumes the all target sites have the uniform chromatin accessibility.

      +
      +
      bean-run sorting variant \
+tests/data/bean_count_${screen_id}_masked.h5ad \
+-o tests/test_res/var/ \
+--fit-negctrl
+
      +
      +
      3. +
    3. +
      No reporter information, assume the same editing efficiency of all gRNAs.

      Use this option if your data don’t have editing outcome information.

      +
      +
      +
      bean-run sorting variant \
+tests/data/bean_count_${screen_id}_masked.h5ad \
+-o tests/test_res/var/ \
+--fit-negctrl \
+--uniform-edit
+
      +
      +
      4. +
    +

    See Subcommands for the full details.

    +
    +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/index.html b/docs/_build/index.html new file mode 100644 index 0000000..06c90d1 --- /dev/null +++ b/docs/_build/index.html @@ -0,0 +1,113 @@ + + + + + + + + title: CRISPR-BEAN — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    layout: default

    +
    +

    title: CRISPR-BEAN¶

    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/index_.html b/docs/_build/index_.html new file mode 100644 index 0000000..bc8e2dc --- /dev/null +++ b/docs/_build/index_.html @@ -0,0 +1,213 @@ + + + + + + + + Welcome to bean’s documentation! — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    Welcome to bean’s documentation!¶

    +
    +

    Workflows¶

    +
    + +
    +
    +
    +

    bean subcommands¶

    +
    + +
    +
    +
    +

    Screen data structure¶

    +
    + +
    +
    +
    +

    Indices and tables¶

    + +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/input.html b/docs/_build/input.html new file mode 100644 index 0000000..b55703d --- /dev/null +++ b/docs/_build/input.html @@ -0,0 +1,171 @@ + + + + + + + + Input file format — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    Input file format¶

    +

    This document describes the input files of bean count-samples.

    +
    +

    sgRNA_info_table.csv¶

    +

    File should contain following columns.

    +
      +

    • name: gRNA ID column

      • +

    • sequence: gRNA sequence

      • +

    • barcode: R2 barcode to help match reporter to gRNA, written in the sense direction (as in R1)

      • +

    • In order to use accessibility in the variant effect quantification, provide accessibility information in one of two options. (For non-targeting guides, provide NA values (empty cell).)

      +
        +

      • Option 1: chrom & genomic_pos: Chromosome (ex. chr19) and genomic position of guide sequence. You will have to provide the path to the bigwig file with matching reference version in bean run.

        • +

      • Option 2: accessibility_signal: ATAC-seq signal value of the target loci of each guide.

        • +
      +
      • +

    • For variant library (gRNAs are designed to target specific variants and ignores bystander edits)

      +
        +

      • target: This column denotes which target variant/element of each gRNA. This is not used in bean count[-samples] but required to run bean run in later steps.

        • +

      • target_group: If negative/positive control gRNA will be considered in bean qc and/or bean run, specify as “NegCtrlâ€/â€PosCtrl†in this column.

        • +

      • target_pos: If --match_target_pos flag is used, input file needs target_pos which specifies 0-based relative position of targeted base within Reporter sequence.

        • +
      +
      • +

    • For tiling library (gRNAs tile coding / noncoding sequences)

      +
        +

      • strand: Specifies gRNA strand information relative to the reference genome.

        • +

      • chrom: Chromosome of gRNA targeted locus.

        • +

      • start_pos: gRNA starting position in the genome. Required when you provide strand column. Should specify the smaller coordinate value among start and end position regardless of gRNA strandedness.

        • +
      +
      • +
    +

    Also see examples for variant library and tiling library.

    +
    +
    +

    sample_list.csv¶

    +

    File should contain following columns with header.

    +
      +

    • R1_filepath: Path to read 1 .fastq[.gz] file

      • +

    • R2_filepath: Path to read 1 .fastq[.gz] file

      • +

    • sample_id: ID of sequencing sample

      • +

    • replicate: Replicate # of this sample (Should NOT contain .)

      • +

    • condition: Name of the sorting bin (ex. top, bot), or label of timepoint (ex. D5, D18)

      • +
    +

    For FACS sorting screens:

    +
      +

    • upper_quantile: FACS sorting upper quantile

      • +

    • lower_quantile: FACS sorting lower quantile

      • +
    +

    For proliferation / survival screens:

    +
      +

    • time: Numeric time following the base editing of each sample.

      • +
    +

    Also see examples for FACS sorting screen.

    +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/objects.inv b/docs/_build/objects.inv new file mode 100644 index 0000000000000000000000000000000000000000..b01eb0a63fe257f5edac5cea636283a2a547048a GIT binary patch literal 761 zcmVNERX>N99Zgg*Qc_4OWa&u{KZXhxWBOp+6Z)#;@bUGknWnpd# zBOq2~a&u{KZaN?eBOp|0Wgv28ZDDC{WMy(7Z)PBLXlZjGW@&6?AZc?TV{dJ6a%FRK zWn>_Ab7^j8AbMYo@{2`yf+LpaGe`l!^kk3~5uXD@joP4jm%z>TDXm zu%pd>hfHUrVwh*7(mn+&ZOm3yOq`IWmyQZTkzu@Fb5<&>t{IU{gGHquiR^?47y^$a zD=8LeSbxtoZdy+Dk2Og2q4#Rc?g43=Uxf|!1Qf-F=A$nu}Gjb0ywE_T~^TkuhGZh+gIuyFi5tBNr5@PBBQX-AOOAx1Qr^|Fwu7D zG7cUqqECmXO~FJh3xi8eS46+e6s?kd?n#o-=M`tPtkHf9qZR9OLoGJ21L5U%AxX{) z@#rpOjlOJ=tdTp;^l7RteTHE7*kA>89TtYwSM-9RQ=wC6UBglJiA61!=rju}#&(O~ z&an)0{h0{oxTHKya2Ld;=;*5yMU9JEss;zyhaQ{HZfJ*9jfZwbV-S1>>fCl)7Wby# zl0yCkrnkd|?BgDTnrfn@JAI|NZ?(BY<@c6$bZX_a=*`${QwIVz0a&dfM}h zhZ4mRO@B#7g=(+4b6Ba*vg#nnmMk%*H>Wf6sQ#m|znZfYp;S1yi+Oe4iE{M2UI4^m zSfd%8027CUIFcTuDOThh;;hZG9j$+lPc+kA>8=D{*)xwZvmuI^MHW$zlpp7wK>f%o rUM$|h$Z2)I+E-A+4L5x46*oPmVH9-#ZAx%j%5US0+~WKPKoQ)5f$d=t literal 0 HcmV?d00001 diff --git a/docs/_build/profile.html b/docs/_build/profile.html new file mode 100644 index 0000000..9d55d75 --- /dev/null +++ b/docs/_build/profile.html @@ -0,0 +1,185 @@ + + + + + + + + bean profile — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    bean profile¶

    +
    +

    bean profile: Profile editing patterns¶

    +
    bean profile my_sorting_screen.h5ad -o output_prefix `# Prefix for editing profile report`
+
    +
    +
    +
    +

    Output¶

    +

    Above command produces prefix_editing_preference.[html,ipynb] as editing preferences (see example).

    +

    Allele translation

    +
    +
    +

    Full parameters¶

    +
    usage: bean profile [-h] [-o OUTPUT_PREFIX] [--replicate-col REPLICATE_COL]
+                    [--condition-col CONDITION_COL] [--pam-col PAM_COL]
+                    [--control-condition CONTROL_CONDITION] [-w WINDOW_LENGTH]
+                    bdata_path
+
    +
    +
    +

    Positional Arguments¶

    +
    +
    bdata_path
    +

    Path to the ReporterScreen object to run QC on

    +
    +
    +
    +
    +

    Named Arguments¶

    +
    +
    -o, --output-prefix
    +

    Output prefix of editing pattern report (prefix.html, prefix.ipynb). If not provided, base name of bdata_path is used.

    +
    +
    --replicate-col
    +

    Column name in bdata.samples that describes replicate ID.

    +

    Default: “replicateâ€

    +
    +
    --condition-col
    +

    Column name in bdata.samples that describes experimental condition. (sorting bin, time, etc.)

    +

    Default: “binâ€

    +
    +
    --pam-col
    +

    Column name describing PAM of each gRNA in bdata.guides.

    +
    +
    --control-condition
    +

    Control condition where editing preference would be profiled at. Pre-filters data where bdata.samples[condition_col] == control_condition.

    +

    Default: “bulkâ€

    +
    +
    -w, --window-length
    +

    Window length of editing window of maximal editing efficiency to be identified. This window is used to quantify context specificity within the window.

    +

    Default: 6

    +
    +
    +
    +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/qc.html b/docs/_build/qc.html new file mode 100644 index 0000000..f08de74 --- /dev/null +++ b/docs/_build/qc.html @@ -0,0 +1,429 @@ + + + + + + + + bean qc — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    bean qc¶

    +
    +

    bean qc: QC of reporter screen data¶

    +
    bean qc \
+  my_sorting_screen.h5ad             `# Input ReporterScreen .h5ad file path` \
+  -o my_sorting_screen_masked.h5ad   `# Output ReporterScreen .h5ad file path` \
+  -r qc_report_my_sorting_screen     `# Prefix for QC report` \
+  --ctrl-cond presort                `# "condition" column in the control sample before selection. Mean gRNA editing rates in these samples are reported. ` \
+# Inspect the output qc_report_my_sorting_screen.html to tweak QC threshold
+
+bean qc \
+  my_sorting_screen.h5ad              \
+  -o my_sorting_screen_masked.h5ad    \
+  -r qc_report_my_sorting_screen      \
+  #[--count-correlation-thres 0.7 ...]\
+  -b
+
    +
    +

    bean qc supports following quality control and masks samples with low quality. Specifically:

    +

    Allele translation

    +
      +

    • Plots guide coverage and the uniformity of coverage

      • +

    • Guide count correlation between samples

      • +

    • Log fold change correlation when positive controls are provided

      • +

    • Plots editing rate distribution

      • +

    • Identify samples with low guide coverage/guide count correlation/editing rate and mask the sample in bdata.samples.mask

      • +

    • Identify outlier guides to filter out

      • +
    +
    +
    +

    Output¶

    +

    Above command produces

    +
      +

    • my_sorting_screen_masked.h5ad without problematic replicate and guides and with sample masks, and

      • +

    • qc_report_my_sorting_screen.[html,ipynb] as QC report.
       +##### Optional arguments:

      • +

    • -o OUT_SCREEN_PATH, --out-screen-path OUT_SCREEN_PATH

      +
      Path where quality-filtered ReporterScreen object to be written to
+
      +
      +
      • +

    • -r OUT_REPORT_PREFIX, --out-report-prefix OUT_REPORT_PREFIX

      +
      Output prefix of qc report (prefix.html, prefix.ipynb)
+
      +
      +
      • +
    +
    +

    QC thresholds:¶

    +
      +

    • --count-correlation-thres COUNT_CORRELATION_THRES

      +
      Correlation threshold to mask out.
+
      +
      +
      • +

    • --edit-rate-thres EDIT_RATE_THRES

      +
      Mean editing rate threshold per sample to mask out.
+
      +
      +
      • +

    • --lfc-thres LFC_THRES

      +
      Positive guides' correlation threshold to filter out.
+
      +
      +
      • +
    +
    +
    +

    Run options:¶

    +
      +

    • -b, --remove-bad-replicates

      +
      Remove replicates with at least two of its samples meet the QC threshold (bean run does not support having only one sorting bin sample for a replicate).
+
      +
      +
      • +

    • -i, --ignore-missing-samples

      +
      If the flag is not provided, if the ReporterScreen object does not contain all condiitons for
+each replicate, make fake empty samples. If the flag is provided, don't add dummy samples.
+
      +
      +
      • +

    • --no-editing Ignore QC about editing. Can be used for QC of other editing modalities.

      • +

    • --dont-recalculate-edits

      +
      When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from
+ReporterScreen.uns['allele_count'].
+
      +
      +
      • +
    +
    +
    +

    Input .h5ad formatting:¶

    +

    Note that these arguements will change the way the QC metrics are calculated for guides, samples, or replicates.

    +
      +

    • --tiling TILING Specify that the guide library is tiling library without ‘n guides per target’ design

      • +

    • --replicate-label REPLICATE_LABEL

      +
      Label of column in `bdata.samples` that describes replicate ID.
+
      +
      +
      • +

    • --sample-covariates SAMPLE_COVARIATES

      +
      Comma-separated list of column names in `bdata.samples` that describes non-selective
+experimental condition. (drug treatment, etc.)
+
      +
      +
      • +

    • --condition-label CONDITION_LABEL

      +
      Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time,
+etc.)
+
      +
      +

      ###### Editing rate calculation

      +
        +

      • --control-condition CTRL_COND

        +
        Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate
+to be calculated. Default is `None`, which considers all samples.
+
        +
        +
        • +

      • --rel-pos-is-reporter

        +
        Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If
+`False`, those are relative to spacer position.
+
        +
        +

        Editing rate is calculated with following parameters in

        +
          +

        • Variant screens:

          +
            +

          • --target-pos-col TARGET_POS_COL

            +
            Target position column in `bdata.guides` specifying target edit position in reporter
+
            +
            +
            • +
          +
          • +

        • tiling screens:

          +
            +

          • --edit-start-pos EDIT_START_POS

            +
            Edit start position to quantify editing rate on, 0-based inclusive.
+
            +
            +
            • +

          • --edit-end-pos EDIT_END_POS

            +
            Edit end position to quantify editing rate on, 0-based exclusive.
+
            +
            +

            ###### LFC of positive controls

            +
            • +
          +
          • +
        +
        • +

      • --posctrl-col POSCTRL_COL

        +
        Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all
+gRNAs, feed empty string ''.
+
        +
        +
        • +

      • --posctrl-val POSCTRL_VAL

        +
        Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the
+positive control in calculating log fold change.
+
        +
        +
        • +

      • --lfc-conds LFC_CONDS

        +
        Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated
+between, delimited by comma
+
        +
        +
        • +
      +
      • +
    +
    +
    +
    +

    Full parameters¶

    +
    usage: bean qc [-h] [--count-correlation-thres COUNT_CORRELATION_THRES]
+               [--edit-rate-thres EDIT_RATE_THRES] [--lfc-thres LFC_THRES]
+               [-o OUT_SCREEN_PATH] [-r OUT_REPORT_PREFIX] [-b] [-i]
+               [--no-editing] [--dont-recalculate-edits] [--tiling TILING]
+               [--replicate-label REPLICATE_LABEL]
+               [--sample-covariates SAMPLE_COVARIATES]
+               [--condition-label CONDITION_LABEL]
+               [--target-pos-col TARGET_POS_COL] [--rel-pos-is-reporter]
+               [--edit-start-pos EDIT_START_POS] [--edit-end-pos EDIT_END_POS]
+               [--posctrl-col POSCTRL_COL] [--posctrl-val POSCTRL_VAL]
+               [--lfc-conds LFC_CONDS] [--control-condition CONTROL_CONDITION]
+               bdata_path
+
    +
    +
    +

    Positional Arguments¶

    +
    +
    bdata_path
    +

    Path to the ReporterScreen object to run QC on

    +
    +
    +
    +
    +

    Named Arguments¶

    +
    +
    -o, --out-screen-path
    +

    Path where quality-filtered ReporterScreen object to be written to

    +
    +
    -r, --out-report-prefix
    +

    Output prefix of qc report (prefix.html, prefix.ipynb)

    +
    +
    +
    +
    +

    QC thresholds¶

    +
    +
    --count-correlation-thres
    +

    Correlation threshold to mask out.

    +

    Default: 0.7

    +
    +
    --edit-rate-thres
    +

    Mean editing rate threshold per sample to mask out.

    +

    Default: 0.1

    +
    +
    --lfc-thres
    +

    Positive guides’ correlation threshold to filter out.

    +

    Default: -0.1

    +
    +
    +
    +
    +

    Run options¶

    +
    +
    -b, --remove-bad-replicates
    +

    Remove replicates with at least two of its samples meet the QC threshold.

    +

    Default: False

    +
    +
    -i, --ignore-missing-samples
    +

    If the flag is not provided, if the ReporterScreen object does not contain all condiitons for each replicate, make fake empty samples. If the flag is provided, don’t add dummy samples.

    +

    Default: False

    +
    +
    --no-editing
    +

    Ignore QC about editing. Can be used for QC of other editing modalities.

    +

    Default: False

    +
    +
    --dont-recalculate-edits
    +

    When ReporterScreen.layers[‘edit_count’] exists, do not recalculate the edit counts from ReporterScreen.uns[‘allele_count’].

    +

    Default: False

    +
    +
    +
    +
    +

    Input .h5ad formatting¶

    +
    +
    --tiling
    +

    Specify that the guide library is tiling library without ‘n guides per target’ design

    +
    +
    --replicate-label
    +

    Label of column in bdata.samples that describes replicate ID.

    +

    Default: “replicateâ€

    +
    +
    --sample-covariates
    +

    Comma-separated list of column names in bdata.samples that describes non-selective experimental condition. (drug treatment, etc.)

    +
    +
    --condition-label
    +

    Label of column in bdata.samples that describes experimental condition. (sorting bin, time, etc.)

    +

    Default: “conditionâ€

    +
    +
    --target-pos-col
    +

    Target position column in bdata.guides specifying target edit position in reporter

    +

    Default: “target_posâ€

    +
    +
    --rel-pos-is-reporter
    +

    Specifies whether edit_start_pos and edit_end_pos are relative to reporter position. If False, those are relative to spacer position.

    +

    Default: False

    +
    +
    --edit-start-pos
    +

    Edit start position to quantify editing rate on, 0-based inclusive.

    +

    Default: 2

    +
    +
    --edit-end-pos
    +

    Edit end position to quantify editing rate on, 0-based exclusive.

    +

    Default: 7

    +
    +
    --posctrl-col
    +

    Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all gRNAs, feed empty string ‘’.

    +

    Default: “target_groupâ€

    +
    +
    --posctrl-val
    +

    Value in ReporterScreen.guides[posctrl_col] that specifies guide will be used as the positive control in calculating log fold change.

    +

    Default: “PosCtrlâ€

    +
    +
    --lfc-conds
    +

    Values in of column in ReporterScreen.samples[condition_label] for LFC will be calculated between, delimited by comma

    +

    Default: “top,botâ€

    +
    +
    --control-condition
    +

    Values in of column in ReporterScreen.samples[condition_label] for guide-level editing rate to be calculated

    +

    Default: “bulkâ€

    +
    +
    +
    +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/run.html b/docs/_build/run.html new file mode 100644 index 0000000..d9e232b --- /dev/null +++ b/docs/_build/run.html @@ -0,0 +1,401 @@ + + + + + + + + bean run — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    bean run¶

    +
    +

    bean run: Quantify variant effects¶

    +

    BEAN uses Bayesian network to incorporate gRNA editing outcome to provide posterior estimate of variant phenotype. The Bayesian network reflects data generation process. Briefly,

    +
      +

    1. Cellular phenotype (either for cells are sorted upon for sorting screen, or log(proliferation rate)) is modeled as the Gaussian mixture distribution of wild-type phenotype and variant phenotype.

      2. +

    2. The weight of the mixture components are inferred from the reporter editing outcome and the chromatin accessibility of the loci.

      3. +

    3. Cells with each gRNA, formulated as the mixture distribution, is sorted by the phenotypic quantile to produce the gRNA counts.

      4. +
    +

    For the full detail, see the method section of the BEAN manuscript.

    +

    model

    +



    +
    +
    +

    Usage example¶

    +
    bean run sorting[survival] variant[tiling] my_sorting_screen_filtered.h5ad \
+[--uniform-edit, --scale-by-acc [--acc-bw-path accessibility_signal.bw, --acc-col accessibility]] \
+-o output_prefix/ \
+--fit-negctrl
+
    +
    +

    See full list of parameters below.

    +
    +
    +

    Input¶

    +

    my_sorting_screen_filtered.h5ad can be produced by one of the following:

    +
      +

    1. ``bean count-samples` <(#bean-count-samples-count-reporter-screen-data>`_) when you have raw .fastq file

      2. +

    2. (Limited to bean run variant mode) bean create-screen when you have flat .csv tables of gRNA metadata table, sample metadata table, gRNA counts table (# guides x # samples), and optionally # edits table. +.. code-block:: bash

      +
      +

      bean create-screen gRNA_info_table.csv sample_info_table.csv gRNA_counts_table.csv [–edits edit_counts_table.csv -o output.h5ad]

      +
      +
        +

      • gRNA_info_table.csv should have following columns.

        +
          +

        • name: gRNA ID column

          • +

        • target: This column denotes which target variant/element of each gRNA.

          • +

        • target_group [Optional]: If negative control gRNA will be used, specify as “NegCtrl†in this column.

          • +
        +
        • +

      • sample_info_table.csv should have following columns.

        +
          +

        • sample_id: ID of sequencing sample

          • +

        • replicate: Replicate # of this sample

          • +

        • bin: Name of the sorting bin

          • +

        • upper_quantile: FACS sorting upper quantile

          • +

        • lower_quantile: FACS sorting lower quantile

          • +
        +
        • +

      • gRNA_counts_table.csv should be formatted as follows.

        +
          +

        • Columns include one of sample_id columns in sample_info_table.csv file.

          • +

        • 1st row (row index) follows name (gRNA ID) in gRNA_info_table.csv file.

          • +
        +
        • +
      +
      3. +

    3. You can manually create the AnnData object with more annotations including allele counts: see API tutorial for full detail.

      4. +
    +
    +
    +

    Output¶

    +

    model

    +

    Above command produces

    +
      +

    • output_prefix/bean_element_result.[model_type].csv with following columns:

      +
        +

      • Estimated variant effect sizes

        +
          +

        • mu (Effect size): Mean of variant phenotype, given the wild type has standard normal phenotype distribution of mu = 0, sd = 1.

          • +

        • mu_sd: Mean of variant phenotype mu is modeled as normal distribution. The column shows fitted standard deviation of mu that quantify the uncertainty of the variant effect.

          • +

        • mu_z: z-score of mu

          • +

        • sd: Standard deviation of variant phenotype, given the wild type has standard normal phenotype distribution of mu = 0, sd = 1.

          • +

        • CI[0.025, 0.975]: Credible interval of mu

          • +

        • When negative control is provided, above columns with _adj suffix are provided, which are the corresponding values adjusted for negative control.

          • +
        +
        • +

      • Metrics on per-variant evidence provided in input (provided in tiling mode)

        +
          +

        • effective_edit_rate: Sum of per-variant editing rates over all alleles observed in the input. Allele-level editing rate is divided by the number of variants observed in the allele prior to summing up.

          • +

        • n_guides: # of guides covering the variant.

          • +

        • n_coocc: # of cooccurring variants with a given variant in any alleles observed in the input.

          • +
        +
        • +
      +
      • +

    • output_prefix/bean_sgRNA_result.[model_type].csv:

      +
        +

      • edit_rate: Estimated editing rate at the target loci.

        • +
      +
      • +
    +
    +
    +

    Full parameters¶

    +

    Run model on data.

    +

    +
    usage: bean run [-h] [--rep-pi] [--uniform-edit] [--scale-by-acc]
+                [--acc-bw-path ACC_BW_PATH] [--acc-col ACC_COL] [--const-pi]
+                [--shrink-alpha] [--condition-col CONDITION_COL]
+                [--time-col TIME_COL] [--control-condition CONTROL_CONDITION]
+                [--include-control-condition-for-inference]
+                [--replicate-col REPLICATE_COL] [--target-col TARGET_COL]
+                [--guide-activity-col GUIDE_ACTIVITY_COL] [--outdir OUTDIR]
+                [--result-suffix RESULT_SUFFIX]
+                [--sorting-bin-upper-quantile-col SORTING_BIN_UPPER_QUANTILE_COL]
+                [--sorting-bin-lower-quantile-col SORTING_BIN_LOWER_QUANTILE_COL]
+                [--alpha-if-overdispersion-fitting-fails ALPHA_IF_OVERDISPERSION_FITTING_FAILS]
+                [--cuda] [--sample-mask-col SAMPLE_MASK_COL] [--fit-negctrl]
+                [--negctrl-col NEGCTRL_COL]
+                [--negctrl-col-value NEGCTRL_COL_VALUE]
+                [--repguide-mask REPGUIDE_MASK] [--device DEVICE]
+                [--ignore-bcmatch] [--allele-df-key ALLELE_DF_KEY]
+                [--splice-site-path SPLICE_SITE_PATH]
+                [--control-guide-tag CONTROL_GUIDE_TAG] [--dont-fit-noise]
+                [--dont-adjust-confidence-by-negative-control]
+                [--n-iter N_ITER] [--load-existing]
+                {sorting,survival} {variant,tiling} bdata_path
+
    +
    +
    +

    Positional Arguments¶

    +
    +
    selection
    +

    Possible choices: sorting, survival

    +

    Screen selection type whether cells are sorted based on continuous phenotype (‘sorting’) or proliferated based on their viability (‘survival’).

    +
    +
    library_design
    +

    Possible choices: variant, tiling

    +

    Library design type whether to run variant or tiling screen model. +Variant library design assumes gRNA has specific target variant and bystander edits are ignored. Tiling library design considers all alleles generated by gRNA in reporter.

    +
    +
    bdata_path
    +

    Path of an ReporterScreen object

    +
    +
    +
    +
    +

    Named Arguments¶

    +
    +
    --rep-pi, -r
    +

    Fit replicate specific scaling factor. Recommended to set as True if you expect variable editing activity across biological replicates.

    +

    Default: False

    +
    +
    --uniform-edit, -p
    +

    Assume uniform editing rate for all guides.

    +

    Default: False

    +
    +
    --scale-by-acc
    +

    Scale guide editing efficiency by the target loci accessibility

    +

    Default: False

    +
    +
    --acc-bw-path
    +

    Accessibility .bigWig file to be used to assign accessibility of guides.

    +
    +
    --acc-col
    +

    Column name in bdata.guides that specify raw ATAC-seq signal.

    +
    +
    --const-pi
    +

    Use constant pi provided in –guide-activity-col (instead of fitting from reporter data)

    +

    Default: False

    +
    +
    --shrink-alpha
    +

    Instead of using the trend-fitted alpha values, use estimated alpha values for each gRNA that are shrunk towards the fitted trend.

    +

    Default: False

    +
    +
    --condition-col
    +

    Column key in bdata.samples that describes experimental condition.

    +

    Default: “conditionâ€

    +
    +
    --time-col
    +

    Column key in bdata.samples that describes time elapsed.

    +

    Default: “timeâ€

    +
    +
    --control-condition
    +

    Value in bdata.samples[condition_col] that indicates control experimental condition.

    +

    Default: “bulkâ€

    +
    +
    --include-control-condition-for-inference, -ic
    +

    Include control conditions for inference. Currently only supported for survival screens.

    +

    Default: False

    +
    +
    --replicate-col
    +

    Column key in bdata.samples that describes experimental replicates.

    +

    Default: “replicateâ€

    +
    +
    --target-col
    +

    Column key in bdata.guides that describes the target element of each guide.

    +

    Default: “targetâ€

    +
    +
    --guide-activity-col, -a
    +

    Column in ReporterScreen.guides DataFrame showing the editing rate estimated via external tools

    +
    +
    --outdir, -o
    +

    Directory to save the run result.

    +

    Default: “.â€

    +
    +
    --result-suffix
    +

    Suffix of the output files

    +

    Default: “â€

    +
    +
    --sorting-bin-upper-quantile-col, -uq
    +

    Column name with upper quantile values of each sorting bin in [Reporter]Screen.samples (or AnnData.var)

    +

    Default: “upper_quantileâ€

    +
    +
    --sorting-bin-lower-quantile-col, -lq
    +

    Column name with lower quantile values of each sorting bin in [Reporter]Screen.samples (or AnnData var)

    +

    Default: “lower_quantileâ€

    +
    +
    --alpha-if-overdispersion-fitting-fails, -af
    +

    Comma-separated regression coefficient (b0, b1) of log(a0) ~ log(q) that will be used if fitting dispersion on the data fails.

    +
    +
    --cuda
    +

    run on GPU

    +

    Default: False

    +
    +
    --sample-mask-col
    +

    Name of the column indicating the sample mask in [Reporter]Screen.samples (or AnnData.var). Sample is ignored if the value in this column is 0. This can be used to mask out low-quality samples.

    +
    +
    --fit-negctrl
    +

    Fit the shared negative control distribution to normalize the fitted parameters

    +

    Default: False

    +
    +
    --negctrl-col
    +

    Column in bdata.obs specifying if a guide is negative control. If the bdata.guides[negctrl_col].lower() == negctrl_col_value, it is treated as negative control guide.

    +

    Default: “target_groupâ€

    +
    +
    --negctrl-col-value
    +

    Column value in bdata.guides specifying if a guide is negative control. If the bdata.guides[negctrl_col].lower() == negctrl_col_value, it is treated as negative control guide.

    +

    Default: “negctrlâ€

    +
    +
    --repguide-mask
    +

    n_replicate x n_guide mask to mask the outlier guides. screen.uns[repguide_mask] will be used.

    +

    Default: repguide_mask

    +
    +
    --device
    +

    Optionally use GPU if provided valid GPU device name (ex. cuda:0)

    +
    +
    --ignore-bcmatch
    +

    If provided, even if the screen object has .X_bcmatch, ignore the count when fitting.

    +

    Default: False

    +
    +
    --allele-df-key
    +

    screen.uns[allele_df_key] will be used as the allele count.

    +
    +
    --splice-site-path
    +

    Path to splicing site

    +
    +
    --control-guide-tag
    +

    If this string is in guide name, treat each guide separately not to mix the position. Used for negative controls.

    +
    +
    --dont-fit-noise
    +

    Default: False

    +
    +
    --dont-adjust-confidence-by-negative-control
    +

    Adjust confidence by negative controls. For variant library_design, this uses negative control variants. For tiling library_design, adjusts confidence by synonymous edits.

    +

    Default: False

    +
    +
    --n-iter
    +

    # of SVI steps taken for inference.

    +

    Default: 2000

    +
    +
    --load-existing
    +

    Load existing .pkl file if present.

    +

    Default: False

    +
    +
    +
    +
    +
    + + +
    + +
    +
    + +
    +
    +
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calculation & Addition": [[0, "lfc-calculation-addition"]], "Getting edit rates from allele counts": [[0, "getting-edit-rates-from-allele-counts"]], "Calculating LFC": [[0, "calculating-lfc"]], "Allele translation": [[0, "allele-translation"]], "Writing": [[0, "writing"]], "Coding sequence tiling library": [[1, "coding-sequence-tiling-library"]], "Tiling sorting screen tutorial": [[1, "tiling-sorting-screen-tutorial"], [21, "tiling-sorting-screen-tutorial"]], "Example workflow": [[1, "example-workflow"], [13, "example-workflow"], [21, "example-workflow"], [22, "example-workflow"]], "1. 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Quantify variant effect (bean run)": [[1, "quantify-variant-effect-run"], [21, "quantify-variant-effect-run"]], "bean count[-samples]: Count (reporter) screen data": [[2, "bean-count-samples-count-reporter-screen-data"], [9, "bean-count-samples-count-reporter-screen-data"], [10, "bean-count-samples-count-reporter-screen-data"]], "Input file format": [[2, "input-file-format"], [9, "input-file-format"], [10, "input-file-format"], [16, "input-file-format"]], "Output file format": [[2, "output-file-format"], [9, "output-file-format"], [10, "output-file-format"]], "bean create-screen: Create ReporterScreen object from flat files": [[3, "bean-create-screen-create-reporterscreen-object-from-flat-files"]], "Input": [[3, "input"], [8, "input"], [19, "input"]], "filter: Filtering (and optionally translating) alleles": [[4, "filter-filtering-and-optionally-translating-alleles"], [12, "filter-filtering-and-optionally-translating-alleles"]], "Output": [[4, "output"], [6, "output"], [7, "output"], [8, "output"], [12, "output"], [17, "output"], [18, "output"], [19, "output"]], "Translating alleles": [[4, "translating-alleles"], [12, "translating-alleles"]], "sgRNA_info_table.csv": [[5, "sgrna-info-table-csv"], [16, "sgrna-info-table-csv"]], "sample_list.csv": [[5, "sample-list-csv"], [16, "sample-list-csv"]], "bean profile: Profile editing patterns": [[6, "bean-profile-profile-editing-patterns"], [17, "bean-profile-profile-editing-patterns"]], "bean qc: QC of reporter screen data": [[7, "bean-qc-qc-of-reporter-screen-data"], [18, "bean-qc-qc-of-reporter-screen-data"]], "QC thresholds:": [[7, "qc-thresholds"], [18, "qc-thresholds"]], "Run options:": [[7, "run-options"], [18, "run-options"]], "Input .h5ad formatting:": [[7, "input-h5ad-formatting"], [18, "input-h5ad-formatting"]], "bean run: Quantify variant effects": [[8, "bean-run-quantify-variant-effects"], [19, "bean-run-quantify-variant-effects"]], "Usage example": [[8, "usage-example"], [19, "usage-example"]], "bean count": [[9, "bean-count"]], "Full parameters": [[9, "full-parameters"], [10, "full-parameters"], [12, "full-parameters"], [17, "full-parameters"], [18, "full-parameters"], [19, "full-parameters"]], "Named Arguments": [[9, "named-arguments"], [10, "named-arguments"], [12, "named-arguments"], [17, "named-arguments"], [18, "named-arguments"], [19, "named-arguments"]], "bean count-samples": [[10, "bean-count-samples"]], "Input .fa file format for bean-filter": [[11, "input-fa-file-format-for-bean-filter"]], "bean filter": [[12, "bean-filter"]], "Positional Arguments": [[12, "positional-arguments"], [17, "positional-arguments"], [18, "positional-arguments"], [19, "positional-arguments"]], "GWAS variant library": [[13, "gwas-variant-library"]], "Variant sorting screen tutorial": [[13, "variant-sorting-screen-tutorial"], [22, "variant-sorting-screen-tutorial"]], "2. QC samples & guides (bean qc)": [[13, "qc-samples-guides-qc"], [22, "qc-samples-guides-qc"]], "3. Quantify variant effect (bean run)": [[13, "quantify-variant-effect-run"], [22, "quantify-variant-effect-run"]], "title: CRISPR-BEAN": [[14, "title-crispr-bean"]], "Welcome to bean\u2019s documentation!": [[15, "welcome-to-bean-s-documentation"]], "Workflows": [[15, "workflows"]], "bean subcommands": [[15, "bean-subcommands"]], "Screen data structure": [[15, "screen-data-structure"]], "Indices and tables": [[15, "indices-and-tables"]], "bean profile": [[17, "bean-profile"]], "bean qc": [[18, "bean-qc"]], "QC thresholds": [[18, "qc-thresholds"]], "Run options": [[18, "run-options"]], "Input .h5ad formatting": [[18, "input-.h5ad-formatting"]], "bean run": [[19, "bean-run"]], "Subcommands": [[20, "subcommands"]]}, "indexentries": {}}) \ No newline at end of file diff --git a/docs/_build/subcommands.html b/docs/_build/subcommands.html new file mode 100644 index 0000000..bb2154a --- /dev/null +++ b/docs/_build/subcommands.html @@ -0,0 +1,168 @@ + + + + + + + + Subcommands — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + + + +
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    Subcommands¶

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    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/tutorials/ldl_cds.html b/docs/_build/tutorials/ldl_cds.html new file mode 100644 index 0000000..09e4c5e --- /dev/null +++ b/docs/_build/tutorials/ldl_cds.html @@ -0,0 +1,261 @@ + + + + + + + + Tiling sorting screen tutorial — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + +
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    Tiling sorting screen tutorial¶

    +

    Tiling screen that tiles gRNA densely across locus or multiple loci, selected based on FACS signal quantiles.

    + + + + + + + + + +
    Library designTiling (gRNAs tile each locus densely)
    tiling library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design



    +
    +

    Example workflow¶

    +
    screen_id=my_sorting_tiling_screen
+
+# 1. Count gRNA & reporter
+bean-count-samples \
+--input tests/data/sample_list_tiling.csv          `# Contains fastq file path; see test file for example.`\
+-b A                                               `# Base A is edited (into G)` \
+-f tests/data/test_guide_info_tiling_chrom.csv     `# Contains gRNA metadata; see test file for example.`\
+-o ./                                              `# Output directory` \
+-r                                                 `# Quantify reporter edits` \
+-n ${screen_id}                                       `# ID of the screen` \
+--tiling
+
+# 2. QC samples & guides
+bean-qc \
+  bean_count_${screen_id}.h5ad           `# Input ReporterScreen .h5ad file path` \
+  -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \
+  -r qc_report_${screen_id}              `# Prefix for QC report` \
+
+# 3. Filter & translate alleles
+bean-filter ./bean_count_${screen_id}_masked.h5ad \
+-o ./bean_count_${screen_id}_alleleFiltered \
+--filter-target-basechange                             `# Filter based on intended base changes. If -b A was provided in bean-count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\
+--filter-window --edit-start-pos 0 --edit-end-pos 19   `# Filter based on editing window in spacer position within reporter.`\
+--filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \
+--translate --translate-genes-list tests/data/gene_symbols.txt
+
+# 4. Quantify variant effect
+bean-run sorting tiling \
+    ./bean_count_${screen_id}_alleleFiltered.h5ad \
+    -o tests/test_res/var/ \
+    --fit-negctrl \
+    --scale-by-acc \
+    --accessibility-col accessibility
+
    +
    +

    See more details below.

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    +
    +

    1. Count gRNA & reporter (bean count-samples)¶

    +
    screen_id=my_sorting_tiling_screen
+
+bean-count-samples \
+--input tests/data/sample_list_tiling.csv          `# Contains fastq file path; see test file for example.`\
+-b A                                               `# Base A is edited (into G)` \
+-f tests/data/test_guide_info_tiling_chrom.csv     `# Contains gRNA metadata; see test file for example.`\
+-o ./                                              `# Output directory` \
+-r                                                 `# Quantify reporter edits` \
+-n ${screen_id}                                       `# ID of the screen` \
+--tiling
+
    +
    +

    Make sure you follow the input file format for seamless downstream steps. This will produce ./bean_count_${screen_id}.h5ad.

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    +

    2. QC (bean qc)¶

    +

    Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the input file guideline, but you can change the parameters with the full argument list of ``bean-qc` <../../README#bean-qc-qc-of-reporter-screen-data>`_. (Common factors you may want to tweak is --ctrl-cond=bulk and --lfc-conds=top,bot if you have different sample condition labels.)

    +
    bean-qc \
+  bean_count_${screen_id}.h5ad           `# Input ReporterScreen .h5ad file path` \
+  -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \
+  -r qc_report_${screen_id}              `# Prefix for QC report` \
+  [--tiling]                          `# Not required if you have passed --tiling in counting step`
+
    +
    +

    If the data does not include reporter editing data, you can provide --no-editing flag to omit the editing rate QC.

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    3. Filter alleles (bean filter)¶

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    As tiling library doesn’t have designated per-gRNA target variant, any base edit observed in reporter may be the candidate variant, while having too many variants with very low editing rate significantly decreases the power. Variants are filtered based on multiple criteria in bean-fitler.

    +

    If the screen targets coding sequence, it’s beneficial to translate edits into coding varaints whenever possible for better power. For translation, provide --translate and one of the following:

    +
    [ --translate-gene-name GENE_SYMBOL OR
+  --translate-genes-list path_to_gene_names_file.txt OR
+  --translate-fasta gene_exon.fa, OR
+  --translate-fastas-csv gene_exon_fas.csv]
+
    +
    +

    where path_to_gene_names_file.txt has one gene symbol per line, and gene symbol uses its MANE transcript (hg38) coordinates of exons. In order to use other reference versions or transcript ID, you’ll need to feed in fasta file. See detailed formatting of fasta file here.

    +

    Example allele filtering given we’re translating based on MANE transcript exons of multiple gene symbols:

    +
    bean-filter ./bean_count_${screen_id}_masked.h5ad \
+-o ./bean_count_${screen_id}_alleleFiltered \
+--filter-target-basechange                             `# Filter based on intended base changes. If -b A was provided in bean-count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\
+--filter-window --edit-start-pos 0 --edit-end-pos 19   `# Filter based on editing window in spacer position within reporter.`\
+--filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \
+--translate --translate-genes-list tests/data/gene_symbols.txt
+
    +
    +

    Ouptut file `` shows number of alleles per guide and number of guides per variant, where we want high enough values for the latter. See the typical output for dataset with good editing coverage & filtering result here.

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    4. Quantify variant effect (bean run)¶

    +

    By default, bean-run [sorting,survival] tiling uses most filtered allele counts table for variant identification and quantification of their effects. **Check allele filtering output** and choose alternative filtered allele counts table if necessary.

    +

    bean-run can take 3 run options to quantify editing rate:

    +
      +
    1. +
      From reporter + accessibility

      1-1. If your gRNA metadata table (tests/data/test_guide_info.csv above) included per-gRNA accessibility score,

      +
      +
      +
         bean-run sorting tiling \
+   ./bean_count_${screen_id}_alleleFiltered.h5ad \
+   -o tests/test_res/var/ \
+   --fit-negctrl \
+   --scale-by-acc \
+   --accessibility-col accessibility
+
+1-2. If your gRNA metadata table (\ ``tests/data/test_guide_info.csv`` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal,
+
      +
      +
      bean-run sorting tiling \
+./bean_count_${screen_id}_alleleFiltered.h5ad \
+-o tests/test_res/var/ \
+--fit-negctrl \
+--scale-by-acc \
+--accessibility-bw accessibility.bw
+
      +
      +
      2. +

    2. From reporter

      +
      bean-run sorting tiling \
+./bean_count_${screen_id}_alleleFiltered.h5ad \
+-o tests/test_res/var/ \
+--fit-negctrl
+
      +
      +
      3. +
    3. +
      No reporter information, assume the same editing efficiency of all gRNAs.

      Use this option if your data don’t have editing rate information.

      +
      +
      +
      bean-run sorting tiling \
+./bean_count_${screen_id}_alleleFiltered.h5ad \
+-o tests/test_res/var/ \
+--fit-negctrl \
+--uniform-edit
+
      +
      +
      4. +
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    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file diff --git a/docs/_build/tutorials/ldl_var.html b/docs/_build/tutorials/ldl_var.html new file mode 100644 index 0000000..d03177f --- /dev/null +++ b/docs/_build/tutorials/ldl_var.html @@ -0,0 +1,232 @@ + + + + + + + + Variant sorting screen tutorial — bean 1.0.0 documentation + + + + + + + + + + + + + + + + + +
    +
    +
    + + +
    + +
    +

    Variant sorting screen tutorial¶

    +

    GWAS variant screen with per-variant gRNA tiling design, selected based on FACS signal quantiles.

    + + + + + + + + + +
    Library designVariant (gRNAs tile each target variant)
    variant library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design



    +
    +

    Example workflow¶

    +
    screen_id=my_sorting_tiling_screen
+
+# 1. Count gRNA & reporter
+bean-count-samples \
+--input tests/data/sample_list.csv    `# Contains fastq file path; see test file for example.`\
+-b A                                  `# Base A is edited (into G)` \
+-f tests/data/test_guide_info.csv     `# Contains gRNA metadata; see test file for example.`\
+-o ./                                 `# Output directory` \
+-r                                    `# Quantify reporter edits` \
+-n ${screen_id}                          `# ID of the screen to be counted`
+
+# 2. QC samples & guides
+bean-qc \
+  bean_count_${screen_id}.h5ad             `# Input ReporterScreen .h5ad file path` \
+  -o bean_count_${screen_id}_masked.h5ad   `# Output ReporterScreen .h5ad file path` \
+  -r qc_report_${screen_id}                `# Prefix for QC report` \
+  -b                                       ` # Remove replicates with no good samples.
+
+# 3. Quantify variant effect
+bean-run sorting variant \
+    tests/data/bean_count_${screen_id}_masked.h5ad \
+    -o tests/test_res/var/ \
+    --fit-negctrl \
+    --scale-by-acc \
+    --accessibility-col accessibility
+
    +
    +

    See more details below.

    +
    +
    +

    1. Count gRNA & reporter (bean count-samples)¶

    +
    screen_id=my_sorting_tiling_screen
+
+# 1. Count gRNA & reporter
+bean-count-samples \
+--input tests/data/sample_list.csv    `# Contains fastq file path; see test file for example.`\
+-b A                                  `# Base A is edited (into G)` \
+-f tests/data/test_guide_info.csv     `# Contains gRNA metadata; see test file for example.`\
+-o ./                                 `# Output directory` \
+-r                                    `# Quantify reporter edits` \
+-n ${screen_id}                          `# ID of the screen to be counted`
+
    +
    +

    Make sure you follow the input file format for seamless downstream steps. This will produce ./bean_count_${screen_id}.h5ad.

    +
    +
    +

    2. QC samples & guides (bean qc)¶

    +

    Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the input file guideline, but you can change the parameters with the full argument list of ``bean-qc` <../../README#bean-qc-qc-of-reporter-screen-data>`_. (Common factors you may want to tweak is --ctrl-cond=bulk and --lfc-conds=top,bot if you have different sample condition labels.)

    +
    bean-qc \
+  bean_count_${screen_id}.h5ad    `# Input ReporterScreen .h5ad file path` \
+  -o bean_count_${screen_id}_masked.h5ad   `# Output ReporterScreen .h5ad file path` \
+  -r qc_report_${screen_id}   `# Prefix for QC report`
+
    +
    +

    If the data does not include reporter editing data, you can provide --no-editing flag to omit the editing rate QC.

    +
    +
    +

    3. Quantify variant effect (bean run)¶

    +

    bean-run can take 3 run options to quantify editing rate:

    +
      +
    1. +
      From reporter + accessibility

      If your gRNA metadata table (tests/data/test_guide_info.csv above) included per-gRNA accessibility score,

      +
      +
      +
         bean-run sorting variant \
+   tests/data/bean_count_${screen_id}_masked.h5ad \
+   -o tests/test_res/var/ \
+   --fit-negctrl \
+   --scale-by-acc \
+   --accessibility-col accessibility
+
+If your gRNA metadata table (\ ``tests/data/test_guide_info.csv`` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal,
+
      +
      +
      bean-run sorting variant \
+tests/data/bean_count_${screen_id}_masked.h5ad \
+-o tests/test_res/var/ \
+--fit-negctrl \
+--scale-by-acc \
+--accessibility-bw accessibility.bw
+
      +
      +
      2. +

    2. From reporter, without accessibility

      +
      +

      This assumes the all target sites have the uniform chromatin accessibility.

      +
      +
      bean-run sorting variant \
+tests/data/bean_count_${screen_id}_masked.h5ad \
+-o tests/test_res/var/ \
+--fit-negctrl
+
      +
      +
      3. +
    3. +
      No reporter information, assume the same editing efficiency of all gRNAs.

      Use this option if your data don’t have editing outcome information.

      +
      +
      +
      bean-run sorting variant \
+tests/data/bean_count_${screen_id}_masked.h5ad \
+-o tests/test_res/var/ \
+--fit-negctrl \
+--uniform-edit
+
      +
      +
      4. +
    +
    +
    + + +
    + +
    +
    + +
    +
    +
    + ©2024, Jayoung Ryu. + + | + Powered by Sphinx 7.2.6 + & Alabaster 0.7.16 + + | + Page source +
    + + + + + + \ No newline at end of file From b954a94b5bccdb33d8ddce002e547a4f53dd3347 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 00:31:30 -0400 Subject: [PATCH 058/107] remove incorrect import --- bean/cli/run.py | 1 - 1 file changed, 1 deletion(-) diff --git a/bean/cli/run.py b/bean/cli/run.py index 064cf46..a028d35 100755 --- a/bean/cli/run.py +++ b/bean/cli/run.py @@ -28,7 +28,6 @@ from bean.model.run import ( run_inference, _get_guide_target_info, - parse_args, check_args, identify_model_guide, identify_negctrl_model_guide, From 2519ccf8996182bc6404c9f0bb53e3b794262d63 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 00:37:46 -0400 Subject: [PATCH 059/107] deploy artifact --- .github/workflows/documentation.yml | 24 ++++++++++++++++-------- 1 file changed, 16 insertions(+), 8 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 60c645e..95ba338 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -16,14 +16,22 @@ jobs: run: | pip install sphinx sphinx_markdown_builder sphinx_rtd_theme sphinx-argparse m2r pandas bio sudo apt-get install python3-distutils - - uses: actions/checkout@v3 - name: Build Documentation working-directory: docs run: sphinx-build . _build - - name: Push documentation - run: | - git config --global user.name 'Your Name' - git config --global user.email 'your-username@users.noreply.github.com' - git add --all docs/_build/ - git commit -m "Documentation generated" - git push + + - uses: actions/upload-artifact@v3 + with: + name: Documentation + path: docs/_build/ + deploy: + environment: + name: github-pages + url: ${{ steps.deployment.outputs.page_url }} + needs: build + runs-on: ubuntu-latest + name: Deploy + steps: + - name: Deploy to GitHub Pages + id: deployment + uses: actions/deploy-pages@v4 From a3fcae3d7df0b5f0a7855f88762414fd52c0dab2 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 00:42:58 -0400 Subject: [PATCH 060/107] add permission --- .github/workflows/documentation.yml | 3 ++- 1 file changed, 2 insertions(+), 1 deletion(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 95ba338..511594f 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -1,7 +1,8 @@ name: "Sphinx: Render docs" on: push - +permissions: + id-token: write jobs: build: runs-on: ubuntu-latest From 15c7cc2e7cf8524e58f4826c40f3d9a89861ec40 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 00:44:55 -0400 Subject: [PATCH 061/107] change name of the artifact --- .github/workflows/documentation.yml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 511594f..114b9ad 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -23,7 +23,7 @@ jobs: - uses: actions/upload-artifact@v3 with: - name: Documentation + name: github-pages path: docs/_build/ deploy: environment: From 45e5ebbfd35004adf33865bd9cb813069c60da8f Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 00:47:21 -0400 Subject: [PATCH 062/107] unify version --- .github/workflows/documentation.yml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 114b9ad..8045c9e 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -21,7 +21,7 @@ jobs: working-directory: docs run: sphinx-build . _build - - uses: actions/upload-artifact@v3 + - uses: actions/upload-artifact@v4 with: name: github-pages path: docs/_build/ From 4e9d6badbb176abfee7e38f01e77f51cce116bdc Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 00:49:40 -0400 Subject: [PATCH 063/107] add permission --- .github/workflows/documentation.yml | 1 + 1 file changed, 1 insertion(+) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 8045c9e..3f45f5f 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -3,6 +3,7 @@ name: "Sphinx: Render docs" on: push permissions: id-token: write + pages: write jobs: build: runs-on: ubuntu-latest From be458a2dac1c8027200711121c5cf9b7c7487818 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 00:59:29 -0400 Subject: [PATCH 064/107] edit --- .github/workflows/documentation.yml | 1 + docs/conf.py | 2 +- docs/{index_.rst => index.rst} | 0 3 files changed, 2 insertions(+), 1 deletion(-) rename docs/{index_.rst => index.rst} (100%) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 3f45f5f..3fc3102 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -26,6 +26,7 @@ jobs: with: name: github-pages path: docs/_build/ + deploy: environment: name: github-pages diff --git a/docs/conf.py b/docs/conf.py index ba6c48a..0a8e58c 100755 --- a/docs/conf.py +++ b/docs/conf.py @@ -19,7 +19,7 @@ templates_path = ["_templates"] exclude_patterns = ["_build", "Thumbs.db", ".DS_Store"] -root_doc = "index_" +root_doc = "index" numpydoc_show_class_members = False # -- Options for HTML output ------------------------------------------------- diff --git a/docs/index_.rst b/docs/index.rst similarity index 100% rename from docs/index_.rst rename to docs/index.rst From 4ba1960c7e130578766c90ced67e32129619344e Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 01:05:42 -0400 Subject: [PATCH 065/107] use correct page artifact --- .github/workflows/documentation.yml | 4 ++-- bean/cli/run.py | 10 +++++----- 2 files changed, 7 insertions(+), 7 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 3fc3102..e3b8c69 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -22,7 +22,7 @@ jobs: working-directory: docs run: sphinx-build . _build - - uses: actions/upload-artifact@v4 + - uses: actions/upload-pages-artifact@v1 with: name: github-pages path: docs/_build/ @@ -37,4 +37,4 @@ jobs: steps: - name: Deploy to GitHub Pages id: deployment - uses: actions/deploy-pages@v4 + uses: actions/deploy-pages@v4 \ No newline at end of file diff --git a/bean/cli/run.py b/bean/cli/run.py index a028d35..74533db 100755 --- a/bean/cli/run.py +++ b/bean/cli/run.py @@ -178,11 +178,11 @@ def main(args): if not os.path.exists(prefix): os.makedirs(prefix) with open(f"{prefix}/{model_label}.result{args.result_suffix}.pkl", "wb") as handle: - try: - pkl.dump(param_history_dict, handle) - except TypeError as exc: - print(exc.message) - # print(param_history_dict) + # try: + pkl.dump(param_history_dict, handle) + # except TypeError as exc: + # print(exc.message) + # print(param_history_dict) write_result_table( target_info_df, param_history_dict, From c7beb55a53ac682697d3d657fcc211b8a5694dd1 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 01:14:45 -0400 Subject: [PATCH 066/107] roll back to deploy-pages v3 --- .github/workflows/documentation.yml | 14 +++++++------- 1 file changed, 7 insertions(+), 7 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index e3b8c69..dbe10e6 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -1,9 +1,7 @@ name: "Sphinx: Render docs" on: push -permissions: - id-token: write - pages: write + jobs: build: runs-on: ubuntu-latest @@ -22,19 +20,21 @@ jobs: working-directory: docs run: sphinx-build . _build - - uses: actions/upload-pages-artifact@v1 + - uses: actions/upload-pages-artifact@v3 with: name: github-pages path: docs/_build/ deploy: + needs: build + permissions: + id-token: write + pages: write environment: name: github-pages url: ${{ steps.deployment.outputs.page_url }} - needs: build runs-on: ubuntu-latest - name: Deploy steps: - name: Deploy to GitHub Pages id: deployment - uses: actions/deploy-pages@v4 \ No newline at end of file + uses: actions/deploy-pages@v3 \ No newline at end of file From d29be47f21558903630b67f6b73a2306c421c516 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 01:20:48 -0400 Subject: [PATCH 067/107] return state instead of paramStore --- bean/cli/run.py | 6 ++++-- bean/model/run.py | 2 +- 2 files changed, 5 insertions(+), 3 deletions(-) diff --git a/bean/cli/run.py b/bean/cli/run.py index 74533db..36e7617 100755 --- a/bean/cli/run.py +++ b/bean/cli/run.py @@ -167,8 +167,10 @@ def main(args): f"Using {len(negctrl_idx)} negative control elements to adjust phenotypic effect sizes..." ) ndata_negctrl = ndata[negctrl_idx] - param_history_dict["negctrl"] = run_inference( - negctrl_model, negctrl_guide, ndata_negctrl, num_steps=args.n_iter + param_history_dict["negctrl"] = deepcopy( + run_inference( + negctrl_model, negctrl_guide, ndata_negctrl, num_steps=args.n_iter + ) ) outfile_path = ( diff --git a/bean/model/run.py b/bean/model/run.py index 74c73ff..f6ccea9 100755 --- a/bean/model/run.py +++ b/bean/model/run.py @@ -189,7 +189,7 @@ def run_inference( ) return { "loss": losses, - "params": pyro.get_param_store(), + "params": pyro.get_param_store().get_state(), } From f075ba06e59e1a45bb0c04c4e58aae870a122137 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 01:25:45 -0400 Subject: [PATCH 068/107] back to v4 --- .github/workflows/documentation.yml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index dbe10e6..f36a502 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -37,4 +37,4 @@ jobs: steps: - name: Deploy to GitHub Pages id: deployment - uses: actions/deploy-pages@v3 \ No newline at end of file + uses: actions/deploy-pages@v4 \ No newline at end of file From 78295c365aad2d637037a275df70e1266a2fb833 Mon Sep 17 00:00:00 2001 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    ReporterScreen API tutorial¶

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    Load the required packages. (Anndata import isn’t required to use the package).

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    import numpy as np
-import pandas as pd
-import anndata as ad
-import seaborn as sns
-import matplotlib.pyplot as plt
-import bean as br
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    bean ReporterScreen object and perturb-seq Screen object are both anndata compatible.

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    adata = ad.read_h5ad("bean_count_07+1021_LDLvar.h5ad")
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    adata
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    -
    -
    AnnData object with n_obs × n_vars = 3455 × 12
-    obs: 'name', 'Unnamed: 0', 'Target gene/variant', 'Target descriptor', 'Arbitrary number', 'gRNA position category', 'Target base position in gRNA', 'Target base position in reporter', 'BE', 'Group', 'sequence', 'Reporter', 'barcode', '5-nt PAM', 'offset', 'target', 'target_pos', 'Group2', 'masked_sequence', 'masked_barcode', 'edit_rate'
-    var: 'index', 'sort', 'replicate'
-    uns: 'allele_counts', 'edit_counts'
-    layers: 'X_bcmatch', 'edits'
-
    -
    -
    cdata = br.read_h5ad("bean_count_07+1021_LDLvar.h5ad")
-
    -
    -
    cdata
-
    -
    -
    Genome Editing Screen comprised of n_guides x n_conditions = 3455 x 12
-   guides:    'name', 'Unnamed: 0', 'Target gene/variant', 'Target descriptor', 'Arbitrary number', 'gRNA position category', 'Target base position in gRNA', 'Target base position in reporter', 'BE', 'Group', 'sequence', 'Reporter', 'barcode', '5-nt PAM', 'offset', 'target', 'target_pos', 'Group2', 'masked_sequence', 'masked_barcode', 'edit_rate'
-   samples:    'index', 'sort', 'replicate'
-   condit_m:
-   condit_p:
-   layers:    'X_bcmatch', 'edits'
-   uns:       'allele_counts', 'edit_counts'
-
    -
    -
      -

    • cdata.X: guide count

      • -

    • cdata.guides: guide metadata

      • -

    • cdata.samples: sample/condition metadata

      • -

    • cdata.layers["X_bcmatch"]: barcode-matched guide counts

      • -

    • cdata.layers["edits"]: edit counts

      • -

    • cdata.uns["allele_counts"]: allele counts per guide and condition

      • -

    • cdata.uns["edit_counts"]: edit counts per guide and condition

      • -
    -

    guides attribute contains the information about each guide.

    -
    cdata.guides
-
    -
    -
    - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
    nameUnnamed: 0Target gene/variantTarget descriptorArbitrary numbergRNA position categoryTarget base position in gRNATarget base position in reporterBEGroup...Reporterbarcode5-nt PAMoffsettargettarget_posGroup2masked_sequencemasked_barcodeedit_rate
    0CONTROL_1_g10CONTROLNaN1g1410ABENegCtrl...CCAAGCCCTACGCGGTAGGGAACTTTGGGAGCGTTTGGGAG-10CONTROL_19NegCtrlCCTGCGCGGTGGGGGGCTTTGTTT0.531163
    1CONTROL_1_g21CONTROLNaN1g2511ABENegCtrl...TCCAAGCCCTACGCGGTAGGGAACTTTGGGAGAACATGGGA-11CONTROL_110NegCtrlCCCTGCGCGGTGGGGGGCTTGGCG0.640765
    2CONTROL_1_g32CONTROLNaN1g3512ABENegCtrl...GTCCAAGCCCTACGCGGTAGGGAACTTTGGGACGCTTTGGG-12CONTROL_111NegCtrlCCCTGCGCGGTGGGGGGCTCGCT0.417709
    3CONTROL_1_g43CONTROLNaN1g4713ABENegCtrl...CGTCCAAGCCCTACGCGGTAGGGAACTTTGGGTGAGTTTGG-13CONTROL_112NegCtrlGGCCCTGCGCGGTGGGGGGCTGGG0.126400
    4CONTROL_1_g54CONTROLNaN1g5814ABENegCtrl...ACGTCCAAGCCCTACGCGGTAGGGAACTTTGGGTATCTTTG-14CONTROL_113NegCtrlGGGCCCTGCGCGGTGGGGGGGTGT0.201104
    ..................................................................
    3450rs9987289_Maj_ABE_347_g13450rs9987289Maj347g1310ABEVariant...TGCTTGGGCATCAATATCACGTGGAACCAGCCCAGTCCAGC-10rs9987289_Maj_ABE_3479VariantGCGTCGGTGTCGCGTGGGGCGGT0.087379
    3451rs9987289_Maj_ABE_347_g23451rs9987289Maj347g2411ABEVariant...ATGCTTGGGCATCAATATCACGTGGAACCAGCTCGCACCAG-11rs9987289_Maj_ABE_34710VariantGGCGTCGGTGTCGCGTGGGTCGC0.299923
    3452rs9987289_Maj_ABE_347_g33452rs9987289Maj347g3612ABEVariant...GATGCTTGGGCATCAATATCACGTGGAACCAGGCACAACCA-12rs9987289_Maj_ABE_34711VariantTGGGCGTCGGTGTCGCGTGGGCGC0.224973
    3453rs9987289_Maj_ABE_347_g43453rs9987289Maj347g4713ABEVariant...AGATGCTTGGGCATCAATATCACGTGGAACCATTGCGAACC-13rs9987289_Maj_ABE_34712VariantTTGGGCGTCGGTGTCGCGTGTTGC0.265378
    3454rs9987289_Maj_ABE_347_g53454rs9987289Maj347g5814ABEVariant...TAGATGCTTGGGCATCAATATCACGTGGAACCGCGAGGAAC-14rs9987289_Maj_ABE_34713VariantCTTGGGCGTCGGTGTCGCGTGCGG0.266573
    -

    3455 rows × 21 columns

    -

    samples attribute contains the sample and condition specific information.

    -
    cdata.samples
-
    -
    -
    - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
    indexsortreplicate
    0rep1_botbotrep1
    1rep2_botbotrep2
    2rep3_VPA_botbotrep3_VPA
    3rep4_VPA_botbotrep4_VPA
    4rep1_bulkbulkrep1
    5rep2_bulkbulkrep2
    6rep3_VPA_bulkbulkrep3_VPA
    7rep4_VPA_bulkbulkrep4_VPA
    8rep1_toptoprep1
    9rep2_toptoprep2
    10rep3_VPA_toptoprep3_VPA
    11rep4_VPA_toptoprep4_VPA
    -

    Allele_counts information is stored in .uns["allele_counts"].

    -
    cdata.uns["allele_counts"]
-
    -
    -
    - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
    guideallelerep1_botrep2_botrep3_VPA_botrep4_VPA_botrep1_bulkrep2_bulkrep3_VPA_bulkrep4_VPA_bulkrep1_toprep2_toprep3_VPA_toprep4_VPA_top
    012:51779544AGA_Maj_ABE_2_g10:9:+:A>G,5:14:+:A>G14201306152172214343
    112:51779544AGA_Maj_ABE_2_g1-4:5:+:A>G,-2:7:+:A>G,5:14:+:A>G,10:19:+:A>G100000000000
    212:51779544AGA_Maj_ABE_2_g1-7:2:+:A>G,0:9:+:A>G,5:14:+:A>G342010520010
    312:51779544AGA_Maj_ABE_2_g1-9:0:+:G>A,-8:1:+:G>A,-7:2:+:A>C,-6:3:+:C>A,-4...100102100010
    412:51779544AGA_Maj_ABE_2_g1-7:2:+:A>G,10:19:+:A>G110000000000
    .............................................
    438407rs9987289_Maj_ABE_347_g54:17:+:A>G,6:19:+:A>G,9:22:+:A>G000000000020
    438408rs9987289_Maj_ABE_347_g5-12:1:+:A>G,6:19:+:A>G,9:22:+:A>G,11:24:+:G>A000000000010
    438409rs9987289_Maj_ABE_347_g5-12:1:+:A>G,6:19:+:A>G,9:22:+:A>G,16:29:+:A>G000000000001
    438410rs9987289_Maj_ABE_347_g5-12:1:+:A>G,0:13:+:A>G,6:19:+:A>G,9:22:+:A>G,1...000000000100
    438411rs9987289_Maj_ABE_347_g5-12:1:+:A>G,6:19:+:A>G,9:22:+:A>G,12:25:+:T>G000000000001
    -

    438412 rows × 14 columns

    -

    Base-level edit counts can be saved at .uns[“edit_countsâ€].

    -
    cdata.uns["edit_counts"]
-
    -
    -
    - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
    guideeditrep1_botrep2_botrep3_VPA_botrep4_VPA_botrep1_bulkrep2_bulkrep3_VPA_bulkrep4_VPA_bulkrep1_toprep2_toprep3_VPA_toprep4_VPA_topref_basealt_base
    012:51779544AGA_Maj_ABE_2_g1-1:8:+:G>A000010000000GA
    112:51779544AGA_Maj_ABE_2_g1-1:8:+:G>C000000001010GC
    212:51779544AGA_Maj_ABE_2_g1-1:8:+:G>T000010000000GT
    312:51779544AGA_Maj_ABE_2_g1-2:7:+:A>C000000002010AC
    412:51779544AGA_Maj_ABE_2_g1-2:7:+:A>G1934404592566768481492AG
    ...................................................
    217563rs9987289_Maj_ABE_347_g58:21:+:C>A070001101000CA
    217564rs9987289_Maj_ABE_347_g58:21:+:C>G002008000180CG
    217565rs9987289_Maj_ABE_347_g58:21:+:C>T007000700000CT
    217566rs9987289_Maj_ABE_347_g59:22:+:A>G92130513746122058235947AG
    217567rs9987289_Maj_ABE_347_g59:22:+:A>T000000070000AT
    -

    217568 rows × 16 columns

    -
    -

    Subsetting & addition¶

    -

    Works as anndata, supports allele & edit count operations.

    -
    -

    Subsetting & selection¶

    -
    cdata_subset = cdata[:10,cdata.samples.sort == "bulk"]
-
    -
    -
    ['rep1_bulk', 'rep2_bulk', 'rep3_VPA_bulk', 'rep4_VPA_bulk']
-
    -
    -
    cdata_subset.uns["allele_counts"]
-
    -
    -
    - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
    guideallelerep1_bulkrep2_bulkrep3_VPA_bulkrep4_VPA_bulk
    14979CONTROL_10_g1-4:5:+:A>G,0:9:+:A>G8130
    14980CONTROL_10_g1-7:2:+:C>T00010
    14981CONTROL_10_g1-4:5:+:A>G2922925
    14982CONTROL_10_g11:10:+:A>G0641
    14983CONTROL_10_g1-4:5:+:A>G,1:10:+:A>G111512
    .....................
    22837CONTROL_1_g5-13:0:+:A>-,-12:1:+:C>T,-9:4:+:C>G,-8:5:+:C>T,...0000
    22838CONTROL_1_g5-6:7:+:A>C,7:20:+:A>G0000
    22839CONTROL_1_g5-13:0:+:A>G,-10:3:+:T>G,0:13:+:A>G,7:20:+:A>G0000
    22840CONTROL_1_g50:13:+:A>T0000
    22841CONTROL_1_g50:13:+:A>G,18:31:+:G>A0000
    -

    1080 rows × 6 columns

    -
    -
    -

    LFC calculation & Addition¶

    -
    cdata1 = br.read_h5ad("/data/pinello/PROJECTS/2021_08_ANBE/data/072121_ABE_topbot/bean_counts/LDLvar/032422_crispresso/bean_count_072121_ABE_topbot_LDLvar.h5ad")
-cdata2 = br.read_h5ad("/data/pinello/PROJECTS/2021_08_ANBE/data/102121_ABE_topbot/bean_counts/LDLvar/032422_crispresso/bean_count_102121_ABE_topbot_LDLvar.h5ad")
-
    -
    -
    cdata1.samples["sort"] = cdata1.samples["index"].map(lambda s: s.rsplit("_", 1)[-1])
-cdata1.samples["replicate"] = cdata1.samples["index"].map(lambda s: s.rsplit("_", 1)[0])
-cdata2.samples["sort"] = cdata2.samples["index"].map(lambda s: s.rsplit("_", 1)[-1])
-cdata2.samples["replicate"] = cdata2.samples["index"].map(lambda s: s.rsplit("_", 1)[0])
-
    -
    -
    cdata1.log_norm()
-lfc1 = cdata1.log_fold_change_reps("bot", "top")
-cdata2.log_norm()
-lfc2 = cdata2.log_fold_change_reps("bot", "top")
-lfcs = lfc1.join(lfc2, lsuffix = "_1", rsuffix = "_2")
-sns.pairplot(lfcs)
-
    -
    -_images/output_20_2.png -

    LFC can be aggregated for biological replicates.

    -
    cdata1.log_fold_change_aggregate("bot", "top", aggregate_condit = "replicate")
-
    -
    -
    cdata1.guides
-
    -
    -
    - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
    nameUnnamed: 0Target gene/variantTarget descriptorArbitrary numbergRNA position categoryTarget base position in gRNATarget base position in reporterBEGroup...Reporterbarcode5-nt PAMoffsettargettarget_posGroup2masked_sequencemasked_barcodebot_top.lfc.median
    0CONTROL_1_g10CONTROLNaN1g1410ABENegCtrl...CCAAGCCCTACGCGGTAGGGAACTTTGGGAGCGTTTGGGAG-10CONTROL_19NegCtrlCCTGCGCGGTGGGGGGCTTTGTTT-0.158787
    1CONTROL_1_g21CONTROLNaN1g2511ABENegCtrl...TCCAAGCCCTACGCGGTAGGGAACTTTGGGAGAACATGGGA-11CONTROL_110NegCtrlCCCTGCGCGGTGGGGGGCTTGGCG-0.212254
    2CONTROL_1_g32CONTROLNaN1g3512ABENegCtrl...GTCCAAGCCCTACGCGGTAGGGAACTTTGGGACGCTTTGGG-12CONTROL_111NegCtrlCCCTGCGCGGTGGGGGGCTCGCT0.186679
    3CONTROL_1_g43CONTROLNaN1g4713ABENegCtrl...CGTCCAAGCCCTACGCGGTAGGGAACTTTGGGTGAGTTTGG-13CONTROL_112NegCtrlGGCCCTGCGCGGTGGGGGGCTGGG-0.022441
    4CONTROL_1_g54CONTROLNaN1g5814ABENegCtrl...ACGTCCAAGCCCTACGCGGTAGGGAACTTTGGGTATCTTTG-14CONTROL_113NegCtrlGGGCCCTGCGCGGTGGGGGGGTGT0.457033
    ..................................................................
    3450rs9987289_Maj_ABE_347_g13450rs9987289Maj347g1310ABEVariant...TGCTTGGGCATCAATATCACGTGGAACCAGCCCAGTCCAGC-10rs9987289_Maj_ABE_3479VariantGCGTCGGTGTCGCGTGGGGCGGT-0.418312
    3451rs9987289_Maj_ABE_347_g23451rs9987289Maj347g2411ABEVariant...ATGCTTGGGCATCAATATCACGTGGAACCAGCTCGCACCAG-11rs9987289_Maj_ABE_34710VariantGGCGTCGGTGTCGCGTGGGTCGC-0.084936
    3452rs9987289_Maj_ABE_347_g33452rs9987289Maj347g3612ABEVariant...GATGCTTGGGCATCAATATCACGTGGAACCAGGCACAACCA-12rs9987289_Maj_ABE_34711VariantTGGGCGTCGGTGTCGCGTGGGCGC-0.339419
    3453rs9987289_Maj_ABE_347_g43453rs9987289Maj347g4713ABEVariant...AGATGCTTGGGCATCAATATCACGTGGAACCATTGCGAACC-13rs9987289_Maj_ABE_34712VariantTTGGGCGTCGGTGTCGCGTGTTGC-0.517138
    3454rs9987289_Maj_ABE_347_g53454rs9987289Maj347g5814ABEVariant...TAGATGCTTGGGCATCAATATCACGTGGAACCGCGAGGAAC-14rs9987289_Maj_ABE_34713VariantCTTGGGCGTCGGTGTCGCGTGCGG0.002245
    -

    3455 rows × 21 columns

    -

    Technical replicates show decent LFC correlation.

    -
    cdata = cdata1 + cdata2
-
    -
    -
    cdata
-
    -
    -
    Genome Editing Screen comprised of n_guides x n_conditions = 3455 x 12
-   guides:    'name', 'Unnamed: 0', 'Target gene/variant', 'Target descriptor', 'Arbitrary number', 'gRNA position category', 'Target base position in gRNA', 'Target base position in reporter', 'BE', 'Group', 'sequence', 'Reporter', 'barcode', '5-nt PAM', 'offset', 'target', 'target_pos', 'Group2', 'masked_sequence', 'masked_barcode', 'bot_top.lfc.median'
-   samples:    'index', 'sort', 'replicate'
-   condit_m:
-   condit_p:
-   layers:    'edits', 'X_bcmatch'
-   uns:       'allele_counts'
-
    -
    -

    You can concatenate different samples with shared guides.

    -
    br.concat((cdata1, cdata2))
-
    -
    -
    Genome Editing Screen comprised of n_guides x n_conditions = 3455 x 24
-   guides:    'name', 'Unnamed: 0', 'Target gene/variant', 'Target descriptor', 'Arbitrary number', 'gRNA position category', 'Target base position in gRNA', 'Target base position in reporter', 'BE', 'Group', 'sequence', 'Reporter', 'barcode', '5-nt PAM', 'offset', 'target', 'target_pos', 'Group2', 'masked_sequence', 'masked_barcode', 'bot_top.lfc.median'
-   samples:    'index', 'sort', 'replicate'
-   condit_m:
-   condit_p:
-   layers:    'X', 'X_bcmatch', 'edits', 'lognorm_counts', 'lognorm_edits'
-   uns:       'allele_counts'
-
    -
    -
    -
    -
    -

    Getting edit rates from allele counts¶

    -
    cdata.get_edit_rate(normalize_by_editable_base = False,
-                   edited_base = "A",
-                   editable_base_start = 3,
-                   editable_base_end = 8,
-                   bcmatch_thres = 10,
-                   prior_weight = 1)
-
    -
    -
    cdata.uns["edit_counts"] = cdata.get_edit_from_allele()
-
    -
    -
    cdata.get_edit_mat_from_uns("A", "G", match_target_position = True)
-cdata.get_edit_rate(edited_base = "A", bcmatch_thres = 10)
-plt.hist(cdata.guides.edit_rate, bins=30)
-plt.show()
-
    -
    -_images/output_34_1.png -
    -

    Calculating LFC¶

    -
    cdata.log_norm()
-cdata.log_fold_change_aggregate("bot", "top", aggregate_condit = "replicate")
-
    -
    -
    cdata.guides
-
    -
    -
    - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
    nameUnnamed: 0Target gene/variantTarget descriptorArbitrary numbergRNA position categoryTarget base position in gRNATarget base position in reporterBEGroup...barcode5-nt PAMoffsettargettarget_posGroup2masked_sequencemasked_barcodebot_top.lfc.medianedit_rate
    0CONTROL_1_g10CONTROLNaN1g1410ABENegCtrl...GTTTGGGAG-10CONTROL_19NegCtrlCCTGCGCGGTGGGGGGCTTTGTTT-0.1355500.531163
    1CONTROL_1_g21CONTROLNaN1g2511ABENegCtrl...AACATGGGA-11CONTROL_110NegCtrlCCCTGCGCGGTGGGGGGCTTGGCG-0.0593910.640765
    2CONTROL_1_g32CONTROLNaN1g3512ABENegCtrl...CGCTTTGGG-12CONTROL_111NegCtrlCCCTGCGCGGTGGGGGGCTCGCT0.1412900.417709
    3CONTROL_1_g43CONTROLNaN1g4713ABENegCtrl...TGAGTTTGG-13CONTROL_112NegCtrlGGCCCTGCGCGGTGGGGGGCTGGG-0.0723580.126400
    4CONTROL_1_g54CONTROLNaN1g5814ABENegCtrl...GTATCTTTG-14CONTROL_113NegCtrlGGGCCCTGCGCGGTGGGGGGGTGT0.2696500.201104
    ..................................................................
    3450rs9987289_Maj_ABE_347_g13450rs9987289Maj347g1310ABEVariant...CAGTCCAGC-10rs9987289_Maj_ABE_3479VariantGCGTCGGTGTCGCGTGGGGCGGT-0.2302640.087379
    3451rs9987289_Maj_ABE_347_g23451rs9987289Maj347g2411ABEVariant...TCGCACCAG-11rs9987289_Maj_ABE_34710VariantGGCGTCGGTGTCGCGTGGGTCGC-0.1821510.299923
    3452rs9987289_Maj_ABE_347_g33452rs9987289Maj347g3612ABEVariant...GCACAACCA-12rs9987289_Maj_ABE_34711VariantTGGGCGTCGGTGTCGCGTGGGCGC-0.1657780.224973
    3453rs9987289_Maj_ABE_347_g43453rs9987289Maj347g4713ABEVariant...TTGCGAACC-13rs9987289_Maj_ABE_34712VariantTTGGGCGTCGGTGTCGCGTGTTGC-0.3405900.265378
    3454rs9987289_Maj_ABE_347_g53454rs9987289Maj347g5814ABEVariant...GCGAGGAAC-14rs9987289_Maj_ABE_34713VariantCTTGGGCGTCGGTGTCGCGTGCGG0.0343650.266573
    -

    3455 rows × 22 columns

    -
    -
    -

    Allele translation¶

    -
    cdata_tiling = br.read_h5ad("../../072121_ABE_topbot/bean_counts/LDLRCDS/032422_crispresso/bean_count_072121_ABE_topbot_LDLRCDS.h5ad")
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    cdata_tiling.uns["allele_counts"].allele
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    0                                         11224415:14:+:A>G
-1                        11224401:0:+:A>G,11224415:14:+:A>G
-2                        11224410:9:+:A>G,11224415:14:+:A>G
-3         11224401:0:+:A>G,11224402:1:+:A>G,11224410:9:+...
-4                                          11224401:0:+:A>G
-                                ...
-438001    11203000:4:+:A>G,11203002:6:+:A>G,11203006:10:...
-438002    11224074:0:+:A>G,11224086:12:+:A>G,11224092:18...
-438003    0:0:+:A>G,3:3:+:A>G,11:11:+:A>G,13:13:+:A>G,17...
-438004                  11217409:23:+:G>-,11217417:31:+:->C
-438005    11226735:30:-:A>G,11226742:23:-:A>G,11226747:1...
-Name: allele, Length: 438006, dtype: object
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    -
    -
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    Writing¶

    -
    cdata.to_Excel("tmp.xlsx")
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    Writing to: tmp.xlsx
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-    Sheet 1:        X
-    Sheet 2:        edits
-    Sheet 3:        X_bcmatch
-    Sheet 4:        lognorm_counts
-    Sheet 5:        lognorm_edits
-    Sheet 6:        guides
-    Sheet 7:        samples
-    Sheet 8:        screen.uns.allele_counts
-    Sheet 9:        screen.uns.edit_counts
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    -
    -
    cdata.to_mageck_input("mageck_input.txt", target_column='target')
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    -
    -
    %%bash
-head mageck_input.txt
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    -
    -
    sgRNA       gene    0       1       2       3       4       5       6       7       8       9       10      11
-CONTROL_1_g1        CONTROL_1       171     451     251     422     573     389     456     420     835     435     794     439
-CONTROL_1_g2        CONTROL_1       145     278     257     206     364     273     389     254     527     498     768     195
-CONTROL_1_g3        CONTROL_1       333     835     488     632     898     899     780     713     1189    626     1146    603
-CONTROL_1_g4        CONTROL_1       246     663     387     448     823     595     705     600     921     595     1143    506
-CONTROL_1_g5        CONTROL_1       243     647     434     529     776     451     700     676     1062    611     928     379
-CONTROL_10_g1       CONTROL_10      138     329     229     213     422     292     432     352     409     243     390     274
-CONTROL_10_g2       CONTROL_10      187     468     402     479     643     369     428     469     796     422     787     404
-CONTROL_10_g3       CONTROL_10      57      126     83      131     281     114     184     115     300     106     299     106
-CONTROL_10_g4       CONTROL_10      66      112     120     136     182     128     169     181     256     144     258     179
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    - ©2024, Jayoung Ryu. - - | - Powered by Sphinx 7.2.6 - & Alabaster 0.7.16 - - | - Page source -
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    nameUnnamed: 0Target gene/variantTarget descriptorArbitrary numbergRNA position categoryTarget base position in gRNATarget base position in reporterBEGroup...Reporterbarcode5-nt PAMoffsettargettarget_posGroup2masked_sequencemasked_barcodeedit_rate
    0CONTROL_1_g10CONTROLNaN1g1410ABENegCtrl...CCAAGCCCTACGCGGTAGGGAACTTTGGGAGCGTTTGGGAG-10CONTROL_19NegCtrlCCTGCGCGGTGGGGGGCTTTGTTT0.531163
    1CONTROL_1_g21CONTROLNaN1g2511ABENegCtrl...TCCAAGCCCTACGCGGTAGGGAACTTTGGGAGAACATGGGA-11CONTROL_110NegCtrlCCCTGCGCGGTGGGGGGCTTGGCG0.640765
    2CONTROL_1_g32CONTROLNaN1g3512ABENegCtrl...GTCCAAGCCCTACGCGGTAGGGAACTTTGGGACGCTTTGGG-12CONTROL_111NegCtrlCCCTGCGCGGTGGGGGGCTCGCT0.417709
    3CONTROL_1_g43CONTROLNaN1g4713ABENegCtrl...CGTCCAAGCCCTACGCGGTAGGGAACTTTGGGTGAGTTTGG-13CONTROL_112NegCtrlGGCCCTGCGCGGTGGGGGGCTGGG0.126400
    4CONTROL_1_g54CONTROLNaN1g5814ABENegCtrl...ACGTCCAAGCCCTACGCGGTAGGGAACTTTGGGTATCTTTG-14CONTROL_113NegCtrlGGGCCCTGCGCGGTGGGGGGGTGT0.201104
    ..................................................................
    3450rs9987289_Maj_ABE_347_g13450rs9987289Maj347g1310ABEVariant...TGCTTGGGCATCAATATCACGTGGAACCAGCCCAGTCCAGC-10rs9987289_Maj_ABE_3479VariantGCGTCGGTGTCGCGTGGGGCGGT0.087379
    3451rs9987289_Maj_ABE_347_g23451rs9987289Maj347g2411ABEVariant...ATGCTTGGGCATCAATATCACGTGGAACCAGCTCGCACCAG-11rs9987289_Maj_ABE_34710VariantGGCGTCGGTGTCGCGTGGGTCGC0.299923
    3452rs9987289_Maj_ABE_347_g33452rs9987289Maj347g3612ABEVariant...GATGCTTGGGCATCAATATCACGTGGAACCAGGCACAACCA-12rs9987289_Maj_ABE_34711VariantTGGGCGTCGGTGTCGCGTGGGCGC0.224973
    3453rs9987289_Maj_ABE_347_g43453rs9987289Maj347g4713ABEVariant...AGATGCTTGGGCATCAATATCACGTGGAACCATTGCGAACC-13rs9987289_Maj_ABE_34712VariantTTGGGCGTCGGTGTCGCGTGTTGC0.265378
    3454rs9987289_Maj_ABE_347_g53454rs9987289Maj347g5814ABEVariant...TAGATGCTTGGGCATCAATATCACGTGGAACCGCGAGGAAC-14rs9987289_Maj_ABE_34713VariantCTTGGGCGTCGGTGTCGCGTGCGG0.266573
    -

    3455 rows × 21 columns

    - - - -:bash:`samples` attribute contains the sample and condition specific information. - -.. code:: ipython3 - - cdata.samples - - - - - -.. raw:: html - -
    - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
    indexsortreplicate
    0rep1_botbotrep1
    1rep2_botbotrep2
    2rep3_VPA_botbotrep3_VPA
    3rep4_VPA_botbotrep4_VPA
    4rep1_bulkbulkrep1
    5rep2_bulkbulkrep2
    6rep3_VPA_bulkbulkrep3_VPA
    7rep4_VPA_bulkbulkrep4_VPA
    8rep1_toptoprep1
    9rep2_toptoprep2
    10rep3_VPA_toptoprep3_VPA
    11rep4_VPA_toptoprep4_VPA
    -
    - - -Allele_counts information is stored in :bash:`.uns["allele_counts"]`. - -.. code:: ipython3 - - cdata.uns["allele_counts"] - - - - - -.. raw:: html - -
    - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
    guideallelerep1_botrep2_botrep3_VPA_botrep4_VPA_botrep1_bulkrep2_bulkrep3_VPA_bulkrep4_VPA_bulkrep1_toprep2_toprep3_VPA_toprep4_VPA_top
    012:51779544AGA_Maj_ABE_2_g10:9:+:A>G,5:14:+:A>G14201306152172214343
    112:51779544AGA_Maj_ABE_2_g1-4:5:+:A>G,-2:7:+:A>G,5:14:+:A>G,10:19:+:A>G100000000000
    212:51779544AGA_Maj_ABE_2_g1-7:2:+:A>G,0:9:+:A>G,5:14:+:A>G342010520010
    312:51779544AGA_Maj_ABE_2_g1-9:0:+:G>A,-8:1:+:G>A,-7:2:+:A>C,-6:3:+:C>A,-4...100102100010
    412:51779544AGA_Maj_ABE_2_g1-7:2:+:A>G,10:19:+:A>G110000000000
    .............................................
    438407rs9987289_Maj_ABE_347_g54:17:+:A>G,6:19:+:A>G,9:22:+:A>G000000000020
    438408rs9987289_Maj_ABE_347_g5-12:1:+:A>G,6:19:+:A>G,9:22:+:A>G,11:24:+:G>A000000000010
    438409rs9987289_Maj_ABE_347_g5-12:1:+:A>G,6:19:+:A>G,9:22:+:A>G,16:29:+:A>G000000000001
    438410rs9987289_Maj_ABE_347_g5-12:1:+:A>G,0:13:+:A>G,6:19:+:A>G,9:22:+:A>G,1...000000000100
    438411rs9987289_Maj_ABE_347_g5-12:1:+:A>G,6:19:+:A>G,9:22:+:A>G,12:25:+:T>G000000000001
    -

    438412 rows × 14 columns

    -
    - - -Base-level edit counts can be saved at `.uns["edit_counts"]`. - -.. code:: ipython3 - - cdata.uns["edit_counts"] - - - - - -.. raw:: html - -
    - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
    guideeditrep1_botrep2_botrep3_VPA_botrep4_VPA_botrep1_bulkrep2_bulkrep3_VPA_bulkrep4_VPA_bulkrep1_toprep2_toprep3_VPA_toprep4_VPA_topref_basealt_base
    012:51779544AGA_Maj_ABE_2_g1-1:8:+:G>A000010000000GA
    112:51779544AGA_Maj_ABE_2_g1-1:8:+:G>C000000001010GC
    212:51779544AGA_Maj_ABE_2_g1-1:8:+:G>T000010000000GT
    312:51779544AGA_Maj_ABE_2_g1-2:7:+:A>C000000002010AC
    412:51779544AGA_Maj_ABE_2_g1-2:7:+:A>G1934404592566768481492AG
    ...................................................
    217563rs9987289_Maj_ABE_347_g58:21:+:C>A070001101000CA
    217564rs9987289_Maj_ABE_347_g58:21:+:C>G002008000180CG
    217565rs9987289_Maj_ABE_347_g58:21:+:C>T007000700000CT
    217566rs9987289_Maj_ABE_347_g59:22:+:A>G92130513746122058235947AG
    217567rs9987289_Maj_ABE_347_g59:22:+:A>T000000070000AT
    -

    217568 rows × 16 columns

    -
    - - - - - -Subsetting & addition ---------------------- - -Works as anndata, supports allele & edit count operations. - -Subsetting & selection -~~~~~~~~~~~~~~~~~~~~~~ - -.. code:: ipython3 - - cdata_subset = cdata[:10,cdata.samples.sort == "bulk"] - - -.. parsed-literal:: - - ['rep1_bulk', 'rep2_bulk', 'rep3_VPA_bulk', 'rep4_VPA_bulk'] - - -.. code:: ipython3 - - cdata_subset.uns["allele_counts"] - - - - -.. raw:: html - -
    - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
    guideallelerep1_bulkrep2_bulkrep3_VPA_bulkrep4_VPA_bulk
    14979CONTROL_10_g1-4:5:+:A>G,0:9:+:A>G8130
    14980CONTROL_10_g1-7:2:+:C>T00010
    14981CONTROL_10_g1-4:5:+:A>G2922925
    14982CONTROL_10_g11:10:+:A>G0641
    14983CONTROL_10_g1-4:5:+:A>G,1:10:+:A>G111512
    .....................
    22837CONTROL_1_g5-13:0:+:A>-,-12:1:+:C>T,-9:4:+:C>G,-8:5:+:C>T,...0000
    22838CONTROL_1_g5-6:7:+:A>C,7:20:+:A>G0000
    22839CONTROL_1_g5-13:0:+:A>G,-10:3:+:T>G,0:13:+:A>G,7:20:+:A>G0000
    22840CONTROL_1_g50:13:+:A>T0000
    22841CONTROL_1_g50:13:+:A>G,18:31:+:G>A0000
    -

    1080 rows × 6 columns

    -
    - - - -LFC calculation & Addition -~~~~~~~~~~~~~~~~~~~~~~~~~~ - -.. code:: ipython3 - - cdata1 = br.read_h5ad("/data/pinello/PROJECTS/2021_08_ANBE/data/072121_ABE_topbot/bean_counts/LDLvar/032422_crispresso/bean_count_072121_ABE_topbot_LDLvar.h5ad") - cdata2 = br.read_h5ad("/data/pinello/PROJECTS/2021_08_ANBE/data/102121_ABE_topbot/bean_counts/LDLvar/032422_crispresso/bean_count_102121_ABE_topbot_LDLvar.h5ad") - - -.. code:: ipython3 - - cdata1.samples["sort"] = cdata1.samples["index"].map(lambda s: s.rsplit("_", 1)[-1]) - cdata1.samples["replicate"] = cdata1.samples["index"].map(lambda s: s.rsplit("_", 1)[0]) - cdata2.samples["sort"] = cdata2.samples["index"].map(lambda s: s.rsplit("_", 1)[-1]) - cdata2.samples["replicate"] = cdata2.samples["index"].map(lambda s: s.rsplit("_", 1)[0]) - -.. code:: ipython3 - - cdata1.log_norm() - lfc1 = cdata1.log_fold_change_reps("bot", "top") - cdata2.log_norm() - lfc2 = cdata2.log_fold_change_reps("bot", "top") - lfcs = lfc1.join(lfc2, lsuffix = "_1", rsuffix = "_2") - sns.pairplot(lfcs) - - -.. image:: ../imgs/output_20_2.png - - -LFC can be aggregated for biological replicates. - -.. code:: ipython3 - - cdata1.log_fold_change_aggregate("bot", "top", aggregate_condit = "replicate") - -.. code:: ipython3 - - cdata1.guides - - - - -.. raw:: html - -
    - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
    nameUnnamed: 0Target gene/variantTarget descriptorArbitrary numbergRNA position categoryTarget base position in gRNATarget base position in reporterBEGroup...Reporterbarcode5-nt PAMoffsettargettarget_posGroup2masked_sequencemasked_barcodebot_top.lfc.median
    0CONTROL_1_g10CONTROLNaN1g1410ABENegCtrl...CCAAGCCCTACGCGGTAGGGAACTTTGGGAGCGTTTGGGAG-10CONTROL_19NegCtrlCCTGCGCGGTGGGGGGCTTTGTTT-0.158787
    1CONTROL_1_g21CONTROLNaN1g2511ABENegCtrl...TCCAAGCCCTACGCGGTAGGGAACTTTGGGAGAACATGGGA-11CONTROL_110NegCtrlCCCTGCGCGGTGGGGGGCTTGGCG-0.212254
    2CONTROL_1_g32CONTROLNaN1g3512ABENegCtrl...GTCCAAGCCCTACGCGGTAGGGAACTTTGGGACGCTTTGGG-12CONTROL_111NegCtrlCCCTGCGCGGTGGGGGGCTCGCT0.186679
    3CONTROL_1_g43CONTROLNaN1g4713ABENegCtrl...CGTCCAAGCCCTACGCGGTAGGGAACTTTGGGTGAGTTTGG-13CONTROL_112NegCtrlGGCCCTGCGCGGTGGGGGGCTGGG-0.022441
    4CONTROL_1_g54CONTROLNaN1g5814ABENegCtrl...ACGTCCAAGCCCTACGCGGTAGGGAACTTTGGGTATCTTTG-14CONTROL_113NegCtrlGGGCCCTGCGCGGTGGGGGGGTGT0.457033
    ..................................................................
    3450rs9987289_Maj_ABE_347_g13450rs9987289Maj347g1310ABEVariant...TGCTTGGGCATCAATATCACGTGGAACCAGCCCAGTCCAGC-10rs9987289_Maj_ABE_3479VariantGCGTCGGTGTCGCGTGGGGCGGT-0.418312
    3451rs9987289_Maj_ABE_347_g23451rs9987289Maj347g2411ABEVariant...ATGCTTGGGCATCAATATCACGTGGAACCAGCTCGCACCAG-11rs9987289_Maj_ABE_34710VariantGGCGTCGGTGTCGCGTGGGTCGC-0.084936
    3452rs9987289_Maj_ABE_347_g33452rs9987289Maj347g3612ABEVariant...GATGCTTGGGCATCAATATCACGTGGAACCAGGCACAACCA-12rs9987289_Maj_ABE_34711VariantTGGGCGTCGGTGTCGCGTGGGCGC-0.339419
    3453rs9987289_Maj_ABE_347_g43453rs9987289Maj347g4713ABEVariant...AGATGCTTGGGCATCAATATCACGTGGAACCATTGCGAACC-13rs9987289_Maj_ABE_34712VariantTTGGGCGTCGGTGTCGCGTGTTGC-0.517138
    3454rs9987289_Maj_ABE_347_g53454rs9987289Maj347g5814ABEVariant...TAGATGCTTGGGCATCAATATCACGTGGAACCGCGAGGAAC-14rs9987289_Maj_ABE_34713VariantCTTGGGCGTCGGTGTCGCGTGCGG0.002245
    -

    3455 rows × 21 columns

    -
    - - - -Technical replicates show decent LFC correlation. - -.. code:: ipython3 - - cdata = cdata1 + cdata2 - - -.. code:: ipython3 - - cdata - - - - - -.. parsed-literal:: - - Genome Editing Screen comprised of n_guides x n_conditions = 3455 x 12 - guides: 'name', 'Unnamed: 0', 'Target gene/variant', 'Target descriptor', 'Arbitrary number', 'gRNA position category', 'Target base position in gRNA', 'Target base position in reporter', 'BE', 'Group', 'sequence', 'Reporter', 'barcode', '5-nt PAM', 'offset', 'target', 'target_pos', 'Group2', 'masked_sequence', 'masked_barcode', 'bot_top.lfc.median' - samples: 'index', 'sort', 'replicate' - condit_m: - condit_p: - layers: 'edits', 'X_bcmatch' - uns: 'allele_counts' - - - -You can concatenate different samples with shared guides. - -.. code:: ipython3 - - br.concat((cdata1, cdata2)) - - -.. parsed-literal:: - - Genome Editing Screen comprised of n_guides x n_conditions = 3455 x 24 - guides: 'name', 'Unnamed: 0', 'Target gene/variant', 'Target descriptor', 'Arbitrary number', 'gRNA position category', 'Target base position in gRNA', 'Target base position in reporter', 'BE', 'Group', 'sequence', 'Reporter', 'barcode', '5-nt PAM', 'offset', 'target', 'target_pos', 'Group2', 'masked_sequence', 'masked_barcode', 'bot_top.lfc.median' - samples: 'index', 'sort', 'replicate' - condit_m: - condit_p: - layers: 'X', 'X_bcmatch', 'edits', 'lognorm_counts', 'lognorm_edits' - uns: 'allele_counts' - - - -Getting edit rates from allele counts -------------------------------------- - -.. code:: ipython3 - - cdata.get_edit_rate(normalize_by_editable_base = False, - edited_base = "A", - editable_base_start = 3, - editable_base_end = 8, - bcmatch_thres = 10, - prior_weight = 1) - - -.. code:: ipython3 - - cdata.uns["edit_counts"] = cdata.get_edit_from_allele() - - - -.. code:: ipython3 - - cdata.get_edit_mat_from_uns("A", "G", match_target_position = True) - cdata.get_edit_rate(edited_base = "A", bcmatch_thres = 10) - plt.hist(cdata.guides.edit_rate, bins=30) - plt.show() - - -.. image:: ../imgs/output_34_1.png - - - -Calculating LFC -~~~~~~~~~~~~~~~ - -.. code:: ipython3 - - cdata.log_norm() - cdata.log_fold_change_aggregate("bot", "top", aggregate_condit = "replicate") - -.. code:: ipython3 - - cdata.guides - - - - -.. raw:: html - -
    - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
    nameUnnamed: 0Target gene/variantTarget descriptorArbitrary numbergRNA position categoryTarget base position in gRNATarget base position in reporterBEGroup...barcode5-nt PAMoffsettargettarget_posGroup2masked_sequencemasked_barcodebot_top.lfc.medianedit_rate
    0CONTROL_1_g10CONTROLNaN1g1410ABENegCtrl...GTTTGGGAG-10CONTROL_19NegCtrlCCTGCGCGGTGGGGGGCTTTGTTT-0.1355500.531163
    1CONTROL_1_g21CONTROLNaN1g2511ABENegCtrl...AACATGGGA-11CONTROL_110NegCtrlCCCTGCGCGGTGGGGGGCTTGGCG-0.0593910.640765
    2CONTROL_1_g32CONTROLNaN1g3512ABENegCtrl...CGCTTTGGG-12CONTROL_111NegCtrlCCCTGCGCGGTGGGGGGCTCGCT0.1412900.417709
    3CONTROL_1_g43CONTROLNaN1g4713ABENegCtrl...TGAGTTTGG-13CONTROL_112NegCtrlGGCCCTGCGCGGTGGGGGGCTGGG-0.0723580.126400
    4CONTROL_1_g54CONTROLNaN1g5814ABENegCtrl...GTATCTTTG-14CONTROL_113NegCtrlGGGCCCTGCGCGGTGGGGGGGTGT0.2696500.201104
    ..................................................................
    3450rs9987289_Maj_ABE_347_g13450rs9987289Maj347g1310ABEVariant...CAGTCCAGC-10rs9987289_Maj_ABE_3479VariantGCGTCGGTGTCGCGTGGGGCGGT-0.2302640.087379
    3451rs9987289_Maj_ABE_347_g23451rs9987289Maj347g2411ABEVariant...TCGCACCAG-11rs9987289_Maj_ABE_34710VariantGGCGTCGGTGTCGCGTGGGTCGC-0.1821510.299923
    3452rs9987289_Maj_ABE_347_g33452rs9987289Maj347g3612ABEVariant...GCACAACCA-12rs9987289_Maj_ABE_34711VariantTGGGCGTCGGTGTCGCGTGGGCGC-0.1657780.224973
    3453rs9987289_Maj_ABE_347_g43453rs9987289Maj347g4713ABEVariant...TTGCGAACC-13rs9987289_Maj_ABE_34712VariantTTGGGCGTCGGTGTCGCGTGTTGC-0.3405900.265378
    3454rs9987289_Maj_ABE_347_g53454rs9987289Maj347g5814ABEVariant...GCGAGGAAC-14rs9987289_Maj_ABE_34713VariantCTTGGGCGTCGGTGTCGCGTGCGG0.0343650.266573
    -

    3455 rows × 22 columns

    -
    - - - -Allele translation -~~~~~~~~~~~~~~~~~~ - -.. code:: ipython3 - - cdata_tiling = br.read_h5ad("../../072121_ABE_topbot/bean_counts/LDLRCDS/032422_crispresso/bean_count_072121_ABE_topbot_LDLRCDS.h5ad") - - -.. code:: ipython3 - - cdata_tiling.uns["allele_counts"].allele - - - - - - -.. parsed-literal:: - - 0 11224415:14:+:A>G - 1 11224401:0:+:A>G,11224415:14:+:A>G - 2 11224410:9:+:A>G,11224415:14:+:A>G - 3 11224401:0:+:A>G,11224402:1:+:A>G,11224410:9:+... - 4 11224401:0:+:A>G - ... - 438001 11203000:4:+:A>G,11203002:6:+:A>G,11203006:10:... - 438002 11224074:0:+:A>G,11224086:12:+:A>G,11224092:18... - 438003 0:0:+:A>G,3:3:+:A>G,11:11:+:A>G,13:13:+:A>G,17... - 438004 11217409:23:+:G>-,11217417:31:+:->C - 438005 11226735:30:-:A>G,11226742:23:-:A>G,11226747:1... - Name: allele, Length: 438006, dtype: object - - - -Writing -~~~~~~~ - -.. code:: ipython3 - - cdata.to_Excel("tmp.xlsx") - - -.. parsed-literal:: - - Writing to: tmp.xlsx - - Sheet 1: X - Sheet 2: edits - Sheet 3: X_bcmatch - Sheet 4: lognorm_counts - Sheet 5: lognorm_edits - Sheet 6: guides - Sheet 7: samples - Sheet 8: screen.uns.allele_counts - Sheet 9: screen.uns.edit_counts - - -.. code:: ipython3 - - cdata.to_mageck_input("mageck_input.txt", target_column='target') - -.. code:: bash - - %%bash - head mageck_input.txt - - -.. parsed-literal:: - - sgRNA gene 0 1 2 3 4 5 6 7 8 9 10 11 - CONTROL_1_g1 CONTROL_1 171 451 251 422 573 389 456 420 835 435 794 439 - CONTROL_1_g2 CONTROL_1 145 278 257 206 364 273 389 254 527 498 768 195 - CONTROL_1_g3 CONTROL_1 333 835 488 632 898 899 780 713 1189 626 1146 603 - CONTROL_1_g4 CONTROL_1 246 663 387 448 823 595 705 600 921 595 1143 506 - CONTROL_1_g5 CONTROL_1 243 647 434 529 776 451 700 676 1062 611 928 379 - CONTROL_10_g1 CONTROL_10 138 329 229 213 422 292 432 352 409 243 390 274 - CONTROL_10_g2 CONTROL_10 187 468 402 479 643 369 428 469 796 422 787 404 - CONTROL_10_g3 CONTROL_10 57 126 83 131 281 114 184 115 300 106 299 106 - CONTROL_10_g4 CONTROL_10 66 112 120 136 182 128 169 181 256 144 258 179 - diff --git a/docs/_build/_sources/cds.rst.txt b/docs/_build/_sources/cds.rst.txt deleted file mode 100644 index 5dabfde..0000000 --- a/docs/_build/_sources/cds.rst.txt +++ /dev/null @@ -1,5 +0,0 @@ -Coding sequence tiling library -*********************** -.. mdinclude:: tutorials/ldl_cds.md - -See :ref:`subcommands` for the full details. diff --git a/docs/_build/_sources/commands/count.md.txt b/docs/_build/_sources/commands/count.md.txt deleted file mode 100644 index 5f04b4c..0000000 --- a/docs/_build/_sources/commands/count.md.txt +++ /dev/null @@ -1,31 +0,0 @@ -# `bean count[-samples]`: Count (reporter) screen data -`bean count-samples` (or `bean count` for a single sample) maps guide into guide counts, **allowing for base transition in spacer sequence**. When the matched reporter information is provided, it can count the **target site edits** and **alleles produced by each guide**. Mapping is efficiently done based on [CRISPResso2](https://github.com/pinellolab/CRISPResso2) modified for base-edit-aware mapping. - - - -```python -bean count-samples \ - --input sample_list.csv `# sample with lines 'R1_filepath,R2_filepath,sample_name\n'` \ - -b A `# base that is being edited (A/G)` \ - -f sgRNA_info_table.csv `# sgRNA information` \ - -o . `# output directory` \ - -r `# read edit/allele information from reporter` \ - -t 12 `# number of threads` \ - --name my_sorting_screen `# name of this sample run` \ -``` -```python -bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r -``` -By default, `bean count[-samples]` assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure. - - Read structuren - -See full detail [below](#full-parameters). - -# Input file format -See :ref:`input` for input file formats. - -# Output file format -`count` or `count-samples` produces `.h5ad` and `.xlsx` file with guide and per-guide allele counts. -* `.h5ad`: This output file follows annotated matrix format compatible with `AnnData` and is based on `Screen` object in [purturb_tools](https://github.com/pinellolab/perturb-tools). See [Data Structure](#data-structure) section for more information. -* `.xlsx`: This output file contains `.guides`, `.samples`, `.X[_bcmatch,_edits]`. (`allele_tables` are often too large to write into an Excel!) diff --git a/docs/_build/_sources/commands/create-screen.md.txt b/docs/_build/_sources/commands/create-screen.md.txt deleted file mode 100644 index d9200b7..0000000 --- a/docs/_build/_sources/commands/create-screen.md.txt +++ /dev/null @@ -1,9 +0,0 @@ -# `bean create-screen`: Create ReporterScreen object from flat files -```bash -bean create-screen gRNA_library.csv sample_list.csv gRNA_counts_table.csv -``` -## Input - * gRNA_library.csv - * sample_list.csv - * gRNA_counts_table.csv: Table with gRNA ID in the first column and sample IDs as the column names (first row) -`gRNA_library.csv` and `sample_list.csv` should be formatted as :ref:`input`. \ No newline at end of file diff --git a/docs/_build/_sources/commands/filter.md.txt b/docs/_build/_sources/commands/filter.md.txt deleted file mode 100644 index 53d6ec1..0000000 --- a/docs/_build/_sources/commands/filter.md.txt +++ /dev/null @@ -1,37 +0,0 @@ -# `filter`: Filtering (and optionally translating) alleles -As `tiling` mode of `bean run` accounts for any robustly observed alleles, `bean filter` filters for such alleles. -```bash -bean filter my_sorting_screen_masked.h5ad \ --o my_sorting_screen_filtered.h5ad `# Output file path` \ -``` - -# Output -Above command produces -* `my_sorting_screen_filtered.h5ad` with filtered alleles stored in `.uns`, -* `my_sorting_screen_filtered.filtered_allele_stats.pdf`, and `my_sorting_screen_filtered.filter_log.txt` that report allele count stats in each filtering step. - -You may want to adjust the flitering parameters to obtain optimal balance between # guides per variant & # variants that are scored. See example outputs of filtering step [here](docs/example_filtering_output/). - - -# Translating alleles -If you want to obtain **amino acid level variant** for coding sequence tiling screens, provide coding sequence positions which variants occuring within the coding sequence will be translated. *This is optional, but **highly recommended** to increase per-(coding)variant support.* - -Allele translation - - -```bash -bean filter my_sorting_screen.h5ad \ --o my_sorting_screen_masked.h5ad \ ---translate `# Translate coding variants` \ -[ --translate-gene-name GENE_SYMBOL OR - --translate-genes-list path_to_gene_names_file.txt OR - --translate-fasta gene_exon.fa, OR - --translate-fastas-csv gene_exon_fas.csv] -``` -* When library covers a single gene, do either of the following: - 1. Feed `--translate-gene-name GENE_SYMBOL` if your `genomic_pos` column of `sgRNA_info_tbl` is compatible with [MANE transcript](https://useast.ensembl.org/info/genome/genebuild/mane.html)'s reference genome. (Per 10/23/2023, GRCh38). This will automatically load the exon positions based on MANE transcript annotation. - 2. To use your custom coding sequence and exon positions, feed `--translate-fasta gene_exon.fa` argument where `gene_exon.fa` is the FASTA file with entries of exons. [See full details here](docs/exon_fa_format.md). -* When library covers multiple genes, do either of the following: - 1. Feed `--translate-genes-list path_to_gene_names_file.txt` where `path_to_gene_names_file.txt` is file with one gene symbol per line. - 2. Feed `--translate-fastas-csv gene_exon_fas.csv` where `gene_exon_fas.csv` is the csv file with lines `gene_id,gene_exon_fasta_path` without header. Each FASTA file in `gene_exon_fasta_path` is formatted [as the single-gene FASTA file](docs/exon_fa_format.md). -* Translation will keep the variants outside the coding sequence as nucleotide-level variants, while aggregating variants leading to the same coding sequence variants. diff --git a/docs/_build/_sources/commands/input.md.txt b/docs/_build/_sources/commands/input.md.txt deleted file mode 100644 index 9fa1de9..0000000 --- a/docs/_build/_sources/commands/input.md.txt +++ /dev/null @@ -1,37 +0,0 @@ -This document describes the input files of :ref:`count_samples`. -## sgRNA_info_table.csv -File should contain following columns. -* `name`: gRNA ID column -* `sequence`: gRNA sequence -* `barcode`: R2 barcode to help match reporter to gRNA, written in the sense direction (as in R1) -* In order to use accessibility in the [variant effect quantification](#bean-run-quantify-variant-effects), provide accessibility information in one of two options. (For non-targeting guides, provide NA values (empty cell).) - * Option 1: `chrom` & `genomic_pos`: Chromosome (ex. `chr19`) and genomic position of guide sequence. You will have to provide the path to the bigwig file with matching reference version in `bean run`. - * Option 2: `accessibility_signal`: ATAC-seq signal value of the target loci of each guide. -* For variant library (gRNAs are designed to target specific variants and ignores bystander edits) - * `target`: This column denotes which target variant/element of each gRNA. This is not used in `bean count[-samples]` but required to run `bean run` in later steps. - * `target_group`: If negative/positive control gRNA will be considered in `bean qc` and/or `bean run`, specify as "NegCtrl"/"PosCtrl" in this column. - * `target_pos`: If `--match_target_pos` flag is used, input file needs `target_pos` which specifies 0-based relative position of targeted base within Reporter sequence. -* For tiling library (gRNAs tile coding / noncoding sequences) - * `strand`: Specifies gRNA strand information relative to the reference genome. - * `chrom`: Chromosome of gRNA targeted locus. - * `start_pos`: gRNA starting position in the genome. Required when you provide `strand` column. Should specify the smaller coordinate value among start and end position regardless of gRNA strandedness. - -Also see examples for [variant library](tests/data/test_guide_info.csv) and [tiling library](tests/data/test_guide_info_tiling.csv). - -## sample_list.csv -File should contain following columns with header. -* `R1_filepath`: Path to read 1 `.fastq[.gz]` file -* `R2_filepath`: Path to read 1 `.fastq[.gz]` file -* `sample_id`: ID of sequencing sample -* `replicate`: Replicate # of this sample (Should NOT contain `.`) -* `condition`: Name of the sorting bin (ex. `top`, `bot`), or label of timepoint (ex. `D5`, `D18`) - -For FACS sorting screens: -* `upper_quantile`: FACS sorting upper quantile -* `lower_quantile`: FACS sorting lower quantile - -For proliferation / survival screens: -* `time`: Numeric time following the base editing of each sample. - - -Also see examples for [FACS sorting screen](tests/data/sample_list.csv). \ No newline at end of file diff --git a/docs/_build/_sources/commands/profile.md.txt b/docs/_build/_sources/commands/profile.md.txt deleted file mode 100644 index bbe8a8c..0000000 --- a/docs/_build/_sources/commands/profile.md.txt +++ /dev/null @@ -1,8 +0,0 @@ -# `bean profile`: Profile editing patterns -```bash -bean profile my_sorting_screen.h5ad -o output_prefix `# Prefix for editing profile report` -``` -# Output -Above command produces `prefix_editing_preference.[html,ipynb]` as editing preferences ([see example](../../notebooks/profile_editing_preference.ipynb)). - -Allele translation \ No newline at end of file diff --git a/docs/_build/_sources/commands/qc.md.txt b/docs/_build/_sources/commands/qc.md.txt deleted file mode 100644 index 82b719e..0000000 --- a/docs/_build/_sources/commands/qc.md.txt +++ /dev/null @@ -1,94 +0,0 @@ -# `bean qc`: QC of reporter screen data -```bash -bean qc \ - my_sorting_screen.h5ad `# Input ReporterScreen .h5ad file path` \ - -o my_sorting_screen_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r qc_report_my_sorting_screen `# Prefix for QC report` \ - --ctrl-cond presort `# "condition" column in the control sample before selection. Mean gRNA editing rates in these samples are reported. ` \ -# Inspect the output qc_report_my_sorting_screen.html to tweak QC threshold - -bean qc \ - my_sorting_screen.h5ad \ - -o my_sorting_screen_masked.h5ad \ - -r qc_report_my_sorting_screen \ - #[--count-correlation-thres 0.7 ...]\ - -b -``` - -`bean qc` supports following quality control and masks samples with low quality. Specifically: - -Allele translation - -* Plots guide coverage and the uniformity of coverage -* Guide count correlation between samples -* Log fold change correlation when positive controls are provided -* Plots editing rate distribution -* Identify samples with low guide coverage/guide count correlation/editing rate and mask the sample in `bdata.samples.mask` -* Identify outlier guides to filter out - -# Output -Above command produces -* `my_sorting_screen_masked.h5ad` without problematic replicate and guides and with sample masks, and -* `qc_report_my_sorting_screen.[html,ipynb]` as QC report. -##### Optional arguments: -* `-o OUT_SCREEN_PATH`, `--out-screen-path OUT_SCREEN_PATH` - Path where quality-filtered ReporterScreen object to be written to -* `-r OUT_REPORT_PREFIX`, `--out-report-prefix OUT_REPORT_PREFIX` - Output prefix of qc report (prefix.html, prefix.ipynb) - -##### QC thresholds: -* `--count-correlation-thres COUNT_CORRELATION_THRES` - Correlation threshold to mask out. -* `--edit-rate-thres EDIT_RATE_THRES` - Mean editing rate threshold per sample to mask out. -* `--lfc-thres LFC_THRES` - Positive guides' correlation threshold to filter out. - -##### Run options: -* `-b`, `--remove-bad-replicates` - Remove replicates with at least two of its samples meet the QC threshold (bean run does not support having only one sorting bin sample for a replicate). -* `-i`, `--ignore-missing-samples` - If the flag is not provided, if the ReporterScreen object does not contain all condiitons for - each replicate, make fake empty samples. If the flag is provided, don't add dummy samples. -* `--no-editing` Ignore QC about editing. Can be used for QC of other editing modalities. -* `--dont-recalculate-edits` - When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from - ReporterScreen.uns['allele_count']. - -##### Input `.h5ad` formatting: -Note that these arguements will change the way the QC metrics are calculated for guides, samples, or replicates. -* `--tiling TILING` Specify that the guide library is tiling library without 'n guides per target' design -* `--replicate-label REPLICATE_LABEL` - Label of column in `bdata.samples` that describes replicate ID. -* `--sample-covariates SAMPLE_COVARIATES` - Comma-separated list of column names in `bdata.samples` that describes non-selective - experimental condition. (drug treatment, etc.) -* `--condition-label CONDITION_LABEL` - Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time, - etc.) -###### Editing rate calculation - * `--control-condition CTRL_COND` - Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate - to be calculated. Default is `None`, which considers all samples. - * `--rel-pos-is-reporter` - Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If - `False`, those are relative to spacer position. - Editing rate is calculated with following parameters in - * Variant screens: - * `--target-pos-col TARGET_POS_COL` - Target position column in `bdata.guides` specifying target edit position in reporter - * tiling screens: - * `--edit-start-pos EDIT_START_POS` - Edit start position to quantify editing rate on, 0-based inclusive. - * `--edit-end-pos EDIT_END_POS` - Edit end position to quantify editing rate on, 0-based exclusive. -###### LFC of positive controls - * `--posctrl-col POSCTRL_COL` - Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all - gRNAs, feed empty string ''. - * `--posctrl-val POSCTRL_VAL` - Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the - positive control in calculating log fold change. - * `--lfc-conds LFC_CONDS` - Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated - between, delimited by comma \ No newline at end of file diff --git a/docs/_build/_sources/commands/run.md.txt b/docs/_build/_sources/commands/run.md.txt deleted file mode 100644 index 4c45259..0000000 --- a/docs/_build/_sources/commands/run.md.txt +++ /dev/null @@ -1,64 +0,0 @@ -# `bean run`: Quantify variant effects -BEAN uses Bayesian network to incorporate gRNA editing outcome to provide posterior estimate of variant phenotype. The Bayesian network reflects data generation process. Briefly, -1. Cellular phenotype (either for cells are sorted upon for sorting screen, or log(proliferation rate)) is modeled as the Gaussian mixture distribution of wild-type phenotype and variant phenotype. -2. The weight of the mixture components are inferred from the reporter editing outcome and the chromatin accessibility of the loci. -3. Cells with each gRNA, formulated as the mixture distribution, is sorted by the phenotypic quantile to produce the gRNA counts. - -For the full detail, see the method section of the [BEAN manuscript](https://www.medrxiv.org/content/10.1101/2023.09.08.23295253v1). - -model - -

    - -# Usage example -```bash -bean run sorting[survival] variant[tiling] my_sorting_screen_filtered.h5ad \ -[--uniform-edit, --scale-by-acc [--acc-bw-path accessibility_signal.bw, --acc-col accessibility]] \ --o output_prefix/ \ ---fit-negctrl -``` -See full list of parameters [below](#full-parameters). - - -# Input -`my_sorting_screen_filtered.h5ad` can be produced by one of the following: -1. [`bean count-samples`]((#bean-count-samples-count-reporter-screen-data)) when you have raw `.fastq` file -2. (Limited to `bean run variant` mode) `bean create-screen` when you have flat `.csv` tables of gRNA metadata table, sample metadata table, gRNA counts table (# guides x # samples), and optionally # edits table. - ```bash - bean create-screen gRNA_info_table.csv sample_info_table.csv gRNA_counts_table.csv \ - [--edits edit_counts_table.csv -o output.h5ad] - ``` - * `gRNA_info_table.csv` should have following columns. - * `name`: gRNA ID column - * `target`: This column denotes which target variant/element of each gRNA. - * `target_group [Optional]`: If negative control gRNA will be used, specify as "NegCtrl" in this column. - * `sample_info_table.csv` should have following columns. - * `sample_id`: ID of sequencing sample - * `replicate`: Replicate # of this sample - * `bin`: Name of the sorting bin - * `upper_quantile`: FACS sorting upper quantile - * `lower_quantile`: FACS sorting lower quantile - * `gRNA_counts_table.csv` should be formatted as follows. - * Columns include one of `sample_id` columns in `sample_info_table.csv` file. - * 1st row (row index) follows `name` (gRNA ID) in `gRNA_info_table.csv` file. -3. You can manually create the `AnnData` object with more annotations including allele counts: see [API tutorial](#using-bean-as-python-module) for full detail. - - -# Output -model - -Above command produces -* `output_prefix/bean_element_result.[model_type].csv` with following columns: - * Estimated variant effect sizes - * `mu` (Effect size): Mean of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. - * `mu_sd`: Mean of variant phenotype `mu` is modeled as normal distribution. The column shows fitted standard deviation of `mu` that quantify the uncertainty of the variant effect. - * `mu_z`: z-score of `mu` - * `sd`: Standard deviation of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. - * `CI[0.025`, `0.975]`: Credible interval of `mu` - * When negative control is provided, above columns with `_adj` suffix are provided, which are the corresponding values adjusted for negative control. - * Metrics on per-variant evidence provided in input (provided in `tiling` mode) - * `effective_edit_rate`: Sum of per-variant editing rates over all alleles observed in the input. Allele-level editing rate is divided by the number of variants observed in the allele prior to summing up. - * `n_guides`: # of guides covering the variant. - * `n_coocc`: # of cooccurring variants with a given variant in any alleles observed in the input. -* `output_prefix/bean_sgRNA_result.[model_type].csv`: - * `edit_rate`: Estimated editing rate at the target loci. diff --git a/docs/_build/_sources/count.rst.txt b/docs/_build/_sources/count.rst.txt deleted file mode 100644 index 206dbd2..0000000 --- a/docs/_build/_sources/count.rst.txt +++ /dev/null @@ -1,10 +0,0 @@ -`bean count` -*********************** -.. mdinclude:: commands/count.md - -Full parameters -================== -.. argparse:: - :filename: ../bean/mapping/utils.py - :func: get_input_parser_count - :prog: bean count \ No newline at end of file diff --git a/docs/_build/_sources/count_samples.rst.txt b/docs/_build/_sources/count_samples.rst.txt deleted file mode 100644 index fbbf8f1..0000000 --- a/docs/_build/_sources/count_samples.rst.txt +++ /dev/null @@ -1,11 +0,0 @@ -.. _count_samples: -`bean count-samples` -*********************** -.. mdinclude:: commands/count.md - -Full parameters -================== -.. argparse:: - :filename: ../bean/mapping/utils.py - :func: get_input_parser - :prog: bean count-samples \ No newline at end of file diff --git a/docs/_build/_sources/exon_fa_format.md.txt b/docs/_build/_sources/exon_fa_format.md.txt deleted file mode 100644 index 2498b39..0000000 --- a/docs/_build/_sources/exon_fa_format.md.txt +++ /dev/null @@ -1,8 +0,0 @@ -# Input .fa file format for `bean-filter` -You can provide custom FASTA file with exon sequence entries. Currently only supports positive strand genes. - -* Exon FASTA files can be downloaded from UCSC Genomic sequences / Table Browser: [see the instruction video](https://www.youtube.com/watch?v=T4E0Ez5Vjz8) -* You can manually format as: - * Header line has ` range=chrom:start-end ` and `strand=+/-` tag that is parsed. - * fasta entry has the sequence of exons, where the first (includes 5'-UTR) and last (includes 3'-UTR) exon sequence has lower-case sequence denoting noncoding sequences. -* See the example .fa [here](../tests/data/ldlr_exons.fa). \ No newline at end of file diff --git a/docs/_build/_sources/filter.rst.txt b/docs/_build/_sources/filter.rst.txt deleted file mode 100644 index ce485ab..0000000 --- a/docs/_build/_sources/filter.rst.txt +++ /dev/null @@ -1,11 +0,0 @@ -.. _filter: -`bean filter` -*********************** -.. mdinclude:: commands/filter.md - -Full parameters -================== -.. argparse:: - :filename: ../bean/annotate/utils.py - :func: parse_args - :prog: bean filter \ No newline at end of file diff --git a/docs/_build/_sources/gwas.rst.txt b/docs/_build/_sources/gwas.rst.txt deleted file mode 100644 index e407ad9..0000000 --- a/docs/_build/_sources/gwas.rst.txt +++ /dev/null @@ -1,5 +0,0 @@ -GWAS variant library -*********************** -.. mdinclude:: tutorials/ldl_var.md - -See :ref:`subcommands` for the full details. diff --git a/docs/_build/_sources/index.md.txt b/docs/_build/_sources/index.md.txt deleted file mode 100644 index 45e2414..0000000 --- a/docs/_build/_sources/index.md.txt +++ /dev/null @@ -1,4 +0,0 @@ ---- -layout: default -title: CRISPR-BEAN ---- diff --git a/docs/_build/_sources/index_.rst.txt b/docs/_build/_sources/index_.rst.txt deleted file mode 100644 index d78bc9a..0000000 --- a/docs/_build/_sources/index_.rst.txt +++ /dev/null @@ -1,38 +0,0 @@ -.. bean documentation master file, created by - sphinx-quickstart on Fri Mar 29 19:10:46 2024. - You can adapt this file completely to your liking, but it should at least - contain the root `toctree` directive. - -Welcome to `bean`'s documentation! -================================ -=================== -Workflows -=================== -.. toctree:: - :maxdepth: 2 - - gwas - cds - input - -=================== -`bean` subcommands -=================== -.. toctree:: - :maxdepth: 3 - - subcommands - -=================== -Screen data structure -=================== -.. toctree:: - ReporterScreen_api - -================== -Indices and tables -================== - -* :ref:`genindex` -* :ref:`modindex` -* :ref:`search` diff --git a/docs/_build/_sources/input.rst.txt b/docs/_build/_sources/input.rst.txt deleted file mode 100644 index d63417e..0000000 --- a/docs/_build/_sources/input.rst.txt +++ /dev/null @@ -1,4 +0,0 @@ -.. _input: -Input file format -*********************** -.. mdinclude:: commands/input.md \ No newline at end of file diff --git a/docs/_build/_sources/profile.rst.txt b/docs/_build/_sources/profile.rst.txt deleted file mode 100644 index 3ee525f..0000000 --- a/docs/_build/_sources/profile.rst.txt +++ /dev/null @@ -1,10 +0,0 @@ -`bean profile` -*********************** -.. mdinclude:: commands/profile.md - -Full parameters -================== -.. argparse:: - :filename: ../bean/plotting/utils.py - :func: parse_args - :prog: bean profile \ No newline at end of file diff --git a/docs/_build/_sources/qc.rst.txt b/docs/_build/_sources/qc.rst.txt deleted file mode 100644 index 604e77a..0000000 --- a/docs/_build/_sources/qc.rst.txt +++ /dev/null @@ -1,11 +0,0 @@ -.. _qc: -`bean qc` -*********************** -.. mdinclude:: commands/qc.md - -Full parameters -================== -.. argparse:: - :filename: ../bean/qc/parser.py - :func: parse_args - :prog: bean qc \ No newline at end of file diff --git a/docs/_build/_sources/run.rst.txt b/docs/_build/_sources/run.rst.txt deleted file mode 100644 index a6dc1fc..0000000 --- a/docs/_build/_sources/run.rst.txt +++ /dev/null @@ -1,11 +0,0 @@ -.. _run: -`bean run` -*********************** -.. mdinclude:: commands/run.md - -Full parameters -================== -.. argparse:: - :filename: ../bean/model/parser.py - :func: parse_args - :prog: bean run \ No newline at end of file diff --git a/docs/_build/_sources/subcommands.rst.txt b/docs/_build/_sources/subcommands.rst.txt deleted file mode 100644 index 56319ca..0000000 --- a/docs/_build/_sources/subcommands.rst.txt +++ /dev/null @@ -1,14 +0,0 @@ -.. _subcommands: -=================== -Subcommands -=================== -.. toctree:: - :maxdepth: 2 - - count - count_samples - profile - qc - filter - run - create_screen \ No newline at end of file diff --git a/docs/_build/_sources/tutorials/ldl_cds.md.txt b/docs/_build/_sources/tutorials/ldl_cds.md.txt deleted file mode 100644 index ba78a00..0000000 --- a/docs/_build/_sources/tutorials/ldl_cds.md.txt +++ /dev/null @@ -1,148 +0,0 @@ -# Tiling sorting screen tutorial -Tiling screen that tiles gRNA densely across locus or multiple loci, selected based on FACS signal quantiles. - - - - - - - - - - -
    Library designTiling (gRNAs tile each locus densely)
    tiling library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design
    - -

    - -## Example workflow -```bash -screen_id=my_sorting_tiling_screen - -# 1. Count gRNA & reporter -bean-count-samples \ ---input tests/data/sample_list_tiling.csv `# Contains fastq file path; see test file for example.`\ --b A `# Base A is edited (into G)` \ --f tests/data/test_guide_info_tiling_chrom.csv `# Contains gRNA metadata; see test file for example.`\ --o ./ `# Output directory` \ --r `# Quantify reporter edits` \ --n ${screen_id} `# ID of the screen` \ ---tiling - -# 2. QC samples & guides -bean-qc \ - bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ - -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r qc_report_${screen_id} `# Prefix for QC report` \ - -# 3. Filter & translate alleles -bean-filter ./bean_count_${screen_id}_masked.h5ad \ --o ./bean_count_${screen_id}_alleleFiltered \ ---filter-target-basechange `# Filter based on intended base changes. If -b A was provided in bean-count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\ ---filter-window --edit-start-pos 0 --edit-end-pos 19 `# Filter based on editing window in spacer position within reporter.`\ ---filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \ ---translate --translate-genes-list tests/data/gene_symbols.txt - -# 4. Quantify variant effect -bean-run sorting tiling \ - ./bean_count_${screen_id}_alleleFiltered.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-col accessibility -``` -See more details below. - -## 1. Count gRNA & reporter (:ref:`count_samples`) -``` -screen_id=my_sorting_tiling_screen - -bean-count-samples \ ---input tests/data/sample_list_tiling.csv `# Contains fastq file path; see test file for example.`\ --b A `# Base A is edited (into G)` \ --f tests/data/test_guide_info_tiling_chrom.csv `# Contains gRNA metadata; see test file for example.`\ --o ./ `# Output directory` \ --r `# Quantify reporter edits` \ --n ${screen_id} `# ID of the screen` \ ---tiling -``` -Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. - -## 2. QC (:ref:`qc`) -Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean-qc`](../../README#bean-qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) -``` -bean-qc \ - bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ - -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r qc_report_${screen_id} `# Prefix for QC report` \ - [--tiling] `# Not required if you have passed --tiling in counting step` -``` - - - -If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. - -## 3. Filter alleles (:ref:`filter`) -As tiling library doesn't have designated per-gRNA target variant, any base edit observed in reporter may be the candidate variant, while having too many variants with very low editing rate significantly decreases the power. Variants are filtered based on multiple criteria in `bean-fitler`. - -If the screen targets coding sequence, it's beneficial to translate edits into coding varaints whenever possible for better power. For translation, provide `--translate` and one of the following: -``` -[ --translate-gene-name GENE_SYMBOL OR - --translate-genes-list path_to_gene_names_file.txt OR - --translate-fasta gene_exon.fa, OR - --translate-fastas-csv gene_exon_fas.csv] -``` -where `path_to_gene_names_file.txt` has one gene symbol per line, and gene symbol uses its MANE transcript (hg38) coordinates of exons. In order to use other reference versions or transcript ID, you'll need to feed in fasta file. See detailed formatting of fasta file [here](../../README#translating-alleles). - -Example allele filtering given we're translating based on MANE transcript exons of multiple gene symbols: - -```bash -bean-filter ./bean_count_${screen_id}_masked.h5ad \ --o ./bean_count_${screen_id}_alleleFiltered \ ---filter-target-basechange `# Filter based on intended base changes. If -b A was provided in bean-count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\ ---filter-window --edit-start-pos 0 --edit-end-pos 19 `# Filter based on editing window in spacer position within reporter.`\ ---filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \ ---translate --translate-genes-list tests/data/gene_symbols.txt -``` - -Ouptut file `` shows number of alleles per guide and number of guides per variant, where we want high enough values for the latter. See the typical output for dataset with good editing coverage & filtering result [here](../example_filtering_ouptut/). - -## 4. Quantify variant effect (:ref:`run`) -By default, `bean-run [sorting,survival] tiling` uses most filtered allele counts table for variant identification and quantification of their effects. **Check [allele filtering output](../example_filtering_ouptut/)** and choose alternative filtered allele counts table if necessary. - -`bean-run` can take 3 run options to quantify editing rate: -1. From **reporter + accessibility** - 1-1. If your gRNA metadata table (`tests/data/test_guide_info.csv` above) included per-gRNA accessibility score, - ``` - bean-run sorting tiling \ - ./bean_count_${screen_id}_alleleFiltered.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-col accessibility - ``` - 1-2. If your gRNA metadata table (`tests/data/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, - ``` - bean-run sorting tiling \ - ./bean_count_${screen_id}_alleleFiltered.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-bw accessibility.bw - ``` - -2. From **reporter** - ``` - bean-run sorting tiling \ - ./bean_count_${screen_id}_alleleFiltered.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl - ``` -3. No reporter information, assume the same editing efficiency of all gRNAs. - Use this option if your data don't have editing rate information. - ``` - bean-run sorting tiling \ - ./bean_count_${screen_id}_alleleFiltered.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl \ - --uniform-edit - ``` \ No newline at end of file diff --git a/docs/_build/_sources/tutorials/ldl_var.md.txt b/docs/_build/_sources/tutorials/ldl_var.md.txt deleted file mode 100644 index 1542536..0000000 --- a/docs/_build/_sources/tutorials/ldl_var.md.txt +++ /dev/null @@ -1,116 +0,0 @@ -# Variant sorting screen tutorial -GWAS variant screen with per-variant gRNA tiling design, selected based on FACS signal quantiles. - - - - - - - - - - -
    Library designVariant (gRNAs tile each target variant)
    variant library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design
    - -

    - -## Example workflow -```bash -screen_id=my_sorting_tiling_screen - -# 1. Count gRNA & reporter -bean-count-samples \ ---input tests/data/sample_list.csv `# Contains fastq file path; see test file for example.`\ --b A `# Base A is edited (into G)` \ --f tests/data/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ --o ./ `# Output directory` \ --r `# Quantify reporter edits` \ --n ${screen_id} `# ID of the screen to be counted` - -# 2. QC samples & guides -bean-qc \ - bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ - -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r qc_report_${screen_id} `# Prefix for QC report` \ - -b ` # Remove replicates with no good samples. - -# 3. Quantify variant effect -bean-run sorting variant \ - tests/data/bean_count_${screen_id}_masked.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-col accessibility -``` -See more details below. - -## 1. Count gRNA & reporter (:ref:`count_samples`) -```bash -screen_id=my_sorting_tiling_screen - -# 1. Count gRNA & reporter -bean-count-samples \ ---input tests/data/sample_list.csv `# Contains fastq file path; see test file for example.`\ --b A `# Base A is edited (into G)` \ --f tests/data/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ --o ./ `# Output directory` \ --r `# Quantify reporter edits` \ --n ${screen_id} `# ID of the screen to be counted` -``` -Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. - -## 2. QC samples & guides (:ref:`qc`) -Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean-qc`](../../README#bean-qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) -``` -bean-qc \ - bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ - -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r qc_report_${screen_id} `# Prefix for QC report` -``` - - - -If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. - - -## 3. Quantify variant effect (:ref:`run`) - -`bean-run` can take 3 run options to quantify editing rate: -1. From **reporter + accessibility** - If your gRNA metadata table (`tests/data/test_guide_info.csv` above) included per-gRNA accessibility score, - ``` - bean-run sorting variant \ - tests/data/bean_count_${screen_id}_masked.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-col accessibility - ``` - If your gRNA metadata table (`tests/data/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, - ``` - bean-run sorting variant \ - tests/data/bean_count_${screen_id}_masked.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-bw accessibility.bw - ``` - -2. From **reporter**, without accessibility - - This assumes the all target sites have the uniform chromatin accessibility. - ``` - bean-run sorting variant \ - tests/data/bean_count_${screen_id}_masked.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl - ``` -3. No reporter information, assume the same editing efficiency of all gRNAs. - Use this option if your data don't have editing outcome information. - ``` - bean-run sorting variant \ - tests/data/bean_count_${screen_id}_masked.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl \ - --uniform-edit - ``` \ No newline at end of file diff --git a/docs/_build/_static/alabaster.css b/docs/_build/_static/alabaster.css deleted file mode 100644 index e3174bf..0000000 --- a/docs/_build/_static/alabaster.css +++ /dev/null @@ -1,708 +0,0 @@ -@import url("basic.css"); - -/* -- page layout ----------------------------------------------------------- */ - -body { - font-family: Georgia, serif; - font-size: 17px; - background-color: #fff; - color: #000; - margin: 0; - padding: 0; -} - - -div.document { - width: 940px; - margin: 30px auto 0 auto; -} - -div.documentwrapper { - float: left; - width: 100%; -} - -div.bodywrapper { - margin: 0 0 0 220px; -} - -div.sphinxsidebar { - width: 220px; - font-size: 14px; 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-} - -div.related li.right { - float: right; - margin-right: 5px; -} - -/* -- sidebar --------------------------------------------------------------- */ - -div.sphinxsidebarwrapper { - padding: 10px 5px 0 10px; -} - -div.sphinxsidebar { - float: left; - width: 230px; - margin-left: -100%; - font-size: 90%; - word-wrap: break-word; - overflow-wrap : break-word; -} - -div.sphinxsidebar ul { - list-style: none; -} - -div.sphinxsidebar ul ul, -div.sphinxsidebar ul.want-points { - margin-left: 20px; - list-style: square; -} - -div.sphinxsidebar ul ul { - margin-top: 0; - margin-bottom: 0; -} - -div.sphinxsidebar form { - margin-top: 10px; -} - -div.sphinxsidebar input { - border: 1px solid #98dbcc; - font-family: sans-serif; - font-size: 1em; -} - -div.sphinxsidebar #searchbox form.search { - overflow: hidden; -} - -div.sphinxsidebar #searchbox input[type="text"] { - float: left; - width: 80%; - padding: 0.25em; - box-sizing: border-box; -} - -div.sphinxsidebar #searchbox input[type="submit"] { - float: left; - width: 20%; - border-left: none; - padding: 0.25em; - box-sizing: border-box; -} - - -img { - border: 0; - max-width: 100%; -} - -/* -- search page ----------------------------------------------------------- */ - -ul.search { - margin: 10px 0 0 20px; - padding: 0; -} - -ul.search li { - padding: 5px 0 5px 20px; - background-image: url(file.png); - background-repeat: no-repeat; - background-position: 0 7px; -} - -ul.search li a { - font-weight: bold; -} - -ul.search li p.context { - color: #888; - margin: 2px 0 0 30px; - text-align: left; -} - -ul.keywordmatches li.goodmatch a { - font-weight: bold; -} - -/* -- index page ------------------------------------------------------------ */ - -table.contentstable { - width: 90%; - margin-left: auto; - margin-right: auto; -} - -table.contentstable p.biglink { - line-height: 150%; -} - -a.biglink { - font-size: 1.3em; -} - -span.linkdescr { - font-style: italic; - padding-top: 5px; - font-size: 90%; -} - -/* -- general index --------------------------------------------------------- */ - -table.indextable { - width: 100%; -} - -table.indextable td { - text-align: left; - vertical-align: top; -} - -table.indextable ul { - margin-top: 0; - margin-bottom: 0; - list-style-type: none; -} - -table.indextable > tbody > tr > td > ul { - padding-left: 0em; -} - -table.indextable tr.pcap { - height: 10px; -} - -table.indextable tr.cap { - margin-top: 10px; - background-color: #f2f2f2; -} - -img.toggler { - margin-right: 3px; - margin-top: 3px; - cursor: pointer; -} - -div.modindex-jumpbox { - border-top: 1px solid #ddd; - border-bottom: 1px solid #ddd; - margin: 1em 0 1em 0; - padding: 0.4em; -} - -div.genindex-jumpbox { - border-top: 1px solid #ddd; - border-bottom: 1px solid #ddd; - margin: 1em 0 1em 0; - padding: 0.4em; -} - -/* -- domain module index --------------------------------------------------- */ - -table.modindextable td { - padding: 2px; - border-collapse: collapse; -} - -/* -- general body styles --------------------------------------------------- */ - -div.body { - min-width: inherit; - max-width: 800px; -} - -div.body p, div.body dd, div.body li, div.body blockquote { - -moz-hyphens: auto; - -ms-hyphens: auto; - -webkit-hyphens: auto; - hyphens: auto; -} - -a.headerlink { - visibility: hidden; -} - -a:visited { - color: #551A8B; -} - -h1:hover > a.headerlink, -h2:hover > a.headerlink, -h3:hover > a.headerlink, -h4:hover > a.headerlink, -h5:hover > a.headerlink, -h6:hover > a.headerlink, -dt:hover > a.headerlink, -caption:hover > a.headerlink, -p.caption:hover > a.headerlink, -div.code-block-caption:hover > a.headerlink { - visibility: visible; -} - -div.body p.caption { - text-align: inherit; -} - -div.body td { - text-align: left; -} - -.first { - margin-top: 0 !important; -} - -p.rubric { - margin-top: 30px; - font-weight: bold; -} - -img.align-left, figure.align-left, .figure.align-left, object.align-left { - clear: left; - float: left; - margin-right: 1em; -} - -img.align-right, figure.align-right, .figure.align-right, object.align-right { - clear: right; - float: right; - margin-left: 1em; -} - -img.align-center, figure.align-center, .figure.align-center, object.align-center { - display: block; - margin-left: auto; - margin-right: auto; -} - -img.align-default, figure.align-default, .figure.align-default { - display: block; - margin-left: auto; - margin-right: auto; -} - -.align-left { - text-align: left; -} - -.align-center { - text-align: center; -} - -.align-default { - text-align: center; -} - -.align-right { - text-align: right; -} - -/* -- sidebars -------------------------------------------------------------- */ - -div.sidebar, -aside.sidebar { - margin: 0 0 0.5em 1em; - border: 1px solid #ddb; - padding: 7px; - background-color: #ffe; - width: 40%; - float: right; - clear: right; - overflow-x: auto; -} - -p.sidebar-title { - font-weight: bold; -} - -nav.contents, -aside.topic, -div.admonition, div.topic, blockquote { - clear: left; -} - -/* -- topics ---------------------------------------------------------------- */ - -nav.contents, -aside.topic, -div.topic { - border: 1px solid #ccc; - padding: 7px; - margin: 10px 0 10px 0; -} - -p.topic-title { - font-size: 1.1em; - font-weight: bold; - margin-top: 10px; -} - -/* -- admonitions ----------------------------------------------------------- */ - -div.admonition { - margin-top: 10px; - margin-bottom: 10px; - padding: 7px; -} - -div.admonition dt { - font-weight: bold; -} - -p.admonition-title { - margin: 0px 10px 5px 0px; - font-weight: bold; -} - -div.body p.centered { - text-align: center; - margin-top: 25px; -} - -/* -- content of sidebars/topics/admonitions -------------------------------- */ - -div.sidebar > :last-child, -aside.sidebar > :last-child, -nav.contents > :last-child, -aside.topic > :last-child, -div.topic > :last-child, -div.admonition > :last-child { - margin-bottom: 0; -} - -div.sidebar::after, -aside.sidebar::after, -nav.contents::after, -aside.topic::after, -div.topic::after, -div.admonition::after, -blockquote::after { - display: block; - content: ''; - clear: both; -} - -/* -- tables ---------------------------------------------------------------- */ - -table.docutils { - margin-top: 10px; - margin-bottom: 10px; - border: 0; - border-collapse: collapse; -} - -table.align-center { - margin-left: auto; - margin-right: auto; -} - -table.align-default { - margin-left: auto; - margin-right: auto; -} - -table caption span.caption-number { - font-style: italic; -} - -table caption span.caption-text { -} - -table.docutils td, table.docutils th { - padding: 1px 8px 1px 5px; - border-top: 0; - border-left: 0; - border-right: 0; - border-bottom: 1px solid #aaa; -} - -th { - text-align: left; - padding-right: 5px; -} - -table.citation { - border-left: solid 1px gray; - margin-left: 1px; -} - -table.citation td { - border-bottom: none; -} - -th > :first-child, -td > :first-child { - margin-top: 0px; -} - -th > :last-child, -td > :last-child { - margin-bottom: 0px; -} - -/* -- figures --------------------------------------------------------------- */ - -div.figure, figure { - margin: 0.5em; - padding: 0.5em; -} - -div.figure p.caption, figcaption { - padding: 0.3em; -} - -div.figure p.caption span.caption-number, -figcaption span.caption-number { - font-style: italic; -} - -div.figure p.caption span.caption-text, -figcaption span.caption-text { -} - -/* -- field list styles ----------------------------------------------------- */ - -table.field-list td, table.field-list th { - border: 0 !important; -} - -.field-list ul { - margin: 0; - padding-left: 1em; -} - -.field-list p { - margin: 0; -} - -.field-name { - -moz-hyphens: manual; - -ms-hyphens: manual; - -webkit-hyphens: manual; - hyphens: manual; -} - -/* -- hlist styles ---------------------------------------------------------- */ - -table.hlist { - margin: 1em 0; -} - -table.hlist td { - vertical-align: top; -} - -/* -- object description styles --------------------------------------------- */ - -.sig { - font-family: 'Consolas', 'Menlo', 'DejaVu Sans Mono', 'Bitstream Vera Sans Mono', monospace; -} - -.sig-name, code.descname { - background-color: transparent; - font-weight: bold; -} - -.sig-name { - font-size: 1.1em; -} - -code.descname { - font-size: 1.2em; -} - -.sig-prename, code.descclassname { - background-color: transparent; -} - -.optional { - font-size: 1.3em; -} - -.sig-paren { - font-size: larger; -} - -.sig-param.n { - font-style: italic; -} - -/* C++ specific styling */ - -.sig-inline.c-texpr, -.sig-inline.cpp-texpr { - font-family: unset; -} - -.sig.c .k, .sig.c .kt, -.sig.cpp .k, .sig.cpp .kt { - color: #0033B3; -} - -.sig.c .m, -.sig.cpp .m { - color: #1750EB; -} - -.sig.c .s, .sig.c .sc, -.sig.cpp .s, .sig.cpp .sc { - color: #067D17; -} - - -/* -- other body styles ----------------------------------------------------- */ - -ol.arabic { - list-style: decimal; -} - -ol.loweralpha { - list-style: lower-alpha; -} - -ol.upperalpha { - list-style: upper-alpha; -} - -ol.lowerroman { - list-style: lower-roman; -} - -ol.upperroman { - list-style: upper-roman; -} - -:not(li) > ol > li:first-child > :first-child, -:not(li) > ul > li:first-child > :first-child { - margin-top: 0px; -} - -:not(li) > ol > li:last-child > :last-child, -:not(li) > ul > li:last-child > :last-child { - margin-bottom: 0px; -} - -ol.simple ol p, -ol.simple ul p, -ul.simple ol p, -ul.simple ul p { - margin-top: 0; -} - -ol.simple > li:not(:first-child) > p, -ul.simple > li:not(:first-child) > p { - margin-top: 0; -} - -ol.simple p, -ul.simple p { - margin-bottom: 0; -} - -aside.footnote > span, -div.citation > span { - float: left; -} -aside.footnote > span:last-of-type, -div.citation > span:last-of-type { - padding-right: 0.5em; -} -aside.footnote > p { - margin-left: 2em; -} -div.citation > p { - margin-left: 4em; -} -aside.footnote > p:last-of-type, -div.citation > p:last-of-type { - margin-bottom: 0em; -} -aside.footnote > p:last-of-type:after, -div.citation > p:last-of-type:after { - content: ""; - clear: both; -} - -dl.field-list { - display: grid; - grid-template-columns: fit-content(30%) auto; -} - -dl.field-list > dt { - font-weight: bold; - word-break: break-word; - padding-left: 0.5em; - padding-right: 5px; -} - -dl.field-list > dd { - padding-left: 0.5em; - margin-top: 0em; - margin-left: 0em; - margin-bottom: 0em; -} - -dl { - margin-bottom: 15px; -} - -dd > :first-child { - margin-top: 0px; -} - -dd ul, dd table { - margin-bottom: 10px; -} - -dd { - margin-top: 3px; - margin-bottom: 10px; - margin-left: 30px; -} - -.sig dd { - margin-top: 0px; - margin-bottom: 0px; -} - -.sig dl { - margin-top: 0px; - margin-bottom: 0px; -} - -dl > dd:last-child, -dl > dd:last-child > :last-child { - margin-bottom: 0; -} - -dt:target, span.highlighted { - background-color: #fbe54e; -} - -rect.highlighted { - fill: #fbe54e; -} - -dl.glossary dt { - font-weight: bold; - font-size: 1.1em; -} - -.versionmodified { - font-style: italic; -} - -.system-message { - background-color: #fda; - padding: 5px; - border: 3px solid red; -} - -.footnote:target { - background-color: #ffa; -} - -.line-block { - display: block; - margin-top: 1em; - margin-bottom: 1em; -} - -.line-block .line-block { - margin-top: 0; - margin-bottom: 0; - margin-left: 1.5em; -} - -.guilabel, .menuselection { - font-family: sans-serif; -} - -.accelerator { - text-decoration: underline; -} - -.classifier { - font-style: oblique; -} - -.classifier:before { - font-style: normal; - margin: 0 0.5em; - content: ":"; - display: inline-block; -} - -abbr, acronym { - border-bottom: dotted 1px; - cursor: help; -} - -.translated { - background-color: rgba(207, 255, 207, 0.2) -} - -.untranslated { - background-color: rgba(255, 207, 207, 0.2) -} - -/* -- code displays --------------------------------------------------------- */ - -pre { - overflow: auto; - overflow-y: hidden; /* fixes display issues on Chrome browsers */ -} - -pre, div[class*="highlight-"] { - clear: both; -} - -span.pre { - -moz-hyphens: none; - -ms-hyphens: none; - -webkit-hyphens: none; - hyphens: none; - white-space: nowrap; -} - -div[class*="highlight-"] { - margin: 1em 0; -} - -td.linenos pre { - border: 0; - background-color: transparent; - color: #aaa; -} - -table.highlighttable { - display: block; -} - -table.highlighttable tbody { - display: block; -} - -table.highlighttable tr { - display: flex; -} - -table.highlighttable td { - margin: 0; - padding: 0; -} - -table.highlighttable td.linenos { - padding-right: 0.5em; -} - -table.highlighttable td.code { - flex: 1; - overflow: hidden; -} - -.highlight .hll { - display: block; -} - -div.highlight pre, -table.highlighttable pre { - margin: 0; -} - -div.code-block-caption + div { - margin-top: 0; -} - -div.code-block-caption { - margin-top: 1em; - padding: 2px 5px; - font-size: small; -} - -div.code-block-caption code { - background-color: transparent; -} - -table.highlighttable td.linenos, -span.linenos, -div.highlight span.gp { /* gp: Generic.Prompt */ - user-select: none; - -webkit-user-select: text; /* Safari fallback only */ - -webkit-user-select: none; /* Chrome/Safari */ - -moz-user-select: none; /* Firefox */ - -ms-user-select: none; /* IE10+ */ -} - -div.code-block-caption span.caption-number { - padding: 0.1em 0.3em; - font-style: italic; -} - -div.code-block-caption span.caption-text { -} - -div.literal-block-wrapper { - margin: 1em 0; -} - -code.xref, a code { - background-color: transparent; - font-weight: bold; -} - -h1 code, h2 code, h3 code, h4 code, h5 code, h6 code { - background-color: transparent; -} - -.viewcode-link { - float: right; -} - -.viewcode-back { - float: right; - font-family: sans-serif; -} - -div.viewcode-block:target { - margin: -1px -10px; - padding: 0 10px; -} - -/* -- math display ---------------------------------------------------------- */ - -img.math { - vertical-align: middle; -} - -div.body div.math p { - text-align: center; -} - -span.eqno { - float: right; -} - -span.eqno a.headerlink { - position: absolute; - z-index: 1; -} - -div.math:hover a.headerlink { - visibility: visible; -} - -/* -- printout stylesheet --------------------------------------------------- */ - -@media print { - div.document, - div.documentwrapper, - div.bodywrapper { - margin: 0 !important; - width: 100%; - } - - div.sphinxsidebar, - div.related, - div.footer, - #top-link { - display: none; - } -} \ No newline at end of file diff --git a/docs/_build/_static/custom.css b/docs/_build/_static/custom.css deleted file mode 100644 index 2a924f1..0000000 --- a/docs/_build/_static/custom.css +++ /dev/null @@ -1 +0,0 @@ -/* This file intentionally left blank. */ diff --git a/docs/_build/_static/doctools.js b/docs/_build/_static/doctools.js deleted file mode 100644 index d06a71d..0000000 --- a/docs/_build/_static/doctools.js +++ /dev/null @@ -1,156 +0,0 @@ -/* - * doctools.js - * ~~~~~~~~~~~ - * - * Base JavaScript utilities for all Sphinx HTML documentation. - * - * :copyright: Copyright 2007-2023 by the Sphinx team, see AUTHORS. - * :license: BSD, see LICENSE for details. - * - */ -"use strict"; - -const BLACKLISTED_KEY_CONTROL_ELEMENTS = new Set([ - "TEXTAREA", - "INPUT", - "SELECT", - "BUTTON", -]); - -const _ready = (callback) => { - if (document.readyState !== "loading") { - callback(); - } else { - document.addEventListener("DOMContentLoaded", callback); - } -}; - -/** - * Small JavaScript module for the documentation. - */ -const Documentation = { - init: () => { - Documentation.initDomainIndexTable(); - Documentation.initOnKeyListeners(); - }, - - /** - * i18n support - */ - TRANSLATIONS: {}, - PLURAL_EXPR: (n) => (n === 1 ? 0 : 1), - LOCALE: "unknown", - - // gettext and ngettext don't access this so that the functions - // can safely bound to a different name (_ = Documentation.gettext) - gettext: (string) => { - const translated = Documentation.TRANSLATIONS[string]; - switch (typeof translated) { - case "undefined": - return string; // no translation - case "string": - return translated; // translation exists - default: - return translated[0]; // (singular, plural) translation tuple exists - } - }, - - ngettext: (singular, plural, n) => { - const translated = Documentation.TRANSLATIONS[singular]; - if (typeof translated !== "undefined") - return translated[Documentation.PLURAL_EXPR(n)]; - return n === 1 ? singular : plural; - }, - - addTranslations: (catalog) => { - Object.assign(Documentation.TRANSLATIONS, catalog.messages); - Documentation.PLURAL_EXPR = new Function( - "n", - `return (${catalog.plural_expr})` - ); - Documentation.LOCALE = catalog.locale; - }, - - /** - * helper function to focus on search bar - */ - focusSearchBar: () => { - document.querySelectorAll("input[name=q]")[0]?.focus(); - }, - - /** - * Initialise the domain index toggle buttons - */ - initDomainIndexTable: () => { - const toggler = (el) => { - const idNumber = el.id.substr(7); - const toggledRows = document.querySelectorAll(`tr.cg-${idNumber}`); - if (el.src.substr(-9) === "minus.png") { - el.src = `${el.src.substr(0, el.src.length - 9)}plus.png`; - toggledRows.forEach((el) => (el.style.display = "none")); - } else { - el.src = `${el.src.substr(0, el.src.length - 8)}minus.png`; - toggledRows.forEach((el) => (el.style.display = "")); - } - }; - - const togglerElements = document.querySelectorAll("img.toggler"); - togglerElements.forEach((el) => - el.addEventListener("click", (event) => toggler(event.currentTarget)) - ); - togglerElements.forEach((el) => (el.style.display = "")); - if (DOCUMENTATION_OPTIONS.COLLAPSE_INDEX) togglerElements.forEach(toggler); - }, - - initOnKeyListeners: () => { - // only install a listener if it is really needed - if ( - !DOCUMENTATION_OPTIONS.NAVIGATION_WITH_KEYS && - !DOCUMENTATION_OPTIONS.ENABLE_SEARCH_SHORTCUTS - ) - return; - - document.addEventListener("keydown", (event) => { - // bail for input elements - if (BLACKLISTED_KEY_CONTROL_ELEMENTS.has(document.activeElement.tagName)) return; - // bail with special keys - if (event.altKey || event.ctrlKey || event.metaKey) return; - - if (!event.shiftKey) { - switch (event.key) { - case "ArrowLeft": - if (!DOCUMENTATION_OPTIONS.NAVIGATION_WITH_KEYS) break; - - const prevLink = document.querySelector('link[rel="prev"]'); - if (prevLink && prevLink.href) { - window.location.href = prevLink.href; - event.preventDefault(); - } - break; - case "ArrowRight": - if (!DOCUMENTATION_OPTIONS.NAVIGATION_WITH_KEYS) break; - - const nextLink = document.querySelector('link[rel="next"]'); - if (nextLink && nextLink.href) { - window.location.href = nextLink.href; - event.preventDefault(); - } - break; - } - } - - // some keyboard layouts may need Shift to get / - switch (event.key) { - case "/": - if (!DOCUMENTATION_OPTIONS.ENABLE_SEARCH_SHORTCUTS) break; - Documentation.focusSearchBar(); - event.preventDefault(); - } - }); - }, -}; - -// quick alias for translations -const _ = Documentation.gettext; - -_ready(Documentation.init); diff --git a/docs/_build/_static/documentation_options.js b/docs/_build/_static/documentation_options.js deleted file mode 100644 index 89435bb..0000000 --- a/docs/_build/_static/documentation_options.js +++ /dev/null @@ -1,13 +0,0 @@ -const DOCUMENTATION_OPTIONS = { - VERSION: '1.0.0', - LANGUAGE: 'en', - COLLAPSE_INDEX: false, - BUILDER: 'html', - FILE_SUFFIX: '.html', - LINK_SUFFIX: '.html', - HAS_SOURCE: true, - SOURCELINK_SUFFIX: '.txt', - NAVIGATION_WITH_KEYS: false, - SHOW_SEARCH_SUMMARY: true, - ENABLE_SEARCH_SHORTCUTS: true, -}; \ No newline at end of file diff --git a/docs/_build/_static/file.png b/docs/_build/_static/file.png deleted file mode 100644 index a858a410e4faa62ce324d814e4b816fff83a6fb3..0000000000000000000000000000000000000000 GIT binary patch literal 0 HcmV?d00001 literal 286 zcmV+(0pb3MP)s`hMrGg#P~ix$^RISR_I47Y|r1 z_CyJOe}D1){SET-^Amu_i71Lt6eYfZjRyw@I6OQAIXXHDfiX^GbOlHe=Ae4>0m)d(f|Me07*qoM6N<$f}vM^LjV8( diff --git a/docs/_build/_static/language_data.js b/docs/_build/_static/language_data.js deleted file mode 100644 index 250f566..0000000 --- a/docs/_build/_static/language_data.js +++ /dev/null @@ -1,199 +0,0 @@ -/* - * language_data.js - * ~~~~~~~~~~~~~~~~ - * - * This script contains the language-specific data used by searchtools.js, - * namely the list of stopwords, stemmer, scorer and splitter. - * - * :copyright: Copyright 2007-2023 by the Sphinx team, see AUTHORS. - * :license: BSD, see LICENSE for details. - * - */ - -var stopwords = ["a", "and", "are", "as", "at", "be", "but", "by", "for", "if", "in", "into", "is", "it", "near", "no", "not", "of", "on", "or", "such", "that", "the", "their", "then", "there", "these", "they", "this", "to", "was", "will", "with"]; - - -/* Non-minified version is copied as a separate JS file, is available */ - -/** - * Porter Stemmer - */ -var Stemmer = function() { - - var step2list = { - ational: 'ate', - tional: 'tion', - enci: 'ence', - anci: 'ance', - izer: 'ize', - bli: 'ble', - alli: 'al', - entli: 'ent', - eli: 'e', - ousli: 'ous', - ization: 'ize', - ation: 'ate', - ator: 'ate', - alism: 'al', - iveness: 'ive', - fulness: 'ful', - ousness: 'ous', - aliti: 'al', - iviti: 'ive', - biliti: 'ble', - logi: 'log' - }; - - var step3list = { - icate: 'ic', - ative: '', - alize: 'al', - iciti: 'ic', - ical: 'ic', - ful: '', - ness: '' - }; - - var c = "[^aeiou]"; // consonant - var v = "[aeiouy]"; // vowel - var C = c + "[^aeiouy]*"; // consonant sequence - var V = v + "[aeiou]*"; // vowel sequence - - var mgr0 = "^(" + C + ")?" + V + C; // [C]VC... is m>0 - var meq1 = "^(" + C + ")?" + V + C + "(" + V + ")?$"; // [C]VC[V] is m=1 - var mgr1 = "^(" + C + ")?" + V + C + V + C; // [C]VCVC... is m>1 - var s_v = "^(" + C + ")?" + v; // vowel in stem - - this.stemWord = function (w) { - var stem; - var suffix; - var firstch; - var origword = w; - - if (w.length < 3) - return w; - - var re; - var re2; - var re3; - var re4; - - firstch = w.substr(0,1); - if (firstch == "y") - w = firstch.toUpperCase() + w.substr(1); - - // Step 1a - re = /^(.+?)(ss|i)es$/; - re2 = /^(.+?)([^s])s$/; - - if (re.test(w)) - w = w.replace(re,"$1$2"); - else if (re2.test(w)) - w = w.replace(re2,"$1$2"); - - // Step 1b - re = /^(.+?)eed$/; - re2 = /^(.+?)(ed|ing)$/; - if (re.test(w)) { - var fp = re.exec(w); - re = new RegExp(mgr0); - if (re.test(fp[1])) { - re = /.$/; - w = w.replace(re,""); - } - } - else if (re2.test(w)) { - var fp = re2.exec(w); - stem = fp[1]; - re2 = new RegExp(s_v); - if (re2.test(stem)) { - w = stem; - re2 = /(at|bl|iz)$/; - re3 = new RegExp("([^aeiouylsz])\\1$"); - re4 = new RegExp("^" + C + v + "[^aeiouwxy]$"); - if (re2.test(w)) - w = w + "e"; - else if (re3.test(w)) { - re = /.$/; - w = w.replace(re,""); - } - else if (re4.test(w)) - w = w + "e"; - } - } - - // Step 1c - re = /^(.+?)y$/; - if (re.test(w)) { - var fp = re.exec(w); - stem = fp[1]; - re = new RegExp(s_v); - if (re.test(stem)) - w = stem + "i"; - } - - // Step 2 - re = /^(.+?)(ational|tional|enci|anci|izer|bli|alli|entli|eli|ousli|ization|ation|ator|alism|iveness|fulness|ousness|aliti|iviti|biliti|logi)$/; - if (re.test(w)) { - var fp = re.exec(w); - stem = fp[1]; - suffix = fp[2]; - re = new RegExp(mgr0); - if (re.test(stem)) - w = stem + step2list[suffix]; - } - - // Step 3 - re = /^(.+?)(icate|ative|alize|iciti|ical|ful|ness)$/; - if (re.test(w)) { - var fp = re.exec(w); - stem = fp[1]; - suffix = fp[2]; - re = new RegExp(mgr0); - if (re.test(stem)) - w = stem + step3list[suffix]; - } - - // Step 4 - re = /^(.+?)(al|ance|ence|er|ic|able|ible|ant|ement|ment|ent|ou|ism|ate|iti|ous|ive|ize)$/; - re2 = /^(.+?)(s|t)(ion)$/; - if (re.test(w)) { - var fp = re.exec(w); - stem = fp[1]; - re = new RegExp(mgr1); - if (re.test(stem)) - w = stem; - } - else if (re2.test(w)) { - var fp = re2.exec(w); - stem = fp[1] + fp[2]; - re2 = new RegExp(mgr1); - if (re2.test(stem)) - w = stem; - } - - // Step 5 - re = /^(.+?)e$/; - if (re.test(w)) { - var fp = re.exec(w); - stem = fp[1]; - re = new RegExp(mgr1); - re2 = new RegExp(meq1); - re3 = new RegExp("^" + C + v + "[^aeiouwxy]$"); - if (re.test(stem) || (re2.test(stem) && !(re3.test(stem)))) - w = stem; - } - re = /ll$/; - re2 = new RegExp(mgr1); - if (re.test(w) && re2.test(w)) { - re = /.$/; - w = w.replace(re,""); - } - - // and turn initial Y back to y - if (firstch == "y") - w = firstch.toLowerCase() + w.substr(1); - return w; - } -} - diff --git a/docs/_build/_static/minus.png b/docs/_build/_static/minus.png deleted file mode 100644 index d96755fdaf8bb2214971e0db9c1fd3077d7c419d..0000000000000000000000000000000000000000 GIT binary patch literal 0 HcmV?d00001 literal 90 zcmeAS@N?(olHy`uVBq!ia0vp^+#t*WBp7;*Yy1LIik>cxAr*|t7R?Mi>2?kWtu=nj kDsEF_5m^0CR;1wuP-*O&G^0G}KYk!hp00i_>zopr08q^qX#fBK diff --git a/docs/_build/_static/plus.png b/docs/_build/_static/plus.png deleted file mode 100644 index 7107cec93a979b9a5f64843235a16651d563ce2d..0000000000000000000000000000000000000000 GIT binary patch literal 0 HcmV?d00001 literal 90 zcmeAS@N?(olHy`uVBq!ia0vp^+#t*WBp7;*Yy1LIik>cxAr*|t7R?Mi>2?kWtu>-2 m3q%Vub%g%s<8sJhVPMczOq}xhg9DJoz~JfX=d#Wzp$Pyb1r*Kz diff --git a/docs/_build/_static/pygments.css b/docs/_build/_static/pygments.css deleted file mode 100644 index 04a4174..0000000 --- a/docs/_build/_static/pygments.css +++ /dev/null @@ -1,84 +0,0 @@ -pre { line-height: 125%; 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- -/** - * Simple result scoring code. - */ -if (typeof Scorer === "undefined") { - var Scorer = { - // Implement the following function to further tweak the score for each result - // The function takes a result array [docname, title, anchor, descr, score, filename] - // and returns the new score. - /* - score: result => { - const [docname, title, anchor, descr, score, filename] = result - return score - }, - */ - - // query matches the full name of an object - objNameMatch: 11, - // or matches in the last dotted part of the object name - objPartialMatch: 6, - // Additive scores depending on the priority of the object - objPrio: { - 0: 15, // used to be importantResults - 1: 5, // used to be objectResults - 2: -5, // used to be unimportantResults - }, - // Used when the priority is not in the mapping. - objPrioDefault: 0, - - // query found in title - title: 15, - partialTitle: 7, - // query found in terms - term: 5, - partialTerm: 2, - }; -} - -const _removeChildren = (element) => { - while (element && element.lastChild) element.removeChild(element.lastChild); -}; - -/** - * See https://developer.mozilla.org/en-US/docs/Web/JavaScript/Guide/Regular_Expressions#escaping - */ -const _escapeRegExp = (string) => - string.replace(/[.*+\-?^${}()|[\]\\]/g, "\\$&"); // $& means the whole matched string - -const _displayItem = (item, searchTerms, highlightTerms) => { - const docBuilder = DOCUMENTATION_OPTIONS.BUILDER; - const docFileSuffix = DOCUMENTATION_OPTIONS.FILE_SUFFIX; - const docLinkSuffix = DOCUMENTATION_OPTIONS.LINK_SUFFIX; - const showSearchSummary = DOCUMENTATION_OPTIONS.SHOW_SEARCH_SUMMARY; - const contentRoot = document.documentElement.dataset.content_root; - - const [docName, title, anchor, descr, score, _filename] = item; - - let listItem = document.createElement("li"); - let requestUrl; - let linkUrl; - if (docBuilder === "dirhtml") { - // dirhtml builder - let dirname = docName + "/"; - if (dirname.match(/\/index\/$/)) - dirname = dirname.substring(0, dirname.length - 6); - else if (dirname === "index/") dirname = ""; - requestUrl = contentRoot + dirname; - linkUrl = requestUrl; - } else { - // normal html builders - requestUrl = contentRoot + docName + docFileSuffix; - linkUrl = docName + docLinkSuffix; - } - let linkEl = listItem.appendChild(document.createElement("a")); - linkEl.href = linkUrl + anchor; - linkEl.dataset.score = score; - linkEl.innerHTML = title; - if (descr) { - listItem.appendChild(document.createElement("span")).innerHTML = - " (" + descr + ")"; - // highlight search terms in the description - if (SPHINX_HIGHLIGHT_ENABLED) // set in sphinx_highlight.js - highlightTerms.forEach((term) => _highlightText(listItem, term, "highlighted")); - } - else if (showSearchSummary) - fetch(requestUrl) - .then((responseData) => responseData.text()) - .then((data) => { - if (data) - listItem.appendChild( - Search.makeSearchSummary(data, searchTerms) - ); - // highlight search terms in the summary - if (SPHINX_HIGHLIGHT_ENABLED) // set in sphinx_highlight.js - highlightTerms.forEach((term) => _highlightText(listItem, term, "highlighted")); - }); - Search.output.appendChild(listItem); -}; -const _finishSearch = (resultCount) => { - Search.stopPulse(); - Search.title.innerText = _("Search Results"); - if (!resultCount) - Search.status.innerText = Documentation.gettext( - "Your search did not match any documents. Please make sure that all words are spelled correctly and that you've selected enough categories." - ); - else - Search.status.innerText = _( - `Search finished, found ${resultCount} page(s) matching the search query.` - ); -}; -const _displayNextItem = ( - results, - resultCount, - searchTerms, - highlightTerms, -) => { - // results left, load the summary and display it - // this is intended to be dynamic (don't sub resultsCount) - if (results.length) { - _displayItem(results.pop(), searchTerms, highlightTerms); - setTimeout( - () => _displayNextItem(results, resultCount, searchTerms, highlightTerms), - 5 - ); - } - // search finished, update title and status message - else _finishSearch(resultCount); -}; - -/** - * Default splitQuery function. Can be overridden in ``sphinx.search`` with a - * custom function per language. - * - * The regular expression works by splitting the string on consecutive characters - * that are not Unicode letters, numbers, underscores, or emoji characters. - * This is the same as ``\W+`` in Python, preserving the surrogate pair area. - */ -if (typeof splitQuery === "undefined") { - var splitQuery = (query) => query - .split(/[^\p{Letter}\p{Number}_\p{Emoji_Presentation}]+/gu) - .filter(term => term) // remove remaining empty strings -} - -/** - * Search Module - */ -const Search = { - _index: null, - _queued_query: null, - _pulse_status: -1, - - htmlToText: (htmlString) => { - const htmlElement = new DOMParser().parseFromString(htmlString, 'text/html'); - htmlElement.querySelectorAll(".headerlink").forEach((el) => { el.remove() }); - const docContent = htmlElement.querySelector('[role="main"]'); - if (docContent !== undefined) return docContent.textContent; - console.warn( - "Content block not found. Sphinx search tries to obtain it via '[role=main]'. Could you check your theme or template." - ); - return ""; - }, - - init: () => { - const query = new URLSearchParams(window.location.search).get("q"); - document - .querySelectorAll('input[name="q"]') - .forEach((el) => (el.value = query)); - if (query) Search.performSearch(query); - }, - - loadIndex: (url) => - (document.body.appendChild(document.createElement("script")).src = url), - - setIndex: (index) => { - Search._index = index; - if (Search._queued_query !== null) { - const query = Search._queued_query; - Search._queued_query = null; - Search.query(query); - } - }, - - hasIndex: () => Search._index !== null, - - deferQuery: (query) => (Search._queued_query = query), - - stopPulse: () => (Search._pulse_status = -1), - - startPulse: () => { - if (Search._pulse_status >= 0) return; - - const pulse = () => { - Search._pulse_status = (Search._pulse_status + 1) % 4; - Search.dots.innerText = ".".repeat(Search._pulse_status); - if (Search._pulse_status >= 0) window.setTimeout(pulse, 500); - }; - pulse(); - }, - - /** - * perform a search for something (or wait until index is loaded) - */ - performSearch: (query) => { - // create the required interface elements - const searchText = document.createElement("h2"); - searchText.textContent = _("Searching"); - const searchSummary = document.createElement("p"); - searchSummary.classList.add("search-summary"); - searchSummary.innerText = ""; - const searchList = document.createElement("ul"); - searchList.classList.add("search"); - - const out = document.getElementById("search-results"); - Search.title = out.appendChild(searchText); - Search.dots = Search.title.appendChild(document.createElement("span")); - Search.status = out.appendChild(searchSummary); - Search.output = out.appendChild(searchList); - - const searchProgress = document.getElementById("search-progress"); - // Some themes don't use the search progress node - if (searchProgress) { - searchProgress.innerText = _("Preparing search..."); - } - Search.startPulse(); - - // index already loaded, the browser was quick! - if (Search.hasIndex()) Search.query(query); - else Search.deferQuery(query); - }, - - /** - * execute search (requires search index to be loaded) - */ - query: (query) => { - const filenames = Search._index.filenames; - const docNames = Search._index.docnames; - const titles = Search._index.titles; - const allTitles = Search._index.alltitles; - const indexEntries = Search._index.indexentries; - - // stem the search terms and add them to the correct list - const stemmer = new Stemmer(); - const searchTerms = new Set(); - const excludedTerms = new Set(); - const highlightTerms = new Set(); - const objectTerms = new Set(splitQuery(query.toLowerCase().trim())); - splitQuery(query.trim()).forEach((queryTerm) => { - const queryTermLower = queryTerm.toLowerCase(); - - // maybe skip this "word" - // stopwords array is from language_data.js - if ( - stopwords.indexOf(queryTermLower) !== -1 || - queryTerm.match(/^\d+$/) - ) - return; - - // stem the word - let word = stemmer.stemWord(queryTermLower); - // select the correct list - if (word[0] === "-") excludedTerms.add(word.substr(1)); - else { - searchTerms.add(word); - highlightTerms.add(queryTermLower); - } - }); - - if (SPHINX_HIGHLIGHT_ENABLED) { // set in sphinx_highlight.js - localStorage.setItem("sphinx_highlight_terms", [...highlightTerms].join(" ")) - } - - // console.debug("SEARCH: searching for:"); - // console.info("required: ", [...searchTerms]); - // console.info("excluded: ", [...excludedTerms]); - - // array of [docname, title, anchor, descr, score, filename] - let results = []; - _removeChildren(document.getElementById("search-progress")); - - const queryLower = query.toLowerCase(); - for (const [title, foundTitles] of Object.entries(allTitles)) { - if (title.toLowerCase().includes(queryLower) && (queryLower.length >= title.length/2)) { - for (const [file, id] of foundTitles) { - let score = Math.round(100 * queryLower.length / title.length) - results.push([ - docNames[file], - titles[file] !== title ? `${titles[file]} > ${title}` : title, - id !== null ? "#" + id : "", - null, - score, - filenames[file], - ]); - } - } - } - - // search for explicit entries in index directives - for (const [entry, foundEntries] of Object.entries(indexEntries)) { - if (entry.includes(queryLower) && (queryLower.length >= entry.length/2)) { - for (const [file, id] of foundEntries) { - let score = Math.round(100 * queryLower.length / entry.length) - results.push([ - docNames[file], - titles[file], - id ? "#" + id : "", - null, - score, - filenames[file], - ]); - } - } - } - - // lookup as object - objectTerms.forEach((term) => - results.push(...Search.performObjectSearch(term, objectTerms)) - ); - - // lookup as search terms in fulltext - results.push(...Search.performTermsSearch(searchTerms, excludedTerms)); - - // let the scorer override scores with a custom scoring function - if (Scorer.score) results.forEach((item) => (item[4] = Scorer.score(item))); - - // now sort the results by score (in opposite order of appearance, since the - // display function below uses pop() to retrieve items) and then - // alphabetically - results.sort((a, b) => { - const leftScore = a[4]; - const rightScore = b[4]; - if (leftScore === rightScore) { - // same score: sort alphabetically - const leftTitle = a[1].toLowerCase(); - const rightTitle = b[1].toLowerCase(); - if (leftTitle === rightTitle) return 0; - return leftTitle > rightTitle ? -1 : 1; // inverted is intentional - } - return leftScore > rightScore ? 1 : -1; - }); - - // remove duplicate search results - // note the reversing of results, so that in the case of duplicates, the highest-scoring entry is kept - let seen = new Set(); - results = results.reverse().reduce((acc, result) => { - let resultStr = result.slice(0, 4).concat([result[5]]).map(v => String(v)).join(','); - if (!seen.has(resultStr)) { - acc.push(result); - seen.add(resultStr); - } - return acc; - }, []); - - results = results.reverse(); - - // for debugging - //Search.lastresults = results.slice(); // a copy - // console.info("search results:", Search.lastresults); - - // print the results - _displayNextItem(results, results.length, searchTerms, highlightTerms); - }, - - /** - * search for object names - */ - performObjectSearch: (object, objectTerms) => { - const filenames = Search._index.filenames; - const docNames = Search._index.docnames; - const objects = Search._index.objects; - const objNames = Search._index.objnames; - const titles = Search._index.titles; - - const results = []; - - const objectSearchCallback = (prefix, match) => { - const name = match[4] - const fullname = (prefix ? prefix + "." : "") + name; - const fullnameLower = fullname.toLowerCase(); - if (fullnameLower.indexOf(object) < 0) return; - - let score = 0; - const parts = fullnameLower.split("."); - - // check for different match types: exact matches of full name or - // "last name" (i.e. last dotted part) - if (fullnameLower === object || parts.slice(-1)[0] === object) - score += Scorer.objNameMatch; - else if (parts.slice(-1)[0].indexOf(object) > -1) - score += Scorer.objPartialMatch; // matches in last name - - const objName = objNames[match[1]][2]; - const title = titles[match[0]]; - - // If more than one term searched for, we require other words to be - // found in the name/title/description - const otherTerms = new Set(objectTerms); - otherTerms.delete(object); - if (otherTerms.size > 0) { - const haystack = `${prefix} ${name} ${objName} ${title}`.toLowerCase(); - if ( - [...otherTerms].some((otherTerm) => haystack.indexOf(otherTerm) < 0) - ) - return; - } - - let anchor = match[3]; - if (anchor === "") anchor = fullname; - else if (anchor === "-") anchor = objNames[match[1]][1] + "-" + fullname; - - const descr = objName + _(", in ") + title; - - // add custom score for some objects according to scorer - if (Scorer.objPrio.hasOwnProperty(match[2])) - score += Scorer.objPrio[match[2]]; - else score += Scorer.objPrioDefault; - - results.push([ - docNames[match[0]], - fullname, - "#" + anchor, - descr, - score, - filenames[match[0]], - ]); - }; - Object.keys(objects).forEach((prefix) => - objects[prefix].forEach((array) => - objectSearchCallback(prefix, array) - ) - ); - return results; - }, - - /** - * search for full-text terms in the index - */ - performTermsSearch: (searchTerms, excludedTerms) => { - // prepare search - const terms = Search._index.terms; - const titleTerms = Search._index.titleterms; - const filenames = Search._index.filenames; - const docNames = Search._index.docnames; - const titles = Search._index.titles; - - const scoreMap = new Map(); - const fileMap = new Map(); - - // perform the search on the required terms - searchTerms.forEach((word) => { - const files = []; - const arr = [ - { files: terms[word], score: Scorer.term }, - { files: titleTerms[word], score: Scorer.title }, - ]; - // add support for partial matches - if (word.length > 2) { - const escapedWord = _escapeRegExp(word); - Object.keys(terms).forEach((term) => { - if (term.match(escapedWord) && !terms[word]) - arr.push({ files: terms[term], score: Scorer.partialTerm }); - }); - Object.keys(titleTerms).forEach((term) => { - if (term.match(escapedWord) && !titleTerms[word]) - arr.push({ files: titleTerms[word], score: Scorer.partialTitle }); - }); - } - - // no match but word was a required one - if (arr.every((record) => record.files === undefined)) return; - - // found search word in contents - arr.forEach((record) => { - if (record.files === undefined) return; - - let recordFiles = record.files; - if (recordFiles.length === undefined) recordFiles = [recordFiles]; - files.push(...recordFiles); - - // set score for the word in each file - recordFiles.forEach((file) => { - if (!scoreMap.has(file)) scoreMap.set(file, {}); - scoreMap.get(file)[word] = record.score; - }); - }); - - // create the mapping - files.forEach((file) => { - if (fileMap.has(file) && fileMap.get(file).indexOf(word) === -1) - fileMap.get(file).push(word); - else fileMap.set(file, [word]); - }); - }); - - // now check if the files don't contain excluded terms - const results = []; - for (const [file, wordList] of fileMap) { - // check if all requirements are matched - - // as search terms with length < 3 are discarded - const filteredTermCount = [...searchTerms].filter( - (term) => term.length > 2 - ).length; - if ( - wordList.length !== searchTerms.size && - wordList.length !== filteredTermCount - ) - continue; - - // ensure that none of the excluded terms is in the search result - if ( - [...excludedTerms].some( - (term) => - terms[term] === file || - titleTerms[term] === file || - (terms[term] || []).includes(file) || - (titleTerms[term] || []).includes(file) - ) - ) - break; - - // select one (max) score for the file. - const score = Math.max(...wordList.map((w) => scoreMap.get(file)[w])); - // add result to the result list - results.push([ - docNames[file], - titles[file], - "", - null, - score, - filenames[file], - ]); - } - return results; - }, - - /** - * helper function to return a node containing the - * search summary for a given text. keywords is a list - * of stemmed words. - */ - makeSearchSummary: (htmlText, keywords) => { - const text = Search.htmlToText(htmlText); - if (text === "") return null; - - const textLower = text.toLowerCase(); - const actualStartPosition = [...keywords] - .map((k) => textLower.indexOf(k.toLowerCase())) - .filter((i) => i > -1) - .slice(-1)[0]; - const startWithContext = Math.max(actualStartPosition - 120, 0); - - const top = startWithContext === 0 ? "" : "..."; - const tail = startWithContext + 240 < text.length ? "..." : ""; - - let summary = document.createElement("p"); - summary.classList.add("context"); - summary.textContent = top + text.substr(startWithContext, 240).trim() + tail; - - return summary; - }, -}; - -_ready(Search.init); diff --git a/docs/_build/_static/sphinx_highlight.js b/docs/_build/_static/sphinx_highlight.js deleted file mode 100644 index 8a96c69..0000000 --- a/docs/_build/_static/sphinx_highlight.js +++ /dev/null @@ -1,154 +0,0 @@ -/* Highlighting utilities for Sphinx HTML documentation. */ -"use strict"; - -const SPHINX_HIGHLIGHT_ENABLED = true - -/** - * highlight a given string on a node by wrapping it in - * span elements with the given class name. - */ -const _highlight = (node, addItems, text, className) => { - if (node.nodeType === Node.TEXT_NODE) { - const val = node.nodeValue; - const parent = node.parentNode; - const pos = val.toLowerCase().indexOf(text); - if ( - pos >= 0 && - !parent.classList.contains(className) && - !parent.classList.contains("nohighlight") - ) { - let span; - - const closestNode = parent.closest("body, svg, foreignObject"); - const isInSVG = closestNode && closestNode.matches("svg"); - if (isInSVG) { - span = document.createElementNS("http://www.w3.org/2000/svg", "tspan"); - } else { - span = document.createElement("span"); - span.classList.add(className); - } - - span.appendChild(document.createTextNode(val.substr(pos, text.length))); - const rest = document.createTextNode(val.substr(pos + text.length)); - parent.insertBefore( - span, - parent.insertBefore( - rest, - node.nextSibling - ) - ); - node.nodeValue = val.substr(0, pos); - /* There may be more occurrences of search term in this node. So call this - * function recursively on the remaining fragment. - */ - _highlight(rest, addItems, text, className); - - if (isInSVG) { - const rect = document.createElementNS( - "http://www.w3.org/2000/svg", - "rect" - ); - const bbox = parent.getBBox(); - rect.x.baseVal.value = bbox.x; - rect.y.baseVal.value = bbox.y; - rect.width.baseVal.value = bbox.width; - rect.height.baseVal.value = bbox.height; - rect.setAttribute("class", className); - addItems.push({ parent: parent, target: rect }); - } - } - } else if (node.matches && !node.matches("button, select, textarea")) { - node.childNodes.forEach((el) => _highlight(el, addItems, text, className)); - } -}; -const _highlightText = (thisNode, text, className) => { - let addItems = []; - _highlight(thisNode, addItems, text, className); - addItems.forEach((obj) => - obj.parent.insertAdjacentElement("beforebegin", obj.target) - ); -}; - -/** - * Small JavaScript module for the documentation. - */ -const SphinxHighlight = { - - /** - * highlight the search words provided in localstorage in the text - */ - highlightSearchWords: () => { - if (!SPHINX_HIGHLIGHT_ENABLED) return; // bail if no highlight - - // get and clear terms from localstorage - const url = new URL(window.location); - const highlight = - localStorage.getItem("sphinx_highlight_terms") - || url.searchParams.get("highlight") - || ""; - localStorage.removeItem("sphinx_highlight_terms") - url.searchParams.delete("highlight"); - window.history.replaceState({}, "", url); - - // get individual terms from highlight string - const terms = highlight.toLowerCase().split(/\s+/).filter(x => x); - if (terms.length === 0) return; // nothing to do - - // There should never be more than one element matching "div.body" - const divBody = document.querySelectorAll("div.body"); - const body = divBody.length ? divBody[0] : document.querySelector("body"); - window.setTimeout(() => { - terms.forEach((term) => _highlightText(body, term, "highlighted")); - }, 10); - - const searchBox = document.getElementById("searchbox"); - if (searchBox === null) return; - searchBox.appendChild( - document - .createRange() - .createContextualFragment( - '

    ' + - '' + - _("Hide Search Matches") + - "

    " - ) - ); - }, - - /** - * helper function to hide the search marks again - */ - hideSearchWords: () => { - document - .querySelectorAll("#searchbox .highlight-link") - .forEach((el) => el.remove()); - document - .querySelectorAll("span.highlighted") - .forEach((el) => el.classList.remove("highlighted")); - localStorage.removeItem("sphinx_highlight_terms") - }, - - initEscapeListener: () => { - // only install a listener if it is really needed - if (!DOCUMENTATION_OPTIONS.ENABLE_SEARCH_SHORTCUTS) return; - - document.addEventListener("keydown", (event) => { - // bail for input elements - if (BLACKLISTED_KEY_CONTROL_ELEMENTS.has(document.activeElement.tagName)) return; - // bail with special keys - if (event.shiftKey || event.altKey || event.ctrlKey || event.metaKey) return; - if (DOCUMENTATION_OPTIONS.ENABLE_SEARCH_SHORTCUTS && (event.key === "Escape")) { - SphinxHighlight.hideSearchWords(); - event.preventDefault(); - } - }); - }, -}; - -_ready(() => { - /* Do not call highlightSearchWords() when we are on the search page. - * It will highlight words from the *previous* search query. - */ - if (typeof Search === "undefined") SphinxHighlight.highlightSearchWords(); - SphinxHighlight.initEscapeListener(); -}); diff --git a/docs/_build/cds.html b/docs/_build/cds.html deleted file mode 100644 index 8b93f22..0000000 --- a/docs/_build/cds.html +++ /dev/null @@ -1,272 +0,0 @@ - - - - - - - - Coding sequence tiling library — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - - - -
    -
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    - -
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    Coding sequence tiling library¶

    -
    -

    Tiling sorting screen tutorial¶

    -

    Tiling screen that tiles gRNA densely across locus or multiple loci, selected based on FACS signal quantiles.

    - - - - - - - - - -
    Library designTiling (gRNAs tile each locus densely)
    tiling library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design



    -
    -

    Example workflow¶

    -
    screen_id=my_sorting_tiling_screen
-
-# 1. Count gRNA & reporter
-bean-count-samples \
---input tests/data/sample_list_tiling.csv          `# Contains fastq file path; see test file for example.`\
--b A                                               `# Base A is edited (into G)` \
--f tests/data/test_guide_info_tiling_chrom.csv     `# Contains gRNA metadata; see test file for example.`\
--o ./                                              `# Output directory` \
--r                                                 `# Quantify reporter edits` \
--n ${screen_id}                                       `# ID of the screen` \
---tiling
-
-# 2. QC samples & guides
-bean-qc \
-  bean_count_${screen_id}.h5ad           `# Input ReporterScreen .h5ad file path` \
-  -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \
-  -r qc_report_${screen_id}              `# Prefix for QC report` \
-
-# 3. Filter & translate alleles
-bean-filter ./bean_count_${screen_id}_masked.h5ad \
--o ./bean_count_${screen_id}_alleleFiltered \
---filter-target-basechange                             `# Filter based on intended base changes. If -b A was provided in bean-count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\
---filter-window --edit-start-pos 0 --edit-end-pos 19   `# Filter based on editing window in spacer position within reporter.`\
---filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \
---translate --translate-genes-list tests/data/gene_symbols.txt
-
-# 4. Quantify variant effect
-bean-run sorting tiling \
-    ./bean_count_${screen_id}_alleleFiltered.h5ad \
-    -o tests/test_res/var/ \
-    --fit-negctrl \
-    --scale-by-acc \
-    --accessibility-col accessibility
-
    -
    -

    See more details below.

    -
    -
    -

    1. Count gRNA & reporter (bean count-samples)¶

    -
    screen_id=my_sorting_tiling_screen
-
-bean-count-samples \
---input tests/data/sample_list_tiling.csv          `# Contains fastq file path; see test file for example.`\
--b A                                               `# Base A is edited (into G)` \
--f tests/data/test_guide_info_tiling_chrom.csv     `# Contains gRNA metadata; see test file for example.`\
--o ./                                              `# Output directory` \
--r                                                 `# Quantify reporter edits` \
--n ${screen_id}                                       `# ID of the screen` \
---tiling
-
    -
    -

    Make sure you follow the input file format for seamless downstream steps. This will produce ./bean_count_${screen_id}.h5ad.

    -
    -
    -

    2. QC (bean qc)¶

    -

    Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the input file guideline, but you can change the parameters with the full argument list of ``bean-qc` <../../README#bean-qc-qc-of-reporter-screen-data>`_. (Common factors you may want to tweak is --ctrl-cond=bulk and --lfc-conds=top,bot if you have different sample condition labels.)

    -
    bean-qc \
-  bean_count_${screen_id}.h5ad           `# Input ReporterScreen .h5ad file path` \
-  -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \
-  -r qc_report_${screen_id}              `# Prefix for QC report` \
-  [--tiling]                          `# Not required if you have passed --tiling in counting step`
-
    -
    -

    If the data does not include reporter editing data, you can provide --no-editing flag to omit the editing rate QC.

    -
    -
    -

    3. Filter alleles (bean filter)¶

    -

    As tiling library doesn’t have designated per-gRNA target variant, any base edit observed in reporter may be the candidate variant, while having too many variants with very low editing rate significantly decreases the power. Variants are filtered based on multiple criteria in bean-fitler.

    -

    If the screen targets coding sequence, it’s beneficial to translate edits into coding varaints whenever possible for better power. For translation, provide --translate and one of the following:

    -
    [ --translate-gene-name GENE_SYMBOL OR
-  --translate-genes-list path_to_gene_names_file.txt OR
-  --translate-fasta gene_exon.fa, OR
-  --translate-fastas-csv gene_exon_fas.csv]
-
    -
    -

    where path_to_gene_names_file.txt has one gene symbol per line, and gene symbol uses its MANE transcript (hg38) coordinates of exons. In order to use other reference versions or transcript ID, you’ll need to feed in fasta file. See detailed formatting of fasta file here.

    -

    Example allele filtering given we’re translating based on MANE transcript exons of multiple gene symbols:

    -
    bean-filter ./bean_count_${screen_id}_masked.h5ad \
--o ./bean_count_${screen_id}_alleleFiltered \
---filter-target-basechange                             `# Filter based on intended base changes. If -b A was provided in bean-count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\
---filter-window --edit-start-pos 0 --edit-end-pos 19   `# Filter based on editing window in spacer position within reporter.`\
---filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \
---translate --translate-genes-list tests/data/gene_symbols.txt
-
    -
    -

    Ouptut file `` shows number of alleles per guide and number of guides per variant, where we want high enough values for the latter. See the typical output for dataset with good editing coverage & filtering result here.

    -
    -
    -

    4. Quantify variant effect (bean run)¶

    -

    By default, bean-run [sorting,survival] tiling uses most filtered allele counts table for variant identification and quantification of their effects. **Check allele filtering output** and choose alternative filtered allele counts table if necessary.

    -

    bean-run can take 3 run options to quantify editing rate:

    -
      -
    1. -
      From reporter + accessibility

      1-1. If your gRNA metadata table (tests/data/test_guide_info.csv above) included per-gRNA accessibility score,

      -
      -
      -
         bean-run sorting tiling \
-   ./bean_count_${screen_id}_alleleFiltered.h5ad \
-   -o tests/test_res/var/ \
-   --fit-negctrl \
-   --scale-by-acc \
-   --accessibility-col accessibility
-
-1-2. If your gRNA metadata table (\ ``tests/data/test_guide_info.csv`` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal,
-
      -
      -
      bean-run sorting tiling \
-./bean_count_${screen_id}_alleleFiltered.h5ad \
--o tests/test_res/var/ \
---fit-negctrl \
---scale-by-acc \
---accessibility-bw accessibility.bw
-
      -
      -
      2. -

    2. From reporter

      -
      bean-run sorting tiling \
-./bean_count_${screen_id}_alleleFiltered.h5ad \
--o tests/test_res/var/ \
---fit-negctrl
-
      -
      -
      3. -
    3. -
      No reporter information, assume the same editing efficiency of all gRNAs.

      Use this option if your data don’t have editing rate information.

      -
      -
      -
      bean-run sorting tiling \
-./bean_count_${screen_id}_alleleFiltered.h5ad \
--o tests/test_res/var/ \
---fit-negctrl \
---uniform-edit
-
      -
      -
      4. -
    -

    See Subcommands for the full details.

    -
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    - -
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    - ©2024, Jayoung Ryu. - - | - Powered by Sphinx 7.2.6 - & Alabaster 0.7.16 - - | - Page source -
    - - - - - - \ No newline at end of file diff --git a/docs/_build/commands/count.html b/docs/_build/commands/count.html deleted file mode 100644 index 4b364ed..0000000 --- a/docs/_build/commands/count.html +++ /dev/null @@ -1,142 +0,0 @@ - - - - - - - - bean count[-samples]: Count (reporter) screen data — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - -
    -
    -
    - - -
    - -
    -

    bean count[-samples]: Count (reporter) screen data¶

    -

    bean count-samples (or bean count for a single sample) maps guide into guide counts, allowing for base transition in spacer sequence. When the matched reporter information is provided, it can count the target site edits and alleles produced by each guide. Mapping is efficiently done based on CRISPResso2 modified for base-edit-aware mapping.

    -
    bean count-samples \
-  --input sample_list.csv   `# sample with lines 'R1_filepath,R2_filepath,sample_name\n'` \
-  -b A                      `# base that is being edited (A/G)` \
-  -f sgRNA_info_table.csv   `# sgRNA information` \
-  -o .                      `# output directory` \
-  -r                        `# read edit/allele information from reporter` \
-  -t 12                     `# number of threads` \
-  --name my_sorting_screen  `# name of this sample run` \
-
    -
    -
    bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r
-
    -
    -

    By default, bean count[-samples] assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure.

    -
    -

    Read structuren

    -
    -

    See full detail below.

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    Input file format¶

    -

    See Input file format for input file formats.

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    -

    Output file format¶

    -

    count or count-samples produces .h5ad and .xlsx file with guide and per-guide allele counts.

    -
      -

    • .h5ad: This output file follows annotated matrix format compatible with AnnData and is based on Screen object in [purturb_tools](https://github.com/pinellolab/perturb-tools). See Data Structure section for more information.

      • -

    • .xlsx: This output file contains .guides, .samples, .X[_bcmatch,_edits]. (allele_tables are often too large to write into an Excel!)

      • -
    -
    - - -
    - -
    -
    - -
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    - ©2024, Jayoung Ryu. - - | - Powered by Sphinx 7.2.6 - & Alabaster 0.7.16 - - | - Page source -
    - - - - - - \ No newline at end of file diff --git a/docs/_build/commands/create-screen.html b/docs/_build/commands/create-screen.html deleted file mode 100644 index 8ea5daf..0000000 --- a/docs/_build/commands/create-screen.html +++ /dev/null @@ -1,123 +0,0 @@ - - - - - - - - bean create-screen: Create ReporterScreen object from flat files — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - -
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    - - -
    - -
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    bean create-screen: Create ReporterScreen object from flat files¶

    -
    bean create-screen gRNA_library.csv sample_list.csv gRNA_counts_table.csv
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    Input¶

    -
      -

    • gRNA_library.csv

      • -

    • sample_list.csv

      • -

    • gRNA_counts_table.csv: Table with gRNA ID in the first column and sample IDs as the column names (first row) -gRNA_library.csv and sample_list.csv should be formatted as Input file format.

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    - ©2024, Jayoung Ryu. - - | - Powered by Sphinx 7.2.6 - & Alabaster 0.7.16 - - | - Page source -
    - - - - - - \ No newline at end of file diff --git a/docs/_build/commands/filter.html b/docs/_build/commands/filter.html deleted file mode 100644 index 22d1c8e..0000000 --- a/docs/_build/commands/filter.html +++ /dev/null @@ -1,154 +0,0 @@ - - - - - - - - filter: Filtering (and optionally translating) alleles — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - -
    -
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    - -
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    filter: Filtering (and optionally translating) alleles¶

    -

    As tiling mode of bean run accounts for any robustly observed alleles, bean filter filters for such alleles.

    -
    bean filter my_sorting_screen_masked.h5ad \
--o my_sorting_screen_filtered.h5ad  `# Output file path` \
-
    -
    -
    -
    -

    Output¶

    -

    Above command produces

    -
      -

    • my_sorting_screen_filtered.h5ad with filtered alleles stored in .uns,

      • -

    • my_sorting_screen_filtered.filtered_allele_stats.pdf, and my_sorting_screen_filtered.filter_log.txt that report allele count stats in each filtering step.

      • -
    -

    You may want to adjust the flitering parameters to obtain optimal balance between # guides per variant & # variants that are scored. See example outputs of filtering step here.

    -
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    Translating alleles¶

    -

    If you want to obtain amino acid level variant for coding sequence tiling screens, provide coding sequence positions which variants occuring within the coding sequence will be translated. This is optional, but **highly recommended* to increase per-(coding)variant support.*

    -

    Allele translation

    -
    bean filter my_sorting_screen.h5ad \
--o my_sorting_screen_masked.h5ad \
---translate   `# Translate coding variants` \
-[ --translate-gene-name GENE_SYMBOL OR
-  --translate-genes-list path_to_gene_names_file.txt OR
-  --translate-fasta gene_exon.fa, OR
-  --translate-fastas-csv gene_exon_fas.csv]
-
    -
    -
      -

    • When library covers a single gene, do either of the following:

      -
        -

      1. Feed --translate-gene-name GENE_SYMBOL if your genomic_pos column of sgRNA_info_tbl is compatible with MANE transcript‘s reference genome. (Per 10/23/2023, GRCh38). This will automatically load the exon positions based on MANE transcript annotation.

        2. -

      2. To use your custom coding sequence and exon positions, feed --translate-fasta gene_exon.fa argument where gene_exon.fa is the FASTA file with entries of exons. See full details here.

        3. -
      -
      • -

    • When library covers multiple genes, do either of the following:

      -
        -

      1. Feed --translate-genes-list path_to_gene_names_file.txt where path_to_gene_names_file.txt is file with one gene symbol per line.

        2. -

      2. Feed --translate-fastas-csv gene_exon_fas.csv where gene_exon_fas.csv is the csv file with lines gene_id,gene_exon_fasta_path without header. Each FASTA file in gene_exon_fasta_path is formatted as the single-gene FASTA file.

        3. -
      -
      • -

    • Translation will keep the variants outside the coding sequence as nucleotide-level variants, while aggregating variants leading to the same coding sequence variants.

      • -
    -
    - - -
    - -
    -
    - -
    -
    -
    - ©2024, Jayoung Ryu. - - | - Powered by Sphinx 7.2.6 - & Alabaster 0.7.16 - - | - Page source -
    - - - - - - \ No newline at end of file diff --git a/docs/_build/commands/input.html b/docs/_build/commands/input.html deleted file mode 100644 index f0b9c24..0000000 --- a/docs/_build/commands/input.html +++ /dev/null @@ -1,160 +0,0 @@ - - - - - - - - sgRNA_info_table.csv — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - -
    -
    -
    - - -
    - -

    This document describes the input files of bean count-samples.

    -
    -

    sgRNA_info_table.csv¶

    -

    File should contain following columns.

    -
      -

    • name: gRNA ID column

      • -

    • sequence: gRNA sequence

      • -

    • barcode: R2 barcode to help match reporter to gRNA, written in the sense direction (as in R1)

      • -

    • In order to use accessibility in the variant effect quantification, provide accessibility information in one of two options. (For non-targeting guides, provide NA values (empty cell).)

      -
        -

      • Option 1: chrom & genomic_pos: Chromosome (ex. chr19) and genomic position of guide sequence. You will have to provide the path to the bigwig file with matching reference version in bean run.

        • -

      • Option 2: accessibility_signal: ATAC-seq signal value of the target loci of each guide.

        • -
      -
      • -

    • For variant library (gRNAs are designed to target specific variants and ignores bystander edits)

      -
        -

      • target: This column denotes which target variant/element of each gRNA. This is not used in bean count[-samples] but required to run bean run in later steps.

        • -

      • target_group: If negative/positive control gRNA will be considered in bean qc and/or bean run, specify as “NegCtrlâ€/â€PosCtrl†in this column.

        • -

      • target_pos: If --match_target_pos flag is used, input file needs target_pos which specifies 0-based relative position of targeted base within Reporter sequence.

        • -
      -
      • -

    • For tiling library (gRNAs tile coding / noncoding sequences)

      -
        -

      • strand: Specifies gRNA strand information relative to the reference genome.

        • -

      • chrom: Chromosome of gRNA targeted locus.

        • -

      • start_pos: gRNA starting position in the genome. Required when you provide strand column. Should specify the smaller coordinate value among start and end position regardless of gRNA strandedness.

        • -
      -
      • -
    -

    Also see examples for variant library and tiling library.

    -
    -
    -

    sample_list.csv¶

    -

    File should contain following columns with header.

    -
      -

    • R1_filepath: Path to read 1 .fastq[.gz] file

      • -

    • R2_filepath: Path to read 1 .fastq[.gz] file

      • -

    • sample_id: ID of sequencing sample

      • -

    • replicate: Replicate # of this sample (Should NOT contain .)

      • -

    • condition: Name of the sorting bin (ex. top, bot), or label of timepoint (ex. D5, D18)

      • -
    -

    For FACS sorting screens:

    -
      -

    • upper_quantile: FACS sorting upper quantile

      • -

    • lower_quantile: FACS sorting lower quantile

      • -
    -

    For proliferation / survival screens:

    -
      -

    • time: Numeric time following the base editing of each sample.

      • -
    -

    Also see examples for FACS sorting screen.

    -
    - - -
    - -
    -
    - -
    -
    -
    - ©2024, Jayoung Ryu. - - | - Powered by Sphinx 7.2.6 - & Alabaster 0.7.16 - - | - Page source -
    - - - - - - \ No newline at end of file diff --git a/docs/_build/commands/profile.html b/docs/_build/commands/profile.html deleted file mode 100644 index 32f78d4..0000000 --- a/docs/_build/commands/profile.html +++ /dev/null @@ -1,119 +0,0 @@ - - - - - - - - bean profile: Profile editing patterns — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - -
    -
    -
    - - -
    - -
    -

    bean profile: Profile editing patterns¶

    -
    bean profile my_sorting_screen.h5ad -o output_prefix `# Prefix for editing profile report`
-
    -
    -
    -
    -

    Output¶

    -

    Above command produces prefix_editing_preference.[html,ipynb] as editing preferences (see example).

    -

    Allele translation

    -
    - - -
    - -
    -
    - -
    -
    -
    - ©2024, Jayoung Ryu. - - | - Powered by Sphinx 7.2.6 - & Alabaster 0.7.16 - - | - Page source -
    - - - - - - \ No newline at end of file diff --git a/docs/_build/commands/qc.html b/docs/_build/commands/qc.html deleted file mode 100644 index 5526162..0000000 --- a/docs/_build/commands/qc.html +++ /dev/null @@ -1,282 +0,0 @@ - - - - - - - - bean qc: QC of reporter screen data — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - -
    -
    -
    - - -
    - -
    -

    bean qc: QC of reporter screen data¶

    -
    bean qc \
-  my_sorting_screen.h5ad             `# Input ReporterScreen .h5ad file path` \
-  -o my_sorting_screen_masked.h5ad   `# Output ReporterScreen .h5ad file path` \
-  -r qc_report_my_sorting_screen     `# Prefix for QC report` \
-  --ctrl-cond presort                `# "condition" column in the control sample before selection. Mean gRNA editing rates in these samples are reported. ` \
-# Inspect the output qc_report_my_sorting_screen.html to tweak QC threshold
-
-bean qc \
-  my_sorting_screen.h5ad              \
-  -o my_sorting_screen_masked.h5ad    \
-  -r qc_report_my_sorting_screen      \
-  #[--count-correlation-thres 0.7 ...]\
-  -b
-
    -
    -

    bean qc supports following quality control and masks samples with low quality. Specifically:

    -

    Allele translation

    -
      -

    • Plots guide coverage and the uniformity of coverage

      • -

    • Guide count correlation between samples

      • -

    • Log fold change correlation when positive controls are provided

      • -

    • Plots editing rate distribution

      • -

    • Identify samples with low guide coverage/guide count correlation/editing rate and mask the sample in bdata.samples.mask

      • -

    • Identify outlier guides to filter out

      • -
    -
    -
    -

    Output¶

    -

    Above command produces

    -
      -

    • my_sorting_screen_masked.h5ad without problematic replicate and guides and with sample masks, and

      • -

    • qc_report_my_sorting_screen.[html,ipynb] as QC report.
       -##### Optional arguments:

      • -

    • -o OUT_SCREEN_PATH, --out-screen-path OUT_SCREEN_PATH

      -
      Path where quality-filtered ReporterScreen object to be written to
-
      -
      -
      • -

    • -r OUT_REPORT_PREFIX, --out-report-prefix OUT_REPORT_PREFIX

      -
      Output prefix of qc report (prefix.html, prefix.ipynb)
-
      -
      -
      • -
    -
    -

    QC thresholds:¶

    -
      -

    • --count-correlation-thres COUNT_CORRELATION_THRES

      -
      Correlation threshold to mask out.
-
      -
      -
      • -

    • --edit-rate-thres EDIT_RATE_THRES

      -
      Mean editing rate threshold per sample to mask out.
-
      -
      -
      • -

    • --lfc-thres LFC_THRES

      -
      Positive guides' correlation threshold to filter out.
-
      -
      -
      • -
    -
    -
    -

    Run options:¶

    -
      -

    • -b, --remove-bad-replicates

      -
      Remove replicates with at least two of its samples meet the QC threshold (bean run does not support having only one sorting bin sample for a replicate).
-
      -
      -
      • -

    • -i, --ignore-missing-samples

      -
      If the flag is not provided, if the ReporterScreen object does not contain all condiitons for
-each replicate, make fake empty samples. If the flag is provided, don't add dummy samples.
-
      -
      -
      • -

    • --no-editing Ignore QC about editing. Can be used for QC of other editing modalities.

      • -

    • --dont-recalculate-edits

      -
      When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from
-ReporterScreen.uns['allele_count'].
-
      -
      -
      • -
    -
    -
    -

    Input .h5ad formatting:¶

    -

    Note that these arguements will change the way the QC metrics are calculated for guides, samples, or replicates.

    -
      -

    • --tiling TILING Specify that the guide library is tiling library without ‘n guides per target’ design

      • -

    • --replicate-label REPLICATE_LABEL

      -
      Label of column in `bdata.samples` that describes replicate ID.
-
      -
      -
      • -

    • --sample-covariates SAMPLE_COVARIATES

      -
      Comma-separated list of column names in `bdata.samples` that describes non-selective
-experimental condition. (drug treatment, etc.)
-
      -
      -
      • -

    • --condition-label CONDITION_LABEL

      -
      Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time,
-etc.)
-
      -
      -

      ###### Editing rate calculation

      -
        -

      • --control-condition CTRL_COND

        -
        Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate
-to be calculated. Default is `None`, which considers all samples.
-
        -
        -
        • -

      • --rel-pos-is-reporter

        -
        Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If
-`False`, those are relative to spacer position.
-
        -
        -

        Editing rate is calculated with following parameters in

        -
          -

        • Variant screens:

          -
            -

          • --target-pos-col TARGET_POS_COL

            -
            Target position column in `bdata.guides` specifying target edit position in reporter
-
            -
            -
            • -
          -
          • -

        • tiling screens:

          -
            -

          • --edit-start-pos EDIT_START_POS

            -
            Edit start position to quantify editing rate on, 0-based inclusive.
-
            -
            -
            • -

          • --edit-end-pos EDIT_END_POS

            -
            Edit end position to quantify editing rate on, 0-based exclusive.
-
            -
            -

            ###### LFC of positive controls

            -
            • -
          -
          • -
        -
        • -

      • --posctrl-col POSCTRL_COL

        -
        Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all
-gRNAs, feed empty string ''.
-
        -
        -
        • -

      • --posctrl-val POSCTRL_VAL

        -
        Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the
-positive control in calculating log fold change.
-
        -
        -
        • -

      • --lfc-conds LFC_CONDS

        -
        Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated
-between, delimited by comma
-
        -
        -
        • -
      -
      • -
    -
    -
    - - -
    - -
    -
    - -
    -
    -
    - ©2024, Jayoung Ryu. - - | - Powered by Sphinx 7.2.6 - & Alabaster 0.7.16 - - | - Page source -
    - - - - - - \ No newline at end of file diff --git a/docs/_build/commands/run.html b/docs/_build/commands/run.html deleted file mode 100644 index 4711982..0000000 --- a/docs/_build/commands/run.html +++ /dev/null @@ -1,201 +0,0 @@ - - - - - - - - bean run: Quantify variant effects — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - -
    -
    -
    - - -
    - -
    -

    bean run: Quantify variant effects¶

    -

    BEAN uses Bayesian network to incorporate gRNA editing outcome to provide posterior estimate of variant phenotype. The Bayesian network reflects data generation process. Briefly,

    -
      -

    1. Cellular phenotype (either for cells are sorted upon for sorting screen, or log(proliferation rate)) is modeled as the Gaussian mixture distribution of wild-type phenotype and variant phenotype.

      2. -

    2. The weight of the mixture components are inferred from the reporter editing outcome and the chromatin accessibility of the loci.

      3. -

    3. Cells with each gRNA, formulated as the mixture distribution, is sorted by the phenotypic quantile to produce the gRNA counts.

      4. -
    -

    For the full detail, see the method section of the BEAN manuscript.

    -

    model

    -



    -
    -
    -

    Usage example¶

    -
    bean run sorting[survival] variant[tiling] my_sorting_screen_filtered.h5ad \
-[--uniform-edit, --scale-by-acc [--acc-bw-path accessibility_signal.bw, --acc-col accessibility]] \
--o output_prefix/ \
---fit-negctrl
-
    -
    -

    See full list of parameters below.

    -
    -
    -

    Input¶

    -

    my_sorting_screen_filtered.h5ad can be produced by one of the following:

    -
      -

    1. ``bean count-samples` <(#bean-count-samples-count-reporter-screen-data>`_) when you have raw .fastq file

      2. -

    2. (Limited to bean run variant mode) bean create-screen when you have flat .csv tables of gRNA metadata table, sample metadata table, gRNA counts table (# guides x # samples), and optionally # edits table. -.. code-block:: bash

      -
      -

      bean create-screen gRNA_info_table.csv sample_info_table.csv gRNA_counts_table.csv [–edits edit_counts_table.csv -o output.h5ad]

      -
      -
        -

      • gRNA_info_table.csv should have following columns.

        -
          -

        • name: gRNA ID column

          • -

        • target: This column denotes which target variant/element of each gRNA.

          • -

        • target_group [Optional]: If negative control gRNA will be used, specify as “NegCtrl†in this column.

          • -
        -
        • -

      • sample_info_table.csv should have following columns.

        -
          -

        • sample_id: ID of sequencing sample

          • -

        • replicate: Replicate # of this sample

          • -

        • bin: Name of the sorting bin

          • -

        • upper_quantile: FACS sorting upper quantile

          • -

        • lower_quantile: FACS sorting lower quantile

          • -
        -
        • -

      • gRNA_counts_table.csv should be formatted as follows.

        -
          -

        • Columns include one of sample_id columns in sample_info_table.csv file.

          • -

        • 1st row (row index) follows name (gRNA ID) in gRNA_info_table.csv file.

          • -
        -
        • -
      -
      3. -

    3. You can manually create the AnnData object with more annotations including allele counts: see API tutorial for full detail.

      4. -
    -
    -
    -

    Output¶

    -

    model

    -

    Above command produces

    -
      -

    • output_prefix/bean_element_result.[model_type].csv with following columns:

      -
        -

      • Estimated variant effect sizes

        -
          -

        • mu (Effect size): Mean of variant phenotype, given the wild type has standard normal phenotype distribution of mu = 0, sd = 1.

          • -

        • mu_sd: Mean of variant phenotype mu is modeled as normal distribution. The column shows fitted standard deviation of mu that quantify the uncertainty of the variant effect.

          • -

        • mu_z: z-score of mu

          • -

        • sd: Standard deviation of variant phenotype, given the wild type has standard normal phenotype distribution of mu = 0, sd = 1.

          • -

        • CI[0.025, 0.975]: Credible interval of mu

          • -

        • When negative control is provided, above columns with _adj suffix are provided, which are the corresponding values adjusted for negative control.

          • -
        -
        • -

      • Metrics on per-variant evidence provided in input (provided in tiling mode)

        -
          -

        • effective_edit_rate: Sum of per-variant editing rates over all alleles observed in the input. Allele-level editing rate is divided by the number of variants observed in the allele prior to summing up.

          • -

        • n_guides: # of guides covering the variant.

          • -

        • n_coocc: # of cooccurring variants with a given variant in any alleles observed in the input.

          • -
        -
        • -
      -
      • -

    • output_prefix/bean_sgRNA_result.[model_type].csv:

      -
        -

      • edit_rate: Estimated editing rate at the target loci.

        • -
      -
      • -
    -
    - - -
    - -
    -
    - -
    -
    -
    - ©2024, Jayoung Ryu. - - | - Powered by Sphinx 7.2.6 - & Alabaster 0.7.16 - - | - Page source -
    - - - - - - \ No newline at end of file diff --git a/docs/_build/count.html b/docs/_build/count.html deleted file mode 100644 index 2aa8294..0000000 --- a/docs/_build/count.html +++ /dev/null @@ -1,299 +0,0 @@ - - - - - - - - bean count — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - - - -
    -
    -
    - - -
    - -
    -

    bean count¶

    -
    -

    bean count[-samples]: Count (reporter) screen data¶

    -

    bean count-samples (or bean count for a single sample) maps guide into guide counts, allowing for base transition in spacer sequence. When the matched reporter information is provided, it can count the target site edits and alleles produced by each guide. Mapping is efficiently done based on CRISPResso2 modified for base-edit-aware mapping.

    -
    bean count-samples \
-  --input sample_list.csv   `# sample with lines 'R1_filepath,R2_filepath,sample_name\n'` \
-  -b A                      `# base that is being edited (A/G)` \
-  -f sgRNA_info_table.csv   `# sgRNA information` \
-  -o .                      `# output directory` \
-  -r                        `# read edit/allele information from reporter` \
-  -t 12                     `# number of threads` \
-  --name my_sorting_screen  `# name of this sample run` \
-
    -
    -
    bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r
-
    -
    -

    By default, bean count[-samples] assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure.

    -
    -

    Read structuren

    -
    -

    See full detail below.

    -
    -
    -

    Input file format¶

    -

    See Input file format for input file formats.

    -
    -
    -

    Output file format¶

    -

    count or count-samples produces .h5ad and .xlsx file with guide and per-guide allele counts.

    -
      -

    • .h5ad: This output file follows annotated matrix format compatible with AnnData and is based on Screen object in [purturb_tools](https://github.com/pinellolab/perturb-tools). See Data Structure section for more information.

      • -

    • .xlsx: This output file contains .guides, .samples, .X[_bcmatch,_edits]. (allele_tables are often too large to write into an Excel!)

      • -
    -
    -
    -

    Full parameters¶

    -
    usage: bean count [-h] --R1 R1 --R2 R2 -b EDITED_BASE -f SGRNA_FILENAME
-                  [--guide-start-seq GUIDE_START_SEQ]
-                  [--guide-end-seq GUIDE_END_SEQ]
-                  [--barcode-start-seq BARCODE_START_SEQ] [-r]
-                  [-q MIN_AVERAGE_READ_QUALITY] [-s MIN_SINGLE_BP_QUALITY]
-                  [-n NAME] [-o OUTPUT_FOLDER] [-l REPORTER_LENGTH]
-                  [--keep-intermediate] [--qstart-R1 QSTART_R1]
-                  [--qend-R1 QEND_R1] [--qstart-R2 QSTART_R2]
-                  [--qend-R2 QEND_R2] [--gstart-reporter GSTART_REPORTER]
-                  [--match-target-pos] [--target-pos-col TARGET_POS_COL]
-                  [--guide-bc GUIDE_BC] [--guide-bc-len GUIDE_BC_LEN]
-                  [--offset] [--align-fasta ALIGN_FASTA] [--string-allele]
-                  [-g] [-m] [--tiling]
-
    -
    -
    -

    Named Arguments¶

    -
    -
    --R1
    -

    FASTQ file path for read 1

    -
    -
    --R2
    -

    FASTQ file path for read 2.

    -
    -
    -b, --edited-base
    -

    For base editors, the base that should be ignored when matching the gRNA sequence

    -
    -
    -f, --sgRNA-filename
    -

    sgRNA description file. The format requires three columns: name, sequence, barcode [ reporter [,strand, target_pos], [start_pos, offset] ].

    -
    -
    --guide-start-seq
    -

    Guide starts after this sequence in R1

    -

    Default: “â€

    -
    -
    --guide-end-seq
    -

    Guide starts after this sequence in R1

    -

    Default: “â€

    -
    -
    --barcode-start-seq
    -

    Barcode + reporter starts after this sequence in R2, denoted as the sense direction (the same sequence direction as R1).

    -

    Default: “â€

    -
    -
    -r, --count-reporter
    -

    Count reporter edits.

    -

    Default: False

    -
    -
    -q, --min-average-read-quality
    -

    Minimum average quality score (phred33) to keep a read

    -

    Default: 30

    -
    -
    -s, --min-single-bp-quality
    -

    Minimum single bp score (phred33) to keep a read

    -

    Default: 0

    -
    -
    -n, --name
    -

    Output name

    -

    Default: “â€

    -
    -
    -o, --output-folder
    -

    Default: “â€

    -
    -
    -l, --reporter-length
    -

    length of the reporter

    -

    Default: 32

    -
    -
    --keep-intermediate
    -

    Keep all the intermediate files

    -

    Default: False

    -
    -
    --qstart-R1
    -

    Start position of the read when filtering for quality score of the read 1

    -

    Default: 0

    -
    -
    --qend-R1
    -

    End position of the read when filtering for quality score of the read 1

    -

    Default: 47

    -
    -
    --qstart-R2
    -

    Same as qstart_R1, for read 2 fastq file

    -

    Default: 0

    -
    -
    --qend-R2
    -

    Same as qstart_R2, for read 2 fastq file

    -

    Default: 36

    -
    -
    --gstart-reporter
    -

    Start position of the guide sequence in the reporter

    -

    Default: 6

    -
    -
    --match-target-pos
    -

    Count the edit in the exact target position.

    -

    Default: False

    -
    -
    --target-pos-col
    -

    Column name specifying the relative target position within reporter sequence.

    -

    Default: “target_posâ€

    -
    -
    --guide-bc
    -

    Construct has guide barcode

    -

    Default: True

    -
    -
    --guide-bc-len
    -

    Guide barcode sequence length at the beginning of the R2

    -

    Default: 4

    -
    -
    --offset
    -

    Guide file has offest column that will be added to the relative position of reporters.

    -

    Default: False

    -
    -
    --align-fasta
    -

    gRNA is aligned to this sequence to infer the offset. Can be used when the exact offset is not provided.

    -

    Default: “â€

    -
    -
    --string-allele
    -

    Store allele as quality filtered string instead of Allele object

    -

    Default: False

    -
    -
    -g, --count-guide-edits
    -

    count the self editing of guides

    -

    Default: False

    -
    -
    -m, --count-guide-reporter-alleles
    -

    count the matched allele of guide and reporter edit

    -

    Default: False

    -
    -
    --tiling
    -

    Specify that the guide library is tiling library without ‘n guides per target’ design

    -

    Default: False

    -
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    bean count-samples¶

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    bean count[-samples]: Count (reporter) screen data¶

    -

    bean count-samples (or bean count for a single sample) maps guide into guide counts, allowing for base transition in spacer sequence. When the matched reporter information is provided, it can count the target site edits and alleles produced by each guide. Mapping is efficiently done based on CRISPResso2 modified for base-edit-aware mapping.

    -
    bean count-samples \
-  --input sample_list.csv   `# sample with lines 'R1_filepath,R2_filepath,sample_name\n'` \
-  -b A                      `# base that is being edited (A/G)` \
-  -f sgRNA_info_table.csv   `# sgRNA information` \
-  -o .                      `# output directory` \
-  -r                        `# read edit/allele information from reporter` \
-  -t 12                     `# number of threads` \
-  --name my_sorting_screen  `# name of this sample run` \
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    bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r
-
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    By default, bean count[-samples] assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure.

    -
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    Read structuren

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    See full detail below.

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    Input file format¶

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    See Input file format for input file formats.

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    Output file format¶

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    count or count-samples produces .h5ad and .xlsx file with guide and per-guide allele counts.

    -
      -

    • .h5ad: This output file follows annotated matrix format compatible with AnnData and is based on Screen object in [purturb_tools](https://github.com/pinellolab/perturb-tools). See Data Structure section for more information.

      • -

    • .xlsx: This output file contains .guides, .samples, .X[_bcmatch,_edits]. (allele_tables are often too large to write into an Excel!)

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    Full parameters¶

    -
    usage: bean count-samples [-h] -i SAMPLE_LIST -b EDITED_BASE -f SGRNA_FILENAME
-                          [--guide-start-seq GUIDE_START_SEQ]
-                          [--guide-end-seq GUIDE_END_SEQ]
-                          [--barcode-start-seq BARCODE_START_SEQ] [-r]
-                          [-q MIN_AVERAGE_READ_QUALITY]
-                          [-s MIN_SINGLE_BP_QUALITY] [-n NAME]
-                          [-o OUTPUT_FOLDER] [-l REPORTER_LENGTH]
-                          [--keep-intermediate] [--qstart-R1 QSTART_R1]
-                          [--qend-R1 QEND_R1] [--qstart-R2 QSTART_R2]
-                          [--qend-R2 QEND_R2]
-                          [--gstart-reporter GSTART_REPORTER]
-                          [--match-target-pos]
-                          [--target-pos-col TARGET_POS_COL]
-                          [--guide-bc GUIDE_BC] [--guide-bc-len GUIDE_BC_LEN]
-                          [--offset] [--align-fasta ALIGN_FASTA]
-                          [--string-allele] [-g] [-m] [--tiling] [-t THREADS]
-                          [--guide-start-seqs-file GUIDE_START_SEQS_FILE]
-                          [--guide-end-seqs-file GUIDE_END_SEQS_FILE]
-                          [--barcode-start-seqs-file BARCODE_START_SEQS_FILE]
-                          [--rerun]
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    -
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    Named Arguments¶

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    -i, --sample-list
    -

    List of fastq and sample ids. Formatted as R1_filepath,R2_filepath,sample_id

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    -b, --edited-base
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    For base editors, the base that should be ignored when matching the gRNA sequence

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    -f, --sgRNA-filename
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    sgRNA description file. The format requires three columns: name, sequence, barcode [ reporter [,strand, target_pos], [start_pos, offset] ].

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    --guide-start-seq
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    Guide starts after this sequence in R1

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    Default: “â€

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    --guide-end-seq
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    Guide starts after this sequence in R1

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    Default: “â€

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    --barcode-start-seq
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    Barcode + reporter starts after this sequence in R2, denoted as the sense direction (the same sequence direction as R1).

    -

    Default: “â€

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    -r, --count-reporter
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    Count reporter edits.

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    Default: False

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    -q, --min-average-read-quality
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    Minimum average quality score (phred33) to keep a read

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    Default: 30

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    -s, --min-single-bp-quality
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    Minimum single bp score (phred33) to keep a read

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    Default: 0

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    -n, --name
    -

    Output name

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    Default: “â€

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    -o, --output-folder
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    Default: “â€

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    -l, --reporter-length
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    length of the reporter

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    Default: 32

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    --keep-intermediate
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    Keep all the intermediate files

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    Default: False

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    --qstart-R1
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    Start position of the read when filtering for quality score of the read 1

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    Default: 0

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    --qend-R1
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    End position of the read when filtering for quality score of the read 1

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    Default: 47

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    --qstart-R2
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    Same as qstart_R1, for read 2 fastq file

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    Default: 0

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    --qend-R2
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    Same as qstart_R2, for read 2 fastq file

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    Default: 36

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    --gstart-reporter
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    Start position of the guide sequence in the reporter

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    Default: 6

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    --match-target-pos
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    Count the edit in the exact target position.

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    Default: False

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    --target-pos-col
    -

    Column name specifying the relative target position within reporter sequence.

    -

    Default: “target_posâ€

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    --guide-bc
    -

    Construct has guide barcode

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    Default: True

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    --guide-bc-len
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    Guide barcode sequence length at the beginning of the R2

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    Default: 4

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    --offset
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    Guide file has offest column that will be added to the relative position of reporters.

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    Default: False

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    --align-fasta
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    gRNA is aligned to this sequence to infer the offset. Can be used when the exact offset is not provided.

    -

    Default: “â€

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    --string-allele
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    Store allele as quality filtered string instead of Allele object

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    Default: False

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    -g, --count-guide-edits
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    count the self editing of guides

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    Default: False

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    -m, --count-guide-reporter-alleles
    -

    count the matched allele of guide and reporter edit

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    Default: False

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    --tiling
    -

    Specify that the guide library is tiling library without ‘n guides per target’ design

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    Default: False

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    -t, --threads
    -

    Number of threads

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    Default: 10

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    --guide-start-seqs-file
    -

    CSV file path with per-sample guide_start_seq to be used.Formatted as sample_id, guide_start_seq

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    --guide-end-seqs-file
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    CSV file path with per-sample guide_end_seq to be used.Formatted as sample_id,guide_end_seq

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    --barcode-start-seqs-file
    -

    CSV file path with per-sample barcode_start_seq to be used.Formatted as sample_id,guide_end_seq

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    --rerun
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    Recount each sample

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    Default: False

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    Input .fa file format for bean-filter¶

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    You can provide custom FASTA file with exon sequence entries. Currently only supports positive strand genes.

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    • Exon FASTA files can be downloaded from UCSC Genomic sequences / Table Browser: see the instruction video

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    • You can manually format as:

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      • Header line has range=chrom:start-end and strand=+/- tag that is parsed.

        • -

      • fasta entry has the sequence of exons, where the first (includes 5’-UTR) and last (includes 3’-UTR) exon sequence has lower-case sequence denoting noncoding sequences.

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    bean filter¶

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    filter: Filtering (and optionally translating) alleles¶

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    As tiling mode of bean run accounts for any robustly observed alleles, bean filter filters for such alleles.

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    bean filter my_sorting_screen_masked.h5ad \
--o my_sorting_screen_filtered.h5ad  `# Output file path` \
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    Output¶

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    Above command produces

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    • my_sorting_screen_filtered.h5ad with filtered alleles stored in .uns,

      • -

    • my_sorting_screen_filtered.filtered_allele_stats.pdf, and my_sorting_screen_filtered.filter_log.txt that report allele count stats in each filtering step.

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    You may want to adjust the flitering parameters to obtain optimal balance between # guides per variant & # variants that are scored. See example outputs of filtering step here.

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    Translating alleles¶

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    If you want to obtain amino acid level variant for coding sequence tiling screens, provide coding sequence positions which variants occuring within the coding sequence will be translated. This is optional, but **highly recommended* to increase per-(coding)variant support.*

    -

    Allele translation

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    bean filter my_sorting_screen.h5ad \
--o my_sorting_screen_masked.h5ad \
---translate   `# Translate coding variants` \
-[ --translate-gene-name GENE_SYMBOL OR
-  --translate-genes-list path_to_gene_names_file.txt OR
-  --translate-fasta gene_exon.fa, OR
-  --translate-fastas-csv gene_exon_fas.csv]
-
    -
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    • When library covers a single gene, do either of the following:

      -
        -

      1. Feed --translate-gene-name GENE_SYMBOL if your genomic_pos column of sgRNA_info_tbl is compatible with MANE transcript‘s reference genome. (Per 10/23/2023, GRCh38). This will automatically load the exon positions based on MANE transcript annotation.

        2. -

      2. To use your custom coding sequence and exon positions, feed --translate-fasta gene_exon.fa argument where gene_exon.fa is the FASTA file with entries of exons. See full details here.

        3. -
      -
      • -

    • When library covers multiple genes, do either of the following:

      -
        -

      1. Feed --translate-genes-list path_to_gene_names_file.txt where path_to_gene_names_file.txt is file with one gene symbol per line.

        2. -

      2. Feed --translate-fastas-csv gene_exon_fas.csv where gene_exon_fas.csv is the csv file with lines gene_id,gene_exon_fasta_path without header. Each FASTA file in gene_exon_fasta_path is formatted as the single-gene FASTA file.

        3. -
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    • Translation will keep the variants outside the coding sequence as nucleotide-level variants, while aggregating variants leading to the same coding sequence variants.

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    Full parameters¶

    -

    Filter alleles based on edit position in spacer and frequency across samples.

    -

    -
    usage: bean filter [-h] [--output-prefix OUTPUT_PREFIX]
-                   [--plasmid-path PLASMID_PATH]
-                   [--edit-start-pos EDIT_START_POS]
-                   [--edit-end-pos EDIT_END_POS]
-                   [--jaccard-threshold JACCARD_THRESHOLD] [--filter-window]
-                   [--keep-indels] [--filter-target-basechange] [--translate]
-                   [--translate-fasta TRANSLATE_FASTA]
-                   [--translate-fastas-csv TRANSLATE_FASTAS_CSV]
-                   [--translate-gene TRANSLATE_GENE]
-                   [--translate-genes-list TRANSLATE_GENES_LIST]
-                   [--filter-allele-proportion FILTER_ALLELE_PROPORTION]
-                   [--filter-allele-count FILTER_ALLELE_COUNT]
-                   [--filter-sample-proportion FILTER_SAMPLE_PROPORTION]
-                   [--load-tmp]
-                   bdata_path
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    Positional Arguments¶

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    bdata_path
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    Input ReporterScreen file of which allele will be filtered out.

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    Named Arguments¶

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    --output-prefix, -o
    -

    Output prefix for log and ReporterScreen file with allele assignment

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    --plasmid-path, -p
    -

    Plasmid ReporterScreen object path. If provided, alleles are filtered based on if a nucleotide edit is more significantly enriched in sample compared to the plasmid data. Negative control data where no edit is expected can be fed in instead of plasmid library.

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    --edit-start-pos, -s
    -

    0-based start posiiton (inclusive) of edit relative to the start of guide spacer.

    -

    Default: 2

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    --edit-end-pos, -e
    -

    0-based end position (exclusive) of edit relative to the start of guide spacer.

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    Default: 7

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    --jaccard-threshold, -j
    -

    Jaccard Index threshold when the alleles are mapped to the most similar alleles. In each filtering step, allele counts of filtered out alleles will be mapped to the most similar allele only if they have Jaccard Index of shared edit higher than this threshold.

    -

    Default: 0.3

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    --filter-window, -w
    -

    Only consider edit within window provided by (edit-start-pos, edit-end-pos). If this flag is not provided, –edit-start-pos and –edit-end-pos flags are ignored.

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    Default: False

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    --keep-indels, -i
    -

    Include indels.

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    Default: False

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    --filter-target-basechange, -b
    -

    Only consider target edit (stored in bdata.uns[‘target_base_change’])

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    Default: False

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    --translate, -t
    -

    Translate alleles

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    Default: False

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    --translate-fasta, -f
    -

    fasta file path with exon positions. If not provided, LDLR hg19 coordinates will be used.

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    --translate-fastas-csv, -fs
    -

    .csv with two columns with gene IDs and FASTA file path corresponding to each gene.

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    --translate-gene, -g
    -

    Gene symbol if a gene is tiled. If not provided, LDLR hg19 coordinates will be used.

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    --translate-genes-list, -gs
    -

    File with gene symbols, one per line, if multiple genes are tiled.

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    --filter-allele-proportion, -ap
    -

    If provided, alleles that exceed filter_allele_proportion in filter-sample-proportion will be retained.

    -

    Default: 0.05

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    --filter-allele-count, -ac
    -

    If provided, alleles that exceed filter_allele_proportion AND filter_allele_count in filter-sample-proportion will be retained.

    -

    Default: 5

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    --filter-sample-proportion, -sp
    -

    If filter_allele_proportion is provided, alleles that exceed filter_allele_proportion in filter-sample-proportion will be retained.

    -

    Default: 0.2

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    --load-tmp
    -

    Load temporary file and work from there.

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    Default: False

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    GWAS variant library¶

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    Variant sorting screen tutorial¶

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    GWAS variant screen with per-variant gRNA tiling design, selected based on FACS signal quantiles.

    - - - - - - - - - -
    Library designVariant (gRNAs tile each target variant)
    variant library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design



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    Example workflow¶

    -
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-
-# 1. Count gRNA & reporter
-bean-count-samples \
---input tests/data/sample_list.csv    `# Contains fastq file path; see test file for example.`\
--b A                                  `# Base A is edited (into G)` \
--f tests/data/test_guide_info.csv     `# Contains gRNA metadata; see test file for example.`\
--o ./                                 `# Output directory` \
--r                                    `# Quantify reporter edits` \
--n ${screen_id}                          `# ID of the screen to be counted`
-
-# 2. QC samples & guides
-bean-qc \
-  bean_count_${screen_id}.h5ad             `# Input ReporterScreen .h5ad file path` \
-  -o bean_count_${screen_id}_masked.h5ad   `# Output ReporterScreen .h5ad file path` \
-  -r qc_report_${screen_id}                `# Prefix for QC report` \
-  -b                                       ` # Remove replicates with no good samples.
-
-# 3. Quantify variant effect
-bean-run sorting variant \
-    tests/data/bean_count_${screen_id}_masked.h5ad \
-    -o tests/test_res/var/ \
-    --fit-negctrl \
-    --scale-by-acc \
-    --accessibility-col accessibility
-
    -
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    See more details below.

    -
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    1. Count gRNA & reporter (bean count-samples)¶

    -
    screen_id=my_sorting_tiling_screen
-
-# 1. Count gRNA & reporter
-bean-count-samples \
---input tests/data/sample_list.csv    `# Contains fastq file path; see test file for example.`\
--b A                                  `# Base A is edited (into G)` \
--f tests/data/test_guide_info.csv     `# Contains gRNA metadata; see test file for example.`\
--o ./                                 `# Output directory` \
--r                                    `# Quantify reporter edits` \
--n ${screen_id}                          `# ID of the screen to be counted`
-
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    Make sure you follow the input file format for seamless downstream steps. This will produce ./bean_count_${screen_id}.h5ad.

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    2. QC samples & guides (bean qc)¶

    -

    Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the input file guideline, but you can change the parameters with the full argument list of ``bean-qc` <../../README#bean-qc-qc-of-reporter-screen-data>`_. (Common factors you may want to tweak is --ctrl-cond=bulk and --lfc-conds=top,bot if you have different sample condition labels.)

    -
    bean-qc \
-  bean_count_${screen_id}.h5ad    `# Input ReporterScreen .h5ad file path` \
-  -o bean_count_${screen_id}_masked.h5ad   `# Output ReporterScreen .h5ad file path` \
-  -r qc_report_${screen_id}   `# Prefix for QC report`
-
    -
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    If the data does not include reporter editing data, you can provide --no-editing flag to omit the editing rate QC.

    -
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    3. Quantify variant effect (bean run)¶

    -

    bean-run can take 3 run options to quantify editing rate:

    -
      -
    1. -
      From reporter + accessibility

      If your gRNA metadata table (tests/data/test_guide_info.csv above) included per-gRNA accessibility score,

      -
      -
      -
         bean-run sorting variant \
-   tests/data/bean_count_${screen_id}_masked.h5ad \
-   -o tests/test_res/var/ \
-   --fit-negctrl \
-   --scale-by-acc \
-   --accessibility-col accessibility
-
-If your gRNA metadata table (\ ``tests/data/test_guide_info.csv`` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal,
-
      -
      -
      bean-run sorting variant \
-tests/data/bean_count_${screen_id}_masked.h5ad \
--o tests/test_res/var/ \
---fit-negctrl \
---scale-by-acc \
---accessibility-bw accessibility.bw
-
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      2. -

    2. From reporter, without accessibility

      -
      -

      This assumes the all target sites have the uniform chromatin accessibility.

      -
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      bean-run sorting variant \
-tests/data/bean_count_${screen_id}_masked.h5ad \
--o tests/test_res/var/ \
---fit-negctrl
-
      -
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      3. -
    3. -
      No reporter information, assume the same editing efficiency of all gRNAs.

      Use this option if your data don’t have editing outcome information.

      -
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      bean-run sorting variant \
-tests/data/bean_count_${screen_id}_masked.h5ad \
--o tests/test_res/var/ \
---fit-negctrl \
---uniform-edit
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    title: CRISPR-BEAN¶

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    - - - - - - \ No newline at end of file diff --git a/docs/_build/index_.html b/docs/_build/index_.html deleted file mode 100644 index bc8e2dc..0000000 --- a/docs/_build/index_.html +++ /dev/null @@ -1,213 +0,0 @@ - - - - - - - - Welcome to bean’s documentation! — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - - -
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    Welcome to bean’s documentation!¶

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    Workflows¶

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    bean subcommands¶

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    Screen data structure¶

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    Indices and tables¶

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    - ©2024, Jayoung Ryu. - - | - Powered by Sphinx 7.2.6 - & Alabaster 0.7.16 - - | - Page source -
    - - - - - - \ No newline at end of file diff --git a/docs/_build/input.html b/docs/_build/input.html deleted file mode 100644 index b55703d..0000000 --- a/docs/_build/input.html +++ /dev/null @@ -1,171 +0,0 @@ - - - - - - - - Input file format — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - - - -
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    Input file format¶

    -

    This document describes the input files of bean count-samples.

    -
    -

    sgRNA_info_table.csv¶

    -

    File should contain following columns.

    -
      -

    • name: gRNA ID column

      • -

    • sequence: gRNA sequence

      • -

    • barcode: R2 barcode to help match reporter to gRNA, written in the sense direction (as in R1)

      • -

    • In order to use accessibility in the variant effect quantification, provide accessibility information in one of two options. (For non-targeting guides, provide NA values (empty cell).)

      -
        -

      • Option 1: chrom & genomic_pos: Chromosome (ex. chr19) and genomic position of guide sequence. You will have to provide the path to the bigwig file with matching reference version in bean run.

        • -

      • Option 2: accessibility_signal: ATAC-seq signal value of the target loci of each guide.

        • -
      -
      • -

    • For variant library (gRNAs are designed to target specific variants and ignores bystander edits)

      -
        -

      • target: This column denotes which target variant/element of each gRNA. This is not used in bean count[-samples] but required to run bean run in later steps.

        • -

      • target_group: If negative/positive control gRNA will be considered in bean qc and/or bean run, specify as “NegCtrlâ€/â€PosCtrl†in this column.

        • -

      • target_pos: If --match_target_pos flag is used, input file needs target_pos which specifies 0-based relative position of targeted base within Reporter sequence.

        • -
      -
      • -

    • For tiling library (gRNAs tile coding / noncoding sequences)

      -
        -

      • strand: Specifies gRNA strand information relative to the reference genome.

        • -

      • chrom: Chromosome of gRNA targeted locus.

        • -

      • start_pos: gRNA starting position in the genome. Required when you provide strand column. Should specify the smaller coordinate value among start and end position regardless of gRNA strandedness.

        • -
      -
      • -
    -

    Also see examples for variant library and tiling library.

    -
    -
    -

    sample_list.csv¶

    -

    File should contain following columns with header.

    -
      -

    • R1_filepath: Path to read 1 .fastq[.gz] file

      • -

    • R2_filepath: Path to read 1 .fastq[.gz] file

      • -

    • sample_id: ID of sequencing sample

      • -

    • replicate: Replicate # of this sample (Should NOT contain .)

      • -

    • condition: Name of the sorting bin (ex. top, bot), or label of timepoint (ex. D5, D18)

      • -
    -

    For FACS sorting screens:

    -
      -

    • upper_quantile: FACS sorting upper quantile

      • -

    • lower_quantile: FACS sorting lower quantile

      • -
    -

    For proliferation / survival screens:

    -
      -

    • time: Numeric time following the base editing of each sample.

      • -
    -

    Also see examples for FACS sorting screen.

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    - ©2024, Jayoung Ryu. - - | - Powered by Sphinx 7.2.6 - & Alabaster 0.7.16 - - | - Page source -
    - - - - - - \ No newline at end of file diff --git a/docs/_build/objects.inv b/docs/_build/objects.inv deleted file mode 100644 index b01eb0a63fe257f5edac5cea636283a2a547048a..0000000000000000000000000000000000000000 GIT binary patch literal 0 HcmV?d00001 literal 761 zcmVNERX>N99Zgg*Qc_4OWa&u{KZXhxWBOp+6Z)#;@bUGknWnpd# zBOq2~a&u{KZaN?eBOp|0Wgv28ZDDC{WMy(7Z)PBLXlZjGW@&6?AZc?TV{dJ6a%FRK zWn>_Ab7^j8AbMYo@{2`yf+LpaGe`l!^kk3~5uXD@joP4jm%z>TDXm zu%pd>hfHUrVwh*7(mn+&ZOm3yOq`IWmyQZTkzu@Fb5<&>t{IU{gGHquiR^?47y^$a zD=8LeSbxtoZdy+Dk2Og2q4#Rc?g43=Uxf|!1Qf-F=A$nu}Gjb0ywE_T~^TkuhGZh+gIuyFi5tBNr5@PBBQX-AOOAx1Qr^|Fwu7D zG7cUqqECmXO~FJh3xi8eS46+e6s?kd?n#o-=M`tPtkHf9qZR9OLoGJ21L5U%AxX{) z@#rpOjlOJ=tdTp;^l7RteTHE7*kA>89TtYwSM-9RQ=wC6UBglJiA61!=rju}#&(O~ z&an)0{h0{oxTHKya2Ld;=;*5yMU9JEss;zyhaQ{HZfJ*9jfZwbV-S1>>fCl)7Wby# zl0yCkrnkd|?BgDTnrfn@JAI|NZ?(BY<@c6$bZX_a=*`${QwIVz0a&dfM}h zhZ4mRO@B#7g=(+4b6Ba*vg#nnmMk%*H>Wf6sQ#m|znZfYp;S1yi+Oe4iE{M2UI4^m zSfd%8027CUIFcTuDOThh;;hZG9j$+lPc+kA>8=D{*)xwZvmuI^MHW$zlpp7wK>f%o rUM$|h$Z2)I+E-A+4L5x46*oPmVH9-#ZAx%j%5US0+~WKPKoQ)5f$d=t diff --git a/docs/_build/profile.html b/docs/_build/profile.html deleted file mode 100644 index 9d55d75..0000000 --- a/docs/_build/profile.html +++ /dev/null @@ -1,185 +0,0 @@ - - - - - - - - bean profile — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - - - -
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    bean profile¶

    -
    -

    bean profile: Profile editing patterns¶

    -
    bean profile my_sorting_screen.h5ad -o output_prefix `# Prefix for editing profile report`
-
    -
    -
    -
    -

    Output¶

    -

    Above command produces prefix_editing_preference.[html,ipynb] as editing preferences (see example).

    -

    Allele translation

    -
    -
    -

    Full parameters¶

    -
    usage: bean profile [-h] [-o OUTPUT_PREFIX] [--replicate-col REPLICATE_COL]
-                    [--condition-col CONDITION_COL] [--pam-col PAM_COL]
-                    [--control-condition CONTROL_CONDITION] [-w WINDOW_LENGTH]
-                    bdata_path
-
    -
    -
    -

    Positional Arguments¶

    -
    -
    bdata_path
    -

    Path to the ReporterScreen object to run QC on

    -
    -
    -
    -
    -

    Named Arguments¶

    -
    -
    -o, --output-prefix
    -

    Output prefix of editing pattern report (prefix.html, prefix.ipynb). If not provided, base name of bdata_path is used.

    -
    -
    --replicate-col
    -

    Column name in bdata.samples that describes replicate ID.

    -

    Default: “replicateâ€

    -
    -
    --condition-col
    -

    Column name in bdata.samples that describes experimental condition. (sorting bin, time, etc.)

    -

    Default: “binâ€

    -
    -
    --pam-col
    -

    Column name describing PAM of each gRNA in bdata.guides.

    -
    -
    --control-condition
    -

    Control condition where editing preference would be profiled at. Pre-filters data where bdata.samples[condition_col] == control_condition.

    -

    Default: “bulkâ€

    -
    -
    -w, --window-length
    -

    Window length of editing window of maximal editing efficiency to be identified. This window is used to quantify context specificity within the window.

    -

    Default: 6

    -
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    - ©2024, Jayoung Ryu. - - | - Powered by Sphinx 7.2.6 - & Alabaster 0.7.16 - - | - Page source -
    - - - - - - \ No newline at end of file diff --git a/docs/_build/qc.html b/docs/_build/qc.html deleted file mode 100644 index f08de74..0000000 --- a/docs/_build/qc.html +++ /dev/null @@ -1,429 +0,0 @@ - - - - - - - - bean qc — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - - - -
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    bean qc¶

    -
    -

    bean qc: QC of reporter screen data¶

    -
    bean qc \
-  my_sorting_screen.h5ad             `# Input ReporterScreen .h5ad file path` \
-  -o my_sorting_screen_masked.h5ad   `# Output ReporterScreen .h5ad file path` \
-  -r qc_report_my_sorting_screen     `# Prefix for QC report` \
-  --ctrl-cond presort                `# "condition" column in the control sample before selection. Mean gRNA editing rates in these samples are reported. ` \
-# Inspect the output qc_report_my_sorting_screen.html to tweak QC threshold
-
-bean qc \
-  my_sorting_screen.h5ad              \
-  -o my_sorting_screen_masked.h5ad    \
-  -r qc_report_my_sorting_screen      \
-  #[--count-correlation-thres 0.7 ...]\
-  -b
-
    -
    -

    bean qc supports following quality control and masks samples with low quality. Specifically:

    -

    Allele translation

    -
      -

    • Plots guide coverage and the uniformity of coverage

      • -

    • Guide count correlation between samples

      • -

    • Log fold change correlation when positive controls are provided

      • -

    • Plots editing rate distribution

      • -

    • Identify samples with low guide coverage/guide count correlation/editing rate and mask the sample in bdata.samples.mask

      • -

    • Identify outlier guides to filter out

      • -
    -
    -
    -

    Output¶

    -

    Above command produces

    -
      -

    • my_sorting_screen_masked.h5ad without problematic replicate and guides and with sample masks, and

      • -

    • qc_report_my_sorting_screen.[html,ipynb] as QC report.
       -##### Optional arguments:

      • -

    • -o OUT_SCREEN_PATH, --out-screen-path OUT_SCREEN_PATH

      -
      Path where quality-filtered ReporterScreen object to be written to
-
      -
      -
      • -

    • -r OUT_REPORT_PREFIX, --out-report-prefix OUT_REPORT_PREFIX

      -
      Output prefix of qc report (prefix.html, prefix.ipynb)
-
      -
      -
      • -
    -
    -

    QC thresholds:¶

    -
      -

    • --count-correlation-thres COUNT_CORRELATION_THRES

      -
      Correlation threshold to mask out.
-
      -
      -
      • -

    • --edit-rate-thres EDIT_RATE_THRES

      -
      Mean editing rate threshold per sample to mask out.
-
      -
      -
      • -

    • --lfc-thres LFC_THRES

      -
      Positive guides' correlation threshold to filter out.
-
      -
      -
      • -
    -
    -
    -

    Run options:¶

    -
      -

    • -b, --remove-bad-replicates

      -
      Remove replicates with at least two of its samples meet the QC threshold (bean run does not support having only one sorting bin sample for a replicate).
-
      -
      -
      • -

    • -i, --ignore-missing-samples

      -
      If the flag is not provided, if the ReporterScreen object does not contain all condiitons for
-each replicate, make fake empty samples. If the flag is provided, don't add dummy samples.
-
      -
      -
      • -

    • --no-editing Ignore QC about editing. Can be used for QC of other editing modalities.

      • -

    • --dont-recalculate-edits

      -
      When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from
-ReporterScreen.uns['allele_count'].
-
      -
      -
      • -
    -
    -
    -

    Input .h5ad formatting:¶

    -

    Note that these arguements will change the way the QC metrics are calculated for guides, samples, or replicates.

    -
      -

    • --tiling TILING Specify that the guide library is tiling library without ‘n guides per target’ design

      • -

    • --replicate-label REPLICATE_LABEL

      -
      Label of column in `bdata.samples` that describes replicate ID.
-
      -
      -
      • -

    • --sample-covariates SAMPLE_COVARIATES

      -
      Comma-separated list of column names in `bdata.samples` that describes non-selective
-experimental condition. (drug treatment, etc.)
-
      -
      -
      • -

    • --condition-label CONDITION_LABEL

      -
      Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time,
-etc.)
-
      -
      -

      ###### Editing rate calculation

      -
        -

      • --control-condition CTRL_COND

        -
        Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate
-to be calculated. Default is `None`, which considers all samples.
-
        -
        -
        • -

      • --rel-pos-is-reporter

        -
        Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If
-`False`, those are relative to spacer position.
-
        -
        -

        Editing rate is calculated with following parameters in

        -
          -

        • Variant screens:

          -
            -

          • --target-pos-col TARGET_POS_COL

            -
            Target position column in `bdata.guides` specifying target edit position in reporter
-
            -
            -
            • -
          -
          • -

        • tiling screens:

          -
            -

          • --edit-start-pos EDIT_START_POS

            -
            Edit start position to quantify editing rate on, 0-based inclusive.
-
            -
            -
            • -

          • --edit-end-pos EDIT_END_POS

            -
            Edit end position to quantify editing rate on, 0-based exclusive.
-
            -
            -

            ###### LFC of positive controls

            -
            • -
          -
          • -
        -
        • -

      • --posctrl-col POSCTRL_COL

        -
        Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all
-gRNAs, feed empty string ''.
-
        -
        -
        • -

      • --posctrl-val POSCTRL_VAL

        -
        Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the
-positive control in calculating log fold change.
-
        -
        -
        • -

      • --lfc-conds LFC_CONDS

        -
        Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated
-between, delimited by comma
-
        -
        -
        • -
      -
      • -
    -
    -
    -
    -

    Full parameters¶

    -
    usage: bean qc [-h] [--count-correlation-thres COUNT_CORRELATION_THRES]
-               [--edit-rate-thres EDIT_RATE_THRES] [--lfc-thres LFC_THRES]
-               [-o OUT_SCREEN_PATH] [-r OUT_REPORT_PREFIX] [-b] [-i]
-               [--no-editing] [--dont-recalculate-edits] [--tiling TILING]
-               [--replicate-label REPLICATE_LABEL]
-               [--sample-covariates SAMPLE_COVARIATES]
-               [--condition-label CONDITION_LABEL]
-               [--target-pos-col TARGET_POS_COL] [--rel-pos-is-reporter]
-               [--edit-start-pos EDIT_START_POS] [--edit-end-pos EDIT_END_POS]
-               [--posctrl-col POSCTRL_COL] [--posctrl-val POSCTRL_VAL]
-               [--lfc-conds LFC_CONDS] [--control-condition CONTROL_CONDITION]
-               bdata_path
-
    -
    -
    -

    Positional Arguments¶

    -
    -
    bdata_path
    -

    Path to the ReporterScreen object to run QC on

    -
    -
    -
    -
    -

    Named Arguments¶

    -
    -
    -o, --out-screen-path
    -

    Path where quality-filtered ReporterScreen object to be written to

    -
    -
    -r, --out-report-prefix
    -

    Output prefix of qc report (prefix.html, prefix.ipynb)

    -
    -
    -
    -
    -

    QC thresholds¶

    -
    -
    --count-correlation-thres
    -

    Correlation threshold to mask out.

    -

    Default: 0.7

    -
    -
    --edit-rate-thres
    -

    Mean editing rate threshold per sample to mask out.

    -

    Default: 0.1

    -
    -
    --lfc-thres
    -

    Positive guides’ correlation threshold to filter out.

    -

    Default: -0.1

    -
    -
    -
    -
    -

    Run options¶

    -
    -
    -b, --remove-bad-replicates
    -

    Remove replicates with at least two of its samples meet the QC threshold.

    -

    Default: False

    -
    -
    -i, --ignore-missing-samples
    -

    If the flag is not provided, if the ReporterScreen object does not contain all condiitons for each replicate, make fake empty samples. If the flag is provided, don’t add dummy samples.

    -

    Default: False

    -
    -
    --no-editing
    -

    Ignore QC about editing. Can be used for QC of other editing modalities.

    -

    Default: False

    -
    -
    --dont-recalculate-edits
    -

    When ReporterScreen.layers[‘edit_count’] exists, do not recalculate the edit counts from ReporterScreen.uns[‘allele_count’].

    -

    Default: False

    -
    -
    -
    -
    -

    Input .h5ad formatting¶

    -
    -
    --tiling
    -

    Specify that the guide library is tiling library without ‘n guides per target’ design

    -
    -
    --replicate-label
    -

    Label of column in bdata.samples that describes replicate ID.

    -

    Default: “replicateâ€

    -
    -
    --sample-covariates
    -

    Comma-separated list of column names in bdata.samples that describes non-selective experimental condition. (drug treatment, etc.)

    -
    -
    --condition-label
    -

    Label of column in bdata.samples that describes experimental condition. (sorting bin, time, etc.)

    -

    Default: “conditionâ€

    -
    -
    --target-pos-col
    -

    Target position column in bdata.guides specifying target edit position in reporter

    -

    Default: “target_posâ€

    -
    -
    --rel-pos-is-reporter
    -

    Specifies whether edit_start_pos and edit_end_pos are relative to reporter position. If False, those are relative to spacer position.

    -

    Default: False

    -
    -
    --edit-start-pos
    -

    Edit start position to quantify editing rate on, 0-based inclusive.

    -

    Default: 2

    -
    -
    --edit-end-pos
    -

    Edit end position to quantify editing rate on, 0-based exclusive.

    -

    Default: 7

    -
    -
    --posctrl-col
    -

    Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all gRNAs, feed empty string ‘’.

    -

    Default: “target_groupâ€

    -
    -
    --posctrl-val
    -

    Value in ReporterScreen.guides[posctrl_col] that specifies guide will be used as the positive control in calculating log fold change.

    -

    Default: “PosCtrlâ€

    -
    -
    --lfc-conds
    -

    Values in of column in ReporterScreen.samples[condition_label] for LFC will be calculated between, delimited by comma

    -

    Default: “top,botâ€

    -
    -
    --control-condition
    -

    Values in of column in ReporterScreen.samples[condition_label] for guide-level editing rate to be calculated

    -

    Default: “bulkâ€

    -
    -
    -
    -
    -
    - - -
    - -
    -
    - -
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    -
    - ©2024, Jayoung Ryu. - - | - Powered by Sphinx 7.2.6 - & Alabaster 0.7.16 - - | - Page source -
    - - - - - - \ No newline at end of file diff --git a/docs/_build/run.html b/docs/_build/run.html deleted file mode 100644 index d9e232b..0000000 --- a/docs/_build/run.html +++ /dev/null @@ -1,401 +0,0 @@ - - - - - - - - bean run — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - - - -
    -
    -
    - - -
    - -
    -

    bean run¶

    -
    -

    bean run: Quantify variant effects¶

    -

    BEAN uses Bayesian network to incorporate gRNA editing outcome to provide posterior estimate of variant phenotype. The Bayesian network reflects data generation process. Briefly,

    -
      -

    1. Cellular phenotype (either for cells are sorted upon for sorting screen, or log(proliferation rate)) is modeled as the Gaussian mixture distribution of wild-type phenotype and variant phenotype.

      2. -

    2. The weight of the mixture components are inferred from the reporter editing outcome and the chromatin accessibility of the loci.

      3. -

    3. Cells with each gRNA, formulated as the mixture distribution, is sorted by the phenotypic quantile to produce the gRNA counts.

      4. -
    -

    For the full detail, see the method section of the BEAN manuscript.

    -

    model

    -



    -
    -
    -

    Usage example¶

    -
    bean run sorting[survival] variant[tiling] my_sorting_screen_filtered.h5ad \
-[--uniform-edit, --scale-by-acc [--acc-bw-path accessibility_signal.bw, --acc-col accessibility]] \
--o output_prefix/ \
---fit-negctrl
-
    -
    -

    See full list of parameters below.

    -
    -
    -

    Input¶

    -

    my_sorting_screen_filtered.h5ad can be produced by one of the following:

    -
      -

    1. ``bean count-samples` <(#bean-count-samples-count-reporter-screen-data>`_) when you have raw .fastq file

      2. -

    2. (Limited to bean run variant mode) bean create-screen when you have flat .csv tables of gRNA metadata table, sample metadata table, gRNA counts table (# guides x # samples), and optionally # edits table. -.. code-block:: bash

      -
      -

      bean create-screen gRNA_info_table.csv sample_info_table.csv gRNA_counts_table.csv [–edits edit_counts_table.csv -o output.h5ad]

      -
      -
        -

      • gRNA_info_table.csv should have following columns.

        -
          -

        • name: gRNA ID column

          • -

        • target: This column denotes which target variant/element of each gRNA.

          • -

        • target_group [Optional]: If negative control gRNA will be used, specify as “NegCtrl†in this column.

          • -
        -
        • -

      • sample_info_table.csv should have following columns.

        -
          -

        • sample_id: ID of sequencing sample

          • -

        • replicate: Replicate # of this sample

          • -

        • bin: Name of the sorting bin

          • -

        • upper_quantile: FACS sorting upper quantile

          • -

        • lower_quantile: FACS sorting lower quantile

          • -
        -
        • -

      • gRNA_counts_table.csv should be formatted as follows.

        -
          -

        • Columns include one of sample_id columns in sample_info_table.csv file.

          • -

        • 1st row (row index) follows name (gRNA ID) in gRNA_info_table.csv file.

          • -
        -
        • -
      -
      3. -

    3. You can manually create the AnnData object with more annotations including allele counts: see API tutorial for full detail.

      4. -
    -
    -
    -

    Output¶

    -

    model

    -

    Above command produces

    -
      -

    • output_prefix/bean_element_result.[model_type].csv with following columns:

      -
        -

      • Estimated variant effect sizes

        -
          -

        • mu (Effect size): Mean of variant phenotype, given the wild type has standard normal phenotype distribution of mu = 0, sd = 1.

          • -

        • mu_sd: Mean of variant phenotype mu is modeled as normal distribution. The column shows fitted standard deviation of mu that quantify the uncertainty of the variant effect.

          • -

        • mu_z: z-score of mu

          • -

        • sd: Standard deviation of variant phenotype, given the wild type has standard normal phenotype distribution of mu = 0, sd = 1.

          • -

        • CI[0.025, 0.975]: Credible interval of mu

          • -

        • When negative control is provided, above columns with _adj suffix are provided, which are the corresponding values adjusted for negative control.

          • -
        -
        • -

      • Metrics on per-variant evidence provided in input (provided in tiling mode)

        -
          -

        • effective_edit_rate: Sum of per-variant editing rates over all alleles observed in the input. Allele-level editing rate is divided by the number of variants observed in the allele prior to summing up.

          • -

        • n_guides: # of guides covering the variant.

          • -

        • n_coocc: # of cooccurring variants with a given variant in any alleles observed in the input.

          • -
        -
        • -
      -
      • -

    • output_prefix/bean_sgRNA_result.[model_type].csv:

      -
        -

      • edit_rate: Estimated editing rate at the target loci.

        • -
      -
      • -
    -
    -
    -

    Full parameters¶

    -

    Run model on data.

    -

    -
    usage: bean run [-h] [--rep-pi] [--uniform-edit] [--scale-by-acc]
-                [--acc-bw-path ACC_BW_PATH] [--acc-col ACC_COL] [--const-pi]
-                [--shrink-alpha] [--condition-col CONDITION_COL]
-                [--time-col TIME_COL] [--control-condition CONTROL_CONDITION]
-                [--include-control-condition-for-inference]
-                [--replicate-col REPLICATE_COL] [--target-col TARGET_COL]
-                [--guide-activity-col GUIDE_ACTIVITY_COL] [--outdir OUTDIR]
-                [--result-suffix RESULT_SUFFIX]
-                [--sorting-bin-upper-quantile-col SORTING_BIN_UPPER_QUANTILE_COL]
-                [--sorting-bin-lower-quantile-col SORTING_BIN_LOWER_QUANTILE_COL]
-                [--alpha-if-overdispersion-fitting-fails ALPHA_IF_OVERDISPERSION_FITTING_FAILS]
-                [--cuda] [--sample-mask-col SAMPLE_MASK_COL] [--fit-negctrl]
-                [--negctrl-col NEGCTRL_COL]
-                [--negctrl-col-value NEGCTRL_COL_VALUE]
-                [--repguide-mask REPGUIDE_MASK] [--device DEVICE]
-                [--ignore-bcmatch] [--allele-df-key ALLELE_DF_KEY]
-                [--splice-site-path SPLICE_SITE_PATH]
-                [--control-guide-tag CONTROL_GUIDE_TAG] [--dont-fit-noise]
-                [--dont-adjust-confidence-by-negative-control]
-                [--n-iter N_ITER] [--load-existing]
-                {sorting,survival} {variant,tiling} bdata_path
-
    -
    -
    -

    Positional Arguments¶

    -
    -
    selection
    -

    Possible choices: sorting, survival

    -

    Screen selection type whether cells are sorted based on continuous phenotype (‘sorting’) or proliferated based on their viability (‘survival’).

    -
    -
    library_design
    -

    Possible choices: variant, tiling

    -

    Library design type whether to run variant or tiling screen model. -Variant library design assumes gRNA has specific target variant and bystander edits are ignored. Tiling library design considers all alleles generated by gRNA in reporter.

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    bdata_path
    -

    Path of an ReporterScreen object

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    Named Arguments¶

    -
    -
    --rep-pi, -r
    -

    Fit replicate specific scaling factor. Recommended to set as True if you expect variable editing activity across biological replicates.

    -

    Default: False

    -
    -
    --uniform-edit, -p
    -

    Assume uniform editing rate for all guides.

    -

    Default: False

    -
    -
    --scale-by-acc
    -

    Scale guide editing efficiency by the target loci accessibility

    -

    Default: False

    -
    -
    --acc-bw-path
    -

    Accessibility .bigWig file to be used to assign accessibility of guides.

    -
    -
    --acc-col
    -

    Column name in bdata.guides that specify raw ATAC-seq signal.

    -
    -
    --const-pi
    -

    Use constant pi provided in –guide-activity-col (instead of fitting from reporter data)

    -

    Default: False

    -
    -
    --shrink-alpha
    -

    Instead of using the trend-fitted alpha values, use estimated alpha values for each gRNA that are shrunk towards the fitted trend.

    -

    Default: False

    -
    -
    --condition-col
    -

    Column key in bdata.samples that describes experimental condition.

    -

    Default: “conditionâ€

    -
    -
    --time-col
    -

    Column key in bdata.samples that describes time elapsed.

    -

    Default: “timeâ€

    -
    -
    --control-condition
    -

    Value in bdata.samples[condition_col] that indicates control experimental condition.

    -

    Default: “bulkâ€

    -
    -
    --include-control-condition-for-inference, -ic
    -

    Include control conditions for inference. Currently only supported for survival screens.

    -

    Default: False

    -
    -
    --replicate-col
    -

    Column key in bdata.samples that describes experimental replicates.

    -

    Default: “replicateâ€

    -
    -
    --target-col
    -

    Column key in bdata.guides that describes the target element of each guide.

    -

    Default: “targetâ€

    -
    -
    --guide-activity-col, -a
    -

    Column in ReporterScreen.guides DataFrame showing the editing rate estimated via external tools

    -
    -
    --outdir, -o
    -

    Directory to save the run result.

    -

    Default: “.â€

    -
    -
    --result-suffix
    -

    Suffix of the output files

    -

    Default: “â€

    -
    -
    --sorting-bin-upper-quantile-col, -uq
    -

    Column name with upper quantile values of each sorting bin in [Reporter]Screen.samples (or AnnData.var)

    -

    Default: “upper_quantileâ€

    -
    -
    --sorting-bin-lower-quantile-col, -lq
    -

    Column name with lower quantile values of each sorting bin in [Reporter]Screen.samples (or AnnData var)

    -

    Default: “lower_quantileâ€

    -
    -
    --alpha-if-overdispersion-fitting-fails, -af
    -

    Comma-separated regression coefficient (b0, b1) of log(a0) ~ log(q) that will be used if fitting dispersion on the data fails.

    -
    -
    --cuda
    -

    run on GPU

    -

    Default: False

    -
    -
    --sample-mask-col
    -

    Name of the column indicating the sample mask in [Reporter]Screen.samples (or AnnData.var). Sample is ignored if the value in this column is 0. This can be used to mask out low-quality samples.

    -
    -
    --fit-negctrl
    -

    Fit the shared negative control distribution to normalize the fitted parameters

    -

    Default: False

    -
    -
    --negctrl-col
    -

    Column in bdata.obs specifying if a guide is negative control. If the bdata.guides[negctrl_col].lower() == negctrl_col_value, it is treated as negative control guide.

    -

    Default: “target_groupâ€

    -
    -
    --negctrl-col-value
    -

    Column value in bdata.guides specifying if a guide is negative control. If the bdata.guides[negctrl_col].lower() == negctrl_col_value, it is treated as negative control guide.

    -

    Default: “negctrlâ€

    -
    -
    --repguide-mask
    -

    n_replicate x n_guide mask to mask the outlier guides. screen.uns[repguide_mask] will be used.

    -

    Default: repguide_mask

    -
    -
    --device
    -

    Optionally use GPU if provided valid GPU device name (ex. cuda:0)

    -
    -
    --ignore-bcmatch
    -

    If provided, even if the screen object has .X_bcmatch, ignore the count when fitting.

    -

    Default: False

    -
    -
    --allele-df-key
    -

    screen.uns[allele_df_key] will be used as the allele count.

    -
    -
    --splice-site-path
    -

    Path to splicing site

    -
    -
    --control-guide-tag
    -

    If this string is in guide name, treat each guide separately not to mix the position. Used for negative controls.

    -
    -
    --dont-fit-noise
    -

    Default: False

    -
    -
    --dont-adjust-confidence-by-negative-control
    -

    Adjust confidence by negative controls. For variant library_design, this uses negative control variants. For tiling library_design, adjusts confidence by synonymous edits.

    -

    Default: False

    -
    -
    --n-iter
    -

    # of SVI steps taken for inference.

    -

    Default: 2000

    -
    -
    --load-existing
    -

    Load existing .pkl file if present.

    -

    Default: False

    -
    -
    -
    -
    -
    - - -
    - -
    -
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    -
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    Search

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    - - - - - - \ No newline at end of file diff --git a/docs/_build/searchindex.js b/docs/_build/searchindex.js deleted file mode 100644 index 0f17e54..0000000 --- a/docs/_build/searchindex.js +++ /dev/null @@ -1 +0,0 @@ -Search.setIndex({"docnames": ["ReporterScreen_api", "cds", "commands/count", "commands/create-screen", "commands/filter", "commands/input", "commands/profile", "commands/qc", "commands/run", "count", "count_samples", "exon_fa_format", "filter", "gwas", "index", "index_", "input", "profile", "qc", "run", "subcommands", "tutorials/ldl_cds", "tutorials/ldl_var"], "filenames": ["ReporterScreen_api.rst", "cds.rst", "commands/count.md", "commands/create-screen.md", "commands/filter.md", "commands/input.md", "commands/profile.md", "commands/qc.md", "commands/run.md", "count.rst", "count_samples.rst", "exon_fa_format.md", "filter.rst", "gwas.rst", "index.md", "index_.rst", "input.rst", "profile.rst", "qc.rst", "run.rst", "subcommands.rst", "tutorials/ldl_cds.md", 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Quantify variant effect (bean run)": [[1, "quantify-variant-effect-run"], [21, "quantify-variant-effect-run"]], "bean count[-samples]: Count (reporter) screen data": [[2, "bean-count-samples-count-reporter-screen-data"], [9, "bean-count-samples-count-reporter-screen-data"], [10, "bean-count-samples-count-reporter-screen-data"]], "Input file format": [[2, "input-file-format"], [9, "input-file-format"], [10, "input-file-format"], [16, "input-file-format"]], "Output file format": [[2, "output-file-format"], [9, "output-file-format"], [10, "output-file-format"]], "bean create-screen: Create ReporterScreen object from flat files": [[3, "bean-create-screen-create-reporterscreen-object-from-flat-files"]], "Input": [[3, "input"], [8, "input"], [19, "input"]], "filter: Filtering (and optionally translating) alleles": [[4, "filter-filtering-and-optionally-translating-alleles"], [12, "filter-filtering-and-optionally-translating-alleles"]], "Output": [[4, "output"], [6, "output"], [7, "output"], [8, "output"], [12, "output"], [17, "output"], [18, "output"], [19, "output"]], "Translating alleles": [[4, "translating-alleles"], [12, "translating-alleles"]], "sgRNA_info_table.csv": [[5, "sgrna-info-table-csv"], [16, "sgrna-info-table-csv"]], "sample_list.csv": [[5, "sample-list-csv"], [16, "sample-list-csv"]], "bean profile: Profile editing patterns": [[6, "bean-profile-profile-editing-patterns"], [17, "bean-profile-profile-editing-patterns"]], "bean qc: QC of reporter screen data": [[7, "bean-qc-qc-of-reporter-screen-data"], [18, "bean-qc-qc-of-reporter-screen-data"]], "QC thresholds:": [[7, "qc-thresholds"], [18, "qc-thresholds"]], "Run options:": [[7, "run-options"], [18, "run-options"]], "Input .h5ad formatting:": [[7, "input-h5ad-formatting"], [18, "input-h5ad-formatting"]], "bean run: Quantify variant effects": [[8, "bean-run-quantify-variant-effects"], [19, "bean-run-quantify-variant-effects"]], "Usage example": [[8, "usage-example"], [19, "usage-example"]], "bean count": [[9, "bean-count"]], "Full parameters": [[9, "full-parameters"], [10, "full-parameters"], [12, "full-parameters"], [17, "full-parameters"], [18, "full-parameters"], [19, "full-parameters"]], "Named Arguments": [[9, "named-arguments"], [10, "named-arguments"], [12, "named-arguments"], [17, "named-arguments"], [18, "named-arguments"], [19, "named-arguments"]], "bean count-samples": [[10, "bean-count-samples"]], "Input .fa file format for bean-filter": [[11, "input-fa-file-format-for-bean-filter"]], "bean filter": [[12, "bean-filter"]], "Positional Arguments": [[12, "positional-arguments"], [17, "positional-arguments"], [18, "positional-arguments"], [19, "positional-arguments"]], "GWAS variant library": [[13, "gwas-variant-library"]], "Variant sorting screen tutorial": [[13, "variant-sorting-screen-tutorial"], [22, "variant-sorting-screen-tutorial"]], "2. QC samples & guides (bean qc)": [[13, "qc-samples-guides-qc"], [22, "qc-samples-guides-qc"]], "3. Quantify variant effect (bean run)": [[13, "quantify-variant-effect-run"], [22, "quantify-variant-effect-run"]], "title: CRISPR-BEAN": [[14, "title-crispr-bean"]], "Welcome to bean\u2019s documentation!": [[15, "welcome-to-bean-s-documentation"]], "Workflows": [[15, "workflows"]], "bean subcommands": [[15, "bean-subcommands"]], "Screen data structure": [[15, "screen-data-structure"]], "Indices and tables": [[15, "indices-and-tables"]], "bean profile": [[17, "bean-profile"]], "bean qc": [[18, "bean-qc"]], "QC thresholds": [[18, "qc-thresholds"]], "Run options": [[18, "run-options"]], "Input .h5ad formatting": [[18, "input-.h5ad-formatting"]], "bean run": [[19, "bean-run"]], "Subcommands": [[20, "subcommands"]]}, "indexentries": {}}) \ No newline at end of file diff --git a/docs/_build/subcommands.html b/docs/_build/subcommands.html deleted file mode 100644 index bb2154a..0000000 --- a/docs/_build/subcommands.html +++ /dev/null @@ -1,168 +0,0 @@ - - - - - - - - Subcommands — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - - - -
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    Subcommands¶

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    - ©2024, Jayoung Ryu. - - | - Powered by Sphinx 7.2.6 - & Alabaster 0.7.16 - - | - Page source -
    - - - - - - \ No newline at end of file diff --git a/docs/_build/tutorials/ldl_cds.html b/docs/_build/tutorials/ldl_cds.html deleted file mode 100644 index 09e4c5e..0000000 --- a/docs/_build/tutorials/ldl_cds.html +++ /dev/null @@ -1,261 +0,0 @@ - - - - - - - - Tiling sorting screen tutorial — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - -
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    Tiling sorting screen tutorial¶

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    Tiling screen that tiles gRNA densely across locus or multiple loci, selected based on FACS signal quantiles.

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    Library designTiling (gRNAs tile each locus densely)
    tiling library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design



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    Example workflow¶

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    screen_id=my_sorting_tiling_screen
-
-# 1. Count gRNA & reporter
-bean-count-samples \
---input tests/data/sample_list_tiling.csv          `# Contains fastq file path; see test file for example.`\
--b A                                               `# Base A is edited (into G)` \
--f tests/data/test_guide_info_tiling_chrom.csv     `# Contains gRNA metadata; see test file for example.`\
--o ./                                              `# Output directory` \
--r                                                 `# Quantify reporter edits` \
--n ${screen_id}                                       `# ID of the screen` \
---tiling
-
-# 2. QC samples & guides
-bean-qc \
-  bean_count_${screen_id}.h5ad           `# Input ReporterScreen .h5ad file path` \
-  -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \
-  -r qc_report_${screen_id}              `# Prefix for QC report` \
-
-# 3. Filter & translate alleles
-bean-filter ./bean_count_${screen_id}_masked.h5ad \
--o ./bean_count_${screen_id}_alleleFiltered \
---filter-target-basechange                             `# Filter based on intended base changes. If -b A was provided in bean-count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\
---filter-window --edit-start-pos 0 --edit-end-pos 19   `# Filter based on editing window in spacer position within reporter.`\
---filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \
---translate --translate-genes-list tests/data/gene_symbols.txt
-
-# 4. Quantify variant effect
-bean-run sorting tiling \
-    ./bean_count_${screen_id}_alleleFiltered.h5ad \
-    -o tests/test_res/var/ \
-    --fit-negctrl \
-    --scale-by-acc \
-    --accessibility-col accessibility
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    See more details below.

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    1. Count gRNA & reporter (bean count-samples)¶

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    screen_id=my_sorting_tiling_screen
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-bean-count-samples \
---input tests/data/sample_list_tiling.csv          `# Contains fastq file path; see test file for example.`\
--b A                                               `# Base A is edited (into G)` \
--f tests/data/test_guide_info_tiling_chrom.csv     `# Contains gRNA metadata; see test file for example.`\
--o ./                                              `# Output directory` \
--r                                                 `# Quantify reporter edits` \
--n ${screen_id}                                       `# ID of the screen` \
---tiling
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    Make sure you follow the input file format for seamless downstream steps. This will produce ./bean_count_${screen_id}.h5ad.

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    2. QC (bean qc)¶

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    Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the input file guideline, but you can change the parameters with the full argument list of ``bean-qc` <../../README#bean-qc-qc-of-reporter-screen-data>`_. (Common factors you may want to tweak is --ctrl-cond=bulk and --lfc-conds=top,bot if you have different sample condition labels.)

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    bean-qc \
-  bean_count_${screen_id}.h5ad           `# Input ReporterScreen .h5ad file path` \
-  -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \
-  -r qc_report_${screen_id}              `# Prefix for QC report` \
-  [--tiling]                          `# Not required if you have passed --tiling in counting step`
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    If the data does not include reporter editing data, you can provide --no-editing flag to omit the editing rate QC.

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    3. Filter alleles (bean filter)¶

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    As tiling library doesn’t have designated per-gRNA target variant, any base edit observed in reporter may be the candidate variant, while having too many variants with very low editing rate significantly decreases the power. Variants are filtered based on multiple criteria in bean-fitler.

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    If the screen targets coding sequence, it’s beneficial to translate edits into coding varaints whenever possible for better power. For translation, provide --translate and one of the following:

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    [ --translate-gene-name GENE_SYMBOL OR
-  --translate-genes-list path_to_gene_names_file.txt OR
-  --translate-fasta gene_exon.fa, OR
-  --translate-fastas-csv gene_exon_fas.csv]
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    where path_to_gene_names_file.txt has one gene symbol per line, and gene symbol uses its MANE transcript (hg38) coordinates of exons. In order to use other reference versions or transcript ID, you’ll need to feed in fasta file. See detailed formatting of fasta file here.

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    Example allele filtering given we’re translating based on MANE transcript exons of multiple gene symbols:

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    bean-filter ./bean_count_${screen_id}_masked.h5ad \
--o ./bean_count_${screen_id}_alleleFiltered \
---filter-target-basechange                             `# Filter based on intended base changes. If -b A was provided in bean-count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\
---filter-window --edit-start-pos 0 --edit-end-pos 19   `# Filter based on editing window in spacer position within reporter.`\
---filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \
---translate --translate-genes-list tests/data/gene_symbols.txt
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    Ouptut file `` shows number of alleles per guide and number of guides per variant, where we want high enough values for the latter. See the typical output for dataset with good editing coverage & filtering result here.

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    4. Quantify variant effect (bean run)¶

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    By default, bean-run [sorting,survival] tiling uses most filtered allele counts table for variant identification and quantification of their effects. **Check allele filtering output** and choose alternative filtered allele counts table if necessary.

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    bean-run can take 3 run options to quantify editing rate:

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      From reporter + accessibility

      1-1. If your gRNA metadata table (tests/data/test_guide_info.csv above) included per-gRNA accessibility score,

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         bean-run sorting tiling \
-   ./bean_count_${screen_id}_alleleFiltered.h5ad \
-   -o tests/test_res/var/ \
-   --fit-negctrl \
-   --scale-by-acc \
-   --accessibility-col accessibility
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-1-2. If your gRNA metadata table (\ ``tests/data/test_guide_info.csv`` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal,
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      bean-run sorting tiling \
-./bean_count_${screen_id}_alleleFiltered.h5ad \
--o tests/test_res/var/ \
---fit-negctrl \
---scale-by-acc \
---accessibility-bw accessibility.bw
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    2. From reporter

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      bean-run sorting tiling \
-./bean_count_${screen_id}_alleleFiltered.h5ad \
--o tests/test_res/var/ \
---fit-negctrl
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      No reporter information, assume the same editing efficiency of all gRNAs.

      Use this option if your data don’t have editing rate information.

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      bean-run sorting tiling \
-./bean_count_${screen_id}_alleleFiltered.h5ad \
--o tests/test_res/var/ \
---fit-negctrl \
---uniform-edit
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    - ©2024, Jayoung Ryu. - - | - Powered by Sphinx 7.2.6 - & Alabaster 0.7.16 - - | - Page source -
    - - - - - - \ No newline at end of file diff --git a/docs/_build/tutorials/ldl_var.html b/docs/_build/tutorials/ldl_var.html deleted file mode 100644 index d03177f..0000000 --- a/docs/_build/tutorials/ldl_var.html +++ /dev/null @@ -1,232 +0,0 @@ - - - - - - - - Variant sorting screen tutorial — bean 1.0.0 documentation - - - - - - - - - - - - - - - - - -
    -
    -
    - - -
    - -
    -

    Variant sorting screen tutorial¶

    -

    GWAS variant screen with per-variant gRNA tiling design, selected based on FACS signal quantiles.

    - - - - - - - - - -
    Library designVariant (gRNAs tile each target variant)
    variant library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design



    -
    -

    Example workflow¶

    -
    screen_id=my_sorting_tiling_screen
-
-# 1. Count gRNA & reporter
-bean-count-samples \
---input tests/data/sample_list.csv    `# Contains fastq file path; see test file for example.`\
--b A                                  `# Base A is edited (into G)` \
--f tests/data/test_guide_info.csv     `# Contains gRNA metadata; see test file for example.`\
--o ./                                 `# Output directory` \
--r                                    `# Quantify reporter edits` \
--n ${screen_id}                          `# ID of the screen to be counted`
-
-# 2. QC samples & guides
-bean-qc \
-  bean_count_${screen_id}.h5ad             `# Input ReporterScreen .h5ad file path` \
-  -o bean_count_${screen_id}_masked.h5ad   `# Output ReporterScreen .h5ad file path` \
-  -r qc_report_${screen_id}                `# Prefix for QC report` \
-  -b                                       ` # Remove replicates with no good samples.
-
-# 3. Quantify variant effect
-bean-run sorting variant \
-    tests/data/bean_count_${screen_id}_masked.h5ad \
-    -o tests/test_res/var/ \
-    --fit-negctrl \
-    --scale-by-acc \
-    --accessibility-col accessibility
-
    -
    -

    See more details below.

    -
    -
    -

    1. Count gRNA & reporter (bean count-samples)¶

    -
    screen_id=my_sorting_tiling_screen
-
-# 1. Count gRNA & reporter
-bean-count-samples \
---input tests/data/sample_list.csv    `# Contains fastq file path; see test file for example.`\
--b A                                  `# Base A is edited (into G)` \
--f tests/data/test_guide_info.csv     `# Contains gRNA metadata; see test file for example.`\
--o ./                                 `# Output directory` \
--r                                    `# Quantify reporter edits` \
--n ${screen_id}                          `# ID of the screen to be counted`
-
    -
    -

    Make sure you follow the input file format for seamless downstream steps. This will produce ./bean_count_${screen_id}.h5ad.

    -
    -
    -

    2. QC samples & guides (bean qc)¶

    -

    Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the input file guideline, but you can change the parameters with the full argument list of ``bean-qc` <../../README#bean-qc-qc-of-reporter-screen-data>`_. (Common factors you may want to tweak is --ctrl-cond=bulk and --lfc-conds=top,bot if you have different sample condition labels.)

    -
    bean-qc \
-  bean_count_${screen_id}.h5ad    `# Input ReporterScreen .h5ad file path` \
-  -o bean_count_${screen_id}_masked.h5ad   `# Output ReporterScreen .h5ad file path` \
-  -r qc_report_${screen_id}   `# Prefix for QC report`
-
    -
    -

    If the data does not include reporter editing data, you can provide --no-editing flag to omit the editing rate QC.

    -
    -
    -

    3. Quantify variant effect (bean run)¶

    -

    bean-run can take 3 run options to quantify editing rate:

    -
      -
    1. -
      From reporter + accessibility

      If your gRNA metadata table (tests/data/test_guide_info.csv above) included per-gRNA accessibility score,

      -
      -
      -
         bean-run sorting variant \
-   tests/data/bean_count_${screen_id}_masked.h5ad \
-   -o tests/test_res/var/ \
-   --fit-negctrl \
-   --scale-by-acc \
-   --accessibility-col accessibility
-
-If your gRNA metadata table (\ ``tests/data/test_guide_info.csv`` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal,
-
      -
      -
      bean-run sorting variant \
-tests/data/bean_count_${screen_id}_masked.h5ad \
--o tests/test_res/var/ \
---fit-negctrl \
---scale-by-acc \
---accessibility-bw accessibility.bw
-
      -
      -
      2. -

    2. From reporter, without accessibility

      -
      -

      This assumes the all target sites have the uniform chromatin accessibility.

      -
      -
      bean-run sorting variant \
-tests/data/bean_count_${screen_id}_masked.h5ad \
--o tests/test_res/var/ \
---fit-negctrl
-
      -
      -
      3. -
    3. -
      No reporter information, assume the same editing efficiency of all gRNAs.

      Use this option if your data don’t have editing outcome information.

      -
      -
      -
      bean-run sorting variant \
-tests/data/bean_count_${screen_id}_masked.h5ad \
--o tests/test_res/var/ \
---fit-negctrl \
---uniform-edit
-
      -
      -
      4. -
    -
    -
    - - -
    - -
    -
    - -
    -
    -
    - ©2024, Jayoung Ryu. - - | - Powered by Sphinx 7.2.6 - & Alabaster 0.7.16 - - | - Page source -
    - - - - - - \ No newline at end of file From 65ad2cb79107d28ade8a8834ee195b6ae60a0c5f Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 01:36:00 -0400 Subject: [PATCH 070/107] remove duplicate index --- docs/index.md | 4 ---- 1 file changed, 4 deletions(-) delete mode 100755 docs/index.md diff --git a/docs/index.md b/docs/index.md deleted file mode 100755 index 45e2414..0000000 --- a/docs/index.md +++ /dev/null @@ -1,4 +0,0 @@ ---- -layout: default -title: CRISPR-BEAN ---- From 8314104e9d1b5249fa3fb56e48b1f4a5c0b5df25 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 01:38:58 -0400 Subject: [PATCH 071/107] use separate save and param dict --- bean/cli/run.py | 6 +++--- bean/model/run.py | 2 +- 2 files changed, 4 insertions(+), 4 deletions(-) diff --git a/bean/cli/run.py b/bean/cli/run.py index 36e7617..81e4bda 100755 --- a/bean/cli/run.py +++ b/bean/cli/run.py @@ -152,7 +152,7 @@ def main(args): with open(f"{prefix}/{model_label}.result.pkl", "rb") as handle: param_history_dict = pkl.load(handle) else: - param_history_dict = deepcopy( + param_history_dict, save_dict = deepcopy( run_inference(model, guide, ndata, num_steps=args.n_iter) ) if args.fit_negctrl: @@ -167,7 +167,7 @@ def main(args): f"Using {len(negctrl_idx)} negative control elements to adjust phenotypic effect sizes..." ) ndata_negctrl = ndata[negctrl_idx] - param_history_dict["negctrl"] = deepcopy( + param_history_dict["negctrl"], save_dict["negctrl"] = deepcopy( run_inference( negctrl_model, negctrl_guide, ndata_negctrl, num_steps=args.n_iter ) @@ -181,7 +181,7 @@ def main(args): os.makedirs(prefix) with open(f"{prefix}/{model_label}.result{args.result_suffix}.pkl", "wb") as handle: # try: - pkl.dump(param_history_dict, handle) + pkl.dump(save_dict, handle) # except TypeError as exc: # print(exc.message) # print(param_history_dict) diff --git a/bean/model/run.py b/bean/model/run.py index f6ccea9..e2347e4 100755 --- a/bean/model/run.py +++ b/bean/model/run.py @@ -187,7 +187,7 @@ def run_inference( raise ValueError( f"Fitting halted for command: {' '.join(sys.argv)} with following error: \n {exc}" ) - return { + return pyro.get_param_store(), { "loss": losses, "params": pyro.get_param_store().get_state(), } From 86e910a42c7fee714990436fb5028b04aaf1d23f Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 01:50:24 -0400 Subject: [PATCH 072/107] fix key for paramStore writing --- README.md | 10 ++++------ bean/model/readwrite.py | 34 ++++++++++++++++------------------ 2 files changed, 20 insertions(+), 24 deletions(-) diff --git a/README.md b/README.md index 945dce5..0dd3ef0 100755 --- a/README.md +++ b/README.md @@ -1,4 +1,4 @@ -# crispr-bean +# crispr-bean [![PyPI pyversions](https://img.shields.io/pypi/pyversions/crispr-bean)](https://pypi.org/project/crispr-bean/) [![PyPI version](https://img.shields.io/pypi/v/crispr-bean)](https://pypi.org/project/crispr-bean/) @@ -25,7 +25,8 @@ ### Screen data is saved as *ReporterScreen* object in the pipeline. BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). See [Data Structure](#data-structure) section for more information. -

    +## Documentaton +See the [documentation](https://pinellolab.github.io/crispr-bean/) for tutorials and API references. ## Tutorials | [Library design](#pipeline-run-options-by-library-design) | Selection | Reporter | Tutorial link | @@ -46,7 +47,6 @@ The `bean filter` and `bean run` steps depend on the type of gRNA library design Ex) tiling library design -

    ## Installation ### Full installation @@ -62,7 +62,7 @@ pip install crispr-bean ``` This wouldn't have variant effect size quantification (`bean run`) functionality. -

    + ## Subcommands See the full detail for each subcommand in the documentation. * [`count`, `count-samples`](docs/count.md): Count (reporter) screen data @@ -94,8 +94,6 @@ cdata = be.read_h5ad("bean_counts_sample.h5ad") ``` Python package `bean` supports multiple data wrangling functionalities for `ReporterScreen` objects. See the [**ReporterScreen API tutorial**](docs/ReporterScreen_api.ipynb) for more detail. -

    - ## Run time * Installation takes 14.4 mins after pytorch installation with pytorch in Dell XPS 13 Ubuntu WSL. * `bean run` takes 4.6 mins with `--scale-by-acc` tag in Dell XPS 13 Ubuntu WSL for variant screen dataset with 3455 guides and 6 replicates with 4 sorting bins. diff --git a/bean/model/readwrite.py b/bean/model/readwrite.py index a36493b..76287f3 100755 --- a/bean/model/readwrite.py +++ b/bean/model/readwrite.py @@ -62,19 +62,19 @@ def write_result_table( return_result: bool = False, ) -> Union[pd.DataFrame, None]: """Combine target information and scores to write result table to a csv file or return it.""" - if param_hist_dict["params"]["mu_loc"].dim() == 2: - mu = param_hist_dict["params"]["mu_loc"].detach()[:, 0].cpu().numpy() - mu_sd = param_hist_dict["params"]["mu_scale"].detach()[:, 0].cpu().numpy() + if param_hist_dict["mu_loc"].dim() == 2: + mu = param_hist_dict["mu_loc"].detach()[:, 0].cpu().numpy() + mu_sd = param_hist_dict["mu_scale"].detach()[:, 0].cpu().numpy() if sd_is_fitted: - sd = param_hist_dict["params"]["sd_loc"].detach().exp()[:, 0].cpu().numpy() - elif param_hist_dict["params"]["mu_loc"].dim() == 1: - mu = param_hist_dict["params"]["mu_loc"].detach().cpu().numpy() - mu_sd = param_hist_dict["params"]["mu_scale"].detach().cpu().numpy() + sd = param_hist_dict["sd_loc"].detach().exp()[:, 0].cpu().numpy() + elif param_hist_dict["mu_loc"].dim() == 1: + mu = param_hist_dict["mu_loc"].detach().cpu().numpy() + mu_sd = param_hist_dict["mu_scale"].detach().cpu().numpy() if sd_is_fitted: - sd = param_hist_dict["params"]["sd_loc"].detach().exp().cpu().numpy() + sd = param_hist_dict["sd_loc"].detach().exp().cpu().numpy() else: raise ValueError( - f'`mu_loc` has invalid shape of {param_hist_dict["params"]["mu_loc"].shape}' + f'`mu_loc` has invalid shape of {param_hist_dict["mu_loc"].shape}' ) param_dict = { "mu": mu, @@ -85,17 +85,15 @@ def write_result_table( param_dict["sd"] = sd if sample_covariates is not None: assert ( - "mu_cov_loc" in param_hist_dict["params"] - and "mu_cov_scale" in param_hist_dict["params"] - ), param_hist_dict["params"].keys() + "mu_cov_loc" in param_hist_dict and "mu_cov_scale" in param_hist_dict + ), param_hist_dict.keys() for i, sample_cov in enumerate(sample_covariates): param_dict[f"mu_{sample_cov}"] = ( - mu + param_hist_dict["params"]["mu_cov_loc"].detach().cpu().numpy()[i] + mu + param_hist_dict["mu_cov_loc"].detach().cpu().numpy()[i] ) param_dict[f"mu_sd_{sample_cov}"] = np.sqrt( mu_sd**2 - + param_hist_dict["params"]["mu_cov_scale"].detach().cpu().numpy()[i] - ** 2 + + param_hist_dict["mu_cov_scale"].detach().cpu().numpy()[i] ** 2 ) param_dict[f"mu_z_{sample_cov}"] = ( param_dict[f"mu_{sample_cov}"] / param_dict[f"mu_sd_{sample_cov}"] @@ -185,10 +183,10 @@ def write_result_table( ) if write_fitted_eff or guide_acc is not None: - if "alpha_pi" not in param_hist_dict["params"].keys(): + if "alpha_pi" not in param_hist_dict.keys(): pi = 1.0 else: - a_fitted = param_hist_dict["params"]["alpha_pi"].detach().cpu().numpy() + a_fitted = param_hist_dict["alpha_pi"].detach().cpu().numpy() pi = a_fitted[..., 1:].sum(axis=1) / a_fitted.sum(axis=1) sgRNA_df = pd.DataFrame({"edit_eff": pi}, index=guide_index) if guide_acc is not None: @@ -196,7 +194,7 @@ def write_result_table( sgRNA_df["scaled_edit_eff"] = _scale_pi( pi, guide_acc, - fitted_noise_logit=param_hist_dict["params"]["noise_scale"] + fitted_noise_logit=param_hist_dict["noise_scale"] .detach() .cpu() .numpy(), From 1dc0593c4fa7bf4c63c3c5dd1305badd249b46a5 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 02:22:57 -0400 Subject: [PATCH 073/107] add images to docs --- README.md | 54 ++++---- docs/commands/count.md | 2 +- docs/commands/filter.md | 2 +- docs/commands/profile.md | 4 +- docs/commands/qc.md | 2 +- docs/commands/run.md | 4 +- imgs/dag_bean_v2.svg | 263 +++++++++++++++++++++++++++++++++++++++ 7 files changed, 291 insertions(+), 40 deletions(-) create mode 100644 imgs/dag_bean_v2.svg diff --git a/README.md b/README.md index 0dd3ef0..adcc6ab 100755 --- a/README.md +++ b/README.md @@ -12,18 +12,29 @@ ## Overview `bean` supports end-to-end analysis of pooled sorting screens, with or without reporter. -dag_bean_v2.svg +dag_bean_v2.svg `bean` subcommands include the following: -1. [`count`, `count-samples`](#bean-count-samples-count-reporter-screen-data): Base-editing-aware **mapping** of guide, optionally with reporter from `.fastq` files. +1. [`count`](https://pinellolab.github.io/crispr-bean/count.html), [`count-samples`](https://pinellolab.github.io/crispr-bean/count_samples.html): Base-editing-aware **mapping** of guide, optionally with reporter from `.fastq` files. * [`create-screen`](#bean-create-screen-create-reporterscreen-object-from-flat-files) creates minimal ReporterScreen object from flat gRNA count file. Note that this way, allele counts are not included and many functionalities involving allele and edit counts are not supported. -2. [`profile`](#bean-profile-profile-editing-patterns): Profile editing preferences of your editor. +2. [`profile`](https://pinellolab.github.io/crispr-bean/profile.html): Profile editing preferences of your editor. 3. [`qc`](#bean-qc-qc-of-reporter-screen-data): Quality control report and filtering out / masking of aberrant sample and guides 4. [`filter`](#bean-filter-filtering-and-optionally-translating-alleles): Filter reporter alleles; essential for `tiling` mode that allows for all alleles generated from gRNA. 5. [`run`](#bean-run-quantify-variant-effects): Quantify targeted variants' effect sizes from screen data. - -### Screen data is saved as *ReporterScreen* object in the pipeline. +* Screen data is saved as *ReporterScreen* object in the pipeline. BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). See [Data Structure](#data-structure) section for more information. +## Installation +First install [PyT}orch](https://pytorch.org/get-started/). +Then download from PyPI: +``` +pip install crispr-bean[model] +``` + +Following installation without PyTorch dependency wouldn't have variant effect size quantification (`bean run`) functionality. +``` +pip install crispr-bean +``` + ## Documentaton See the [documentation](https://pinellolab.github.io/crispr-bean/) for tutorials and API references. @@ -37,7 +48,7 @@ See the [documentation](https://pinellolab.github.io/crispr-bean/) for tutorials | Coding sequence tiling libarary | Survival / Proliferation | Yes/No | TKO simulated, tiling (Coming soon!) -### Pipeline run options by library design +### Library design: variant or tiling? The `bean filter` and `bean run` steps depend on the type of gRNA library design, where BEAN supports two modes of running. 1. `variant` library: Several gRNAs tile each of the targeted variants Ex) @@ -47,32 +58,6 @@ The `bean filter` and `bean run` steps depend on the type of gRNA library design Ex) tiling library design - -## Installation -### Full installation -First install [pytorch](https://pytorch.org/) >=12.1,<2. -Then download from PyPI: -``` -pip install crispr-bean[model] -``` - -### Mapping and data wrangling, without variant effect quantification -``` -pip install crispr-bean -``` -This wouldn't have variant effect size quantification (`bean run`) functionality. - - -## Subcommands -See the full detail for each subcommand in the documentation. -* [`count`, `count-samples`](docs/count.md): Count (reporter) screen data -* [`profile`](docs/profile.md) -* [`qc`](docs/qc.md) -* [`filter`](docs/commands/filter.md) -* [`run`](docs/commands/run.md) -* [`create-screen`](docs/commands/create-screen.md) - - ## Data Structure ### ReporterScreen object BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). @@ -87,7 +72,7 @@ Following attributes are included if matched reporter is provided and you chose * `.X_edits [Optional]`: If target position of each guide is specified as `target_pos` in input `gRNA_library.csv` file and `--match-target-position` option is provided, the result has the matrix with the number of target edit at the specified positions. * `.allele_tables [Optional]`: Dictionary with a single allele count table that counts per guide and allele combination, what is the count per sample. -### Using BEAN as Python module +## Using BEAN as Python module ``` import bean as be cdata = be.read_h5ad("bean_counts_sample.h5ad") @@ -99,6 +84,9 @@ Python package `bean` supports multiple data wrangling functionalities for `Repo * `bean run` takes 4.6 mins with `--scale-by-acc` tag in Dell XPS 13 Ubuntu WSL for variant screen dataset with 3455 guides and 6 replicates with 4 sorting bins. * Full pipeline takes 90.1s in GitHub Action for toy dataset of 2 replicates and 30 guides. +## Contribution +If you have questions or feature request, please open an issue. Please feel free to send a pull request. + ## Citation If you have used BEAN for your analysis, please cite: Ryu, J. et al. Joint genotypic and phenotypic outcome modeling improves base editing variant effect quantification. medRxiv (2023) doi:10.1101/2023.09.08.23295253 diff --git a/docs/commands/count.md b/docs/commands/count.md index 5f04b4c..9dac49e 100755 --- a/docs/commands/count.md +++ b/docs/commands/count.md @@ -18,7 +18,7 @@ bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r ``` By default, `bean count[-samples]` assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure. - Read structuren + Read structuren See full detail [below](#full-parameters). diff --git a/docs/commands/filter.md b/docs/commands/filter.md index 53d6ec1..2809251 100755 --- a/docs/commands/filter.md +++ b/docs/commands/filter.md @@ -16,7 +16,7 @@ You may want to adjust the flitering parameters to obtain optimal balance betwee # Translating alleles If you want to obtain **amino acid level variant** for coding sequence tiling screens, provide coding sequence positions which variants occuring within the coding sequence will be translated. *This is optional, but **highly recommended** to increase per-(coding)variant support.* -Allele translation +Allele translation ```bash diff --git a/docs/commands/profile.md b/docs/commands/profile.md index bbe8a8c..7886ff5 100755 --- a/docs/commands/profile.md +++ b/docs/commands/profile.md @@ -3,6 +3,6 @@ bean profile my_sorting_screen.h5ad -o output_prefix `# Prefix for editing profile report` ``` # Output -Above command produces `prefix_editing_preference.[html,ipynb]` as editing preferences ([see example](../../notebooks/profile_editing_preference.ipynb)). +Above command produces `prefix_editing_preference.[html,ipynb]` as editing preferences ([see example](../notebooks/profile_editing_preference.ipynb)). -Allele translation \ No newline at end of file +Allele translation \ No newline at end of file diff --git a/docs/commands/qc.md b/docs/commands/qc.md index 82b719e..d2ac1d5 100755 --- a/docs/commands/qc.md +++ b/docs/commands/qc.md @@ -17,7 +17,7 @@ bean qc \ `bean qc` supports following quality control and masks samples with low quality. Specifically: -Allele translation +Allele translation * Plots guide coverage and the uniformity of coverage * Guide count correlation between samples diff --git a/docs/commands/run.md b/docs/commands/run.md index 4c45259..3274276 100755 --- a/docs/commands/run.md +++ b/docs/commands/run.md @@ -6,7 +6,7 @@ BEAN uses Bayesian network to incorporate gRNA editing outcome to provide poster For the full detail, see the method section of the [BEAN manuscript](https://www.medrxiv.org/content/10.1101/2023.09.08.23295253v1). -model +model

    @@ -45,7 +45,7 @@ See full list of parameters [below](#full-parameters). # Output -model +model Above command produces * `output_prefix/bean_element_result.[model_type].csv` with following columns: diff --git a/imgs/dag_bean_v2.svg b/imgs/dag_bean_v2.svg new file mode 100644 index 0000000..6f89f99 --- /dev/null +++ b/imgs/dag_bean_v2.svg @@ -0,0 +1,263 @@ + + + + + + + + + + + + + + + + run + +qc +filter + +count-samples +R1.fastq + + + +R2.fastq +Coding regions oftargeted genes + + + + + + + + + + + Input + + + + Input(Optional) + +Legend + + +AnnData-based ReporterScreen object + + + + + + + + + + + + + + + + + guides + + samples + + + gRNA counts + + + + + + + + + + + gRNA + Allele + + samples + + + Allele counts + + + + + + + + + + + + + + gRNA1 + Allele1 + Allele2 + ... + + + + + + + + + + + + + + + + + + + + + + + + + + +qc_report.[html,ipynb] + + + + + + + + + + +filter_log.txtfiltered_allele_stats.pdf + + + + + + + + + + +bean_element_result.csvbean_sgRNA_result.csv + + + + + Output + + + + + + + + + + + + +profile + + + + + + + + + + +editing_preferences.[html,ipynb] + +sample_list.csv + +gRNA_info.csv + + + + + + + + + + + + + From fb92a650b0c0025cc2825b3f163e02a8965d03f4 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 02:27:43 -0400 Subject: [PATCH 074/107] add negctrl as separate arg --- README.md | 4 ++-- bean/cli/run.py | 3 ++- bean/model/readwrite.py | 13 ++++--------- 3 files changed, 8 insertions(+), 12 deletions(-) diff --git a/README.md b/README.md index adcc6ab..9fc7106 100755 --- a/README.md +++ b/README.md @@ -12,7 +12,7 @@ ## Overview `bean` supports end-to-end analysis of pooled sorting screens, with or without reporter. -dag_bean_v2.svg +dag_bean_v2.svg `bean` subcommands include the following: 1. [`count`](https://pinellolab.github.io/crispr-bean/count.html), [`count-samples`](https://pinellolab.github.io/crispr-bean/count_samples.html): Base-editing-aware **mapping** of guide, optionally with reporter from `.fastq` files. @@ -24,7 +24,7 @@ * Screen data is saved as *ReporterScreen* object in the pipeline. BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). See [Data Structure](#data-structure) section for more information. ## Installation -First install [PyT}orch](https://pytorch.org/get-started/). +First install [PyTorch](https://pytorch.org/get-started/). Then download from PyPI: ``` pip install crispr-bean[model] diff --git a/bean/cli/run.py b/bean/cli/run.py index 81e4bda..55d8a59 100755 --- a/bean/cli/run.py +++ b/bean/cli/run.py @@ -167,7 +167,7 @@ def main(args): f"Using {len(negctrl_idx)} negative control elements to adjust phenotypic effect sizes..." ) ndata_negctrl = ndata[negctrl_idx] - param_history_dict["negctrl"], save_dict["negctrl"] = deepcopy( + param_history_dict_negctrl, save_dict["negctrl"] = deepcopy( run_inference( negctrl_model, negctrl_guide, ndata_negctrl, num_steps=args.n_iter ) @@ -188,6 +188,7 @@ def main(args): write_result_table( target_info_df, param_history_dict, + negctrl_params=param_history_dict_negctrl, model_label=model_label, prefix=f"{prefix}/", suffix=args.result_suffix, diff --git a/bean/model/readwrite.py b/bean/model/readwrite.py index 76287f3..b566790 100755 --- a/bean/model/readwrite.py +++ b/bean/model/readwrite.py @@ -52,6 +52,7 @@ def write_result_table( model_label: str, prefix: str = "", suffix: str = "", + negctrl_params=None, write_fitted_eff: bool = True, adjust_confidence_by_negative_control: bool = True, adjust_confidence_negatives: np.ndarray = None, @@ -100,17 +101,11 @@ def write_result_table( ) fit_df = pd.DataFrame(param_dict) - if "negctrl" in param_hist_dict.keys(): + if negctrl_params is not None: print("Normalizing with common negative control distribution") - mu0 = param_hist_dict["negctrl"]["params"]["mu_loc"].detach().cpu().numpy() + mu0 = negctrl_params["params"]["mu_loc"].detach().cpu().numpy() if sd_is_fitted: - sd0 = ( - param_hist_dict["negctrl"]["params"]["sd_loc"] - .detach() - .exp() - .cpu() - .numpy() - ) + sd0 = negctrl_params["params"]["sd_loc"].detach().exp().cpu().numpy() else: sd0 = 1.0 print(f"Fitted mu0={mu0}" + (f", sd0={sd0}." if sd_is_fitted else "")) From cde6b27b6f241a2419146da819c14319bce6921e Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 02:30:16 -0400 Subject: [PATCH 075/107] add doc --- README.md | 2 +- docs/create_screen.rst | 10 ++++++++++ 2 files changed, 11 insertions(+), 1 deletion(-) create mode 100755 docs/create_screen.rst diff --git a/README.md b/README.md index 9fc7106..c76a361 100755 --- a/README.md +++ b/README.md @@ -12,7 +12,7 @@ ## Overview `bean` supports end-to-end analysis of pooled sorting screens, with or without reporter. -dag_bean_v2.svg +dag_bean_v2.svg `bean` subcommands include the following: 1. [`count`](https://pinellolab.github.io/crispr-bean/count.html), [`count-samples`](https://pinellolab.github.io/crispr-bean/count_samples.html): Base-editing-aware **mapping** of guide, optionally with reporter from `.fastq` files. diff --git a/docs/create_screen.rst b/docs/create_screen.rst new file mode 100755 index 0000000..6ffa9bf --- /dev/null +++ b/docs/create_screen.rst @@ -0,0 +1,10 @@ +`bean create-screen` +*********************** +.. mdinclude:: commands/create-screen.md + +Full parameters +================== +.. argparse:: + :filename: ../bean/framework/parser.py + :func: get_input_parser + :prog: bean create-screen \ No newline at end of file From 8fec7eaf699508e2ab8655b58820e92a8bc505be Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 02:30:52 -0400 Subject: [PATCH 076/107] add doc --- README.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/README.md b/README.md index c76a361..5ec4a85 100755 --- a/README.md +++ b/README.md @@ -16,7 +16,7 @@ `bean` subcommands include the following: 1. [`count`](https://pinellolab.github.io/crispr-bean/count.html), [`count-samples`](https://pinellolab.github.io/crispr-bean/count_samples.html): Base-editing-aware **mapping** of guide, optionally with reporter from `.fastq` files. - * [`create-screen`](#bean-create-screen-create-reporterscreen-object-from-flat-files) creates minimal ReporterScreen object from flat gRNA count file. Note that this way, allele counts are not included and many functionalities involving allele and edit counts are not supported. + * [`create-screen`](https://pinellolab.github.io/crispr-bean/create_screen.html) creates minimal ReporterScreen object from flat gRNA count file. Note that this way, allele counts are not included and many functionalities involving allele and edit counts are not supported. 2. [`profile`](https://pinellolab.github.io/crispr-bean/profile.html): Profile editing preferences of your editor. 3. [`qc`](#bean-qc-qc-of-reporter-screen-data): Quality control report and filtering out / masking of aberrant sample and guides 4. [`filter`](#bean-filter-filtering-and-optionally-translating-alleles): Filter reporter alleles; essential for `tiling` mode that allows for all alleles generated from gRNA. From 076679eee51999c8884199116bf5af2def0b4e32 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 02:36:01 -0400 Subject: [PATCH 077/107] change title --- README.md | 2 +- imgs/bean_title2.svg | 127 +++++++++++++++++++++++++++++++++++++++++++ 2 files changed, 128 insertions(+), 1 deletion(-) create mode 100644 imgs/bean_title2.svg diff --git a/README.md b/README.md index 5ec4a85..d3cc224 100755 --- a/README.md +++ b/README.md @@ -1,4 +1,4 @@ -# crispr-bean +# crispr-bean [![PyPI pyversions](https://img.shields.io/pypi/pyversions/crispr-bean)](https://pypi.org/project/crispr-bean/) [![PyPI version](https://img.shields.io/pypi/v/crispr-bean)](https://pypi.org/project/crispr-bean/) diff --git a/imgs/bean_title2.svg b/imgs/bean_title2.svg new file mode 100644 index 0000000..037818a --- /dev/null +++ b/imgs/bean_title2.svg @@ -0,0 +1,127 @@ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + From a51d1ba5d39ab458600340945f892a6992832cd4 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 02:42:57 -0400 Subject: [PATCH 078/107] fix unbounderror --- README.md | 6 +++--- bean/cli/run.py | 2 ++ bean/model/readwrite.py | 4 ++-- 3 files changed, 7 insertions(+), 5 deletions(-) diff --git a/README.md b/README.md index d3cc224..e9e6f4f 100755 --- a/README.md +++ b/README.md @@ -18,9 +18,9 @@ 1. [`count`](https://pinellolab.github.io/crispr-bean/count.html), [`count-samples`](https://pinellolab.github.io/crispr-bean/count_samples.html): Base-editing-aware **mapping** of guide, optionally with reporter from `.fastq` files. * [`create-screen`](https://pinellolab.github.io/crispr-bean/create_screen.html) creates minimal ReporterScreen object from flat gRNA count file. Note that this way, allele counts are not included and many functionalities involving allele and edit counts are not supported. 2. [`profile`](https://pinellolab.github.io/crispr-bean/profile.html): Profile editing preferences of your editor. -3. [`qc`](#bean-qc-qc-of-reporter-screen-data): Quality control report and filtering out / masking of aberrant sample and guides -4. [`filter`](#bean-filter-filtering-and-optionally-translating-alleles): Filter reporter alleles; essential for `tiling` mode that allows for all alleles generated from gRNA. -5. [`run`](#bean-run-quantify-variant-effects): Quantify targeted variants' effect sizes from screen data. +3. [`qc`](https://pinellolab.github.io/crispr-bean/qc.html): Quality control report and filtering out / masking of aberrant sample and guides +4. [`filter`](https://pinellolab.github.io/crispr-bean/filter.html): Filter reporter alleles; essential for `tiling` mode that allows for all alleles generated from gRNA. +5. [`run`](https://pinellolab.github.io/crispr-bean/run.html): Quantify targeted variants' effect sizes from screen data. * Screen data is saved as *ReporterScreen* object in the pipeline. BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). See [Data Structure](#data-structure) section for more information. ## Installation diff --git a/bean/cli/run.py b/bean/cli/run.py index 55d8a59..b91457a 100755 --- a/bean/cli/run.py +++ b/bean/cli/run.py @@ -172,6 +172,8 @@ def main(args): negctrl_model, negctrl_guide, ndata_negctrl, num_steps=args.n_iter ) ) + else: + param_history_dict_negctrl = None outfile_path = ( f"{prefix}/bean_element[sgRNA]_result.{model_label}{args.result_suffix}.csv" diff --git a/bean/model/readwrite.py b/bean/model/readwrite.py index b566790..c7cf181 100755 --- a/bean/model/readwrite.py +++ b/bean/model/readwrite.py @@ -103,9 +103,9 @@ def write_result_table( fit_df = pd.DataFrame(param_dict) if negctrl_params is not None: print("Normalizing with common negative control distribution") - mu0 = negctrl_params["params"]["mu_loc"].detach().cpu().numpy() + mu0 = negctrl_params["mu_loc"].detach().cpu().numpy() if sd_is_fitted: - sd0 = negctrl_params["params"]["sd_loc"].detach().exp().cpu().numpy() + sd0 = negctrl_params["sd_loc"].detach().exp().cpu().numpy() else: sd0 = 1.0 print(f"Fitted mu0={mu0}" + (f", sd0={sd0}." if sd_is_fitted else "")) From d242944c0cb39b23baf215d2dd08e2abbb536740 Mon Sep 17 00:00:00 2001 From: Jayoung Kim Ryu Date: Sat, 30 Mar 2024 02:44:43 -0400 Subject: [PATCH 079/107] Update README.md --- README.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/README.md b/README.md index e9e6f4f..35cc89f 100755 --- a/README.md +++ b/README.md @@ -84,7 +84,7 @@ Python package `bean` supports multiple data wrangling functionalities for `Repo * `bean run` takes 4.6 mins with `--scale-by-acc` tag in Dell XPS 13 Ubuntu WSL for variant screen dataset with 3455 guides and 6 replicates with 4 sorting bins. * Full pipeline takes 90.1s in GitHub Action for toy dataset of 2 replicates and 30 guides. -## Contribution +## Contributing If you have questions or feature request, please open an issue. Please feel free to send a pull request. ## Citation From 360acb309c7afd4fde7aad7ff9de3e1c09238857 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 02:52:35 -0400 Subject: [PATCH 080/107] add data structure --- README.md | 14 -------------- docs/commands/reporterscreen.md | 11 +++++++++++ docs/index.rst | 3 ++- docs/reporterscreen.rst | 3 +++ 4 files changed, 16 insertions(+), 15 deletions(-) create mode 100644 docs/commands/reporterscreen.md create mode 100644 docs/reporterscreen.rst diff --git a/README.md b/README.md index e9e6f4f..dfe141c 100755 --- a/README.md +++ b/README.md @@ -58,20 +58,6 @@ The `bean filter` and `bean run` steps depend on the type of gRNA library design Ex) tiling library design -## Data Structure -### ReporterScreen object -BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). - -ReporterScreen object structure - - * `.guides`: guide information provided in input (`gRNA_library.csv` in above example) - * `.samples`: sample information provided in input (`sample_list.csv` in above example) - * `.X`: Main guide count matrix, where row corresponds to each guide in `.guides` and columns correspond to samples in `.samples`. -Following attributes are included if matched reporter is provided and you chose to read edit/allele information from the reporter using `-r` option. - * `.X_bcmatch [Optional]`: Contains information about number of barcode-matched reads. Information about R2 barcode should be specified as `barcode` column in your `gRNA_library.csv` file. - * `.X_edits [Optional]`: If target position of each guide is specified as `target_pos` in input `gRNA_library.csv` file and `--match-target-position` option is provided, the result has the matrix with the number of target edit at the specified positions. - * `.allele_tables [Optional]`: Dictionary with a single allele count table that counts per guide and allele combination, what is the count per sample. - ## Using BEAN as Python module ``` import bean as be diff --git a/docs/commands/reporterscreen.md b/docs/commands/reporterscreen.md new file mode 100644 index 0000000..d939b5e --- /dev/null +++ b/docs/commands/reporterscreen.md @@ -0,0 +1,11 @@ +BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). + +ReporterScreen object structure + + * `.guides`: guide information provided in input (`gRNA_library.csv` in above example) + * `.samples`: sample information provided in input (`sample_list.csv` in above example) + * `.X`: Main guide count matrix, where row corresponds to each guide in `.guides` and columns correspond to samples in `.samples`. +Following attributes are included if matched reporter is provided and you chose to read edit/allele information from the reporter using `-r` option. + * `.X_bcmatch [Optional]`: Contains information about number of barcode-matched reads. Information about R2 barcode should be specified as `barcode` column in your `gRNA_library.csv` file. + * `.X_edits [Optional]`: If target position of each guide is specified as `target_pos` in input `gRNA_library.csv` file and `--match-target-position` option is provided, the result has the matrix with the number of target edit at the specified positions. + * `.allele_tables [Optional]`: Dictionary with a single allele count table that counts per guide and allele combination, what is the count per sample. \ No newline at end of file diff --git a/docs/index.rst b/docs/index.rst index d78bc9a..9f04032 100755 --- a/docs/index.rst +++ b/docs/index.rst @@ -16,7 +16,7 @@ Workflows input =================== -`bean` subcommands +API references =================== .. toctree:: :maxdepth: 3 @@ -27,6 +27,7 @@ Workflows Screen data structure =================== .. toctree:: + reporterscreen ReporterScreen_api ================== diff --git a/docs/reporterscreen.rst b/docs/reporterscreen.rst new file mode 100644 index 0000000..aa3c79d --- /dev/null +++ b/docs/reporterscreen.rst @@ -0,0 +1,3 @@ +ReporterScreen object +*********************** +.. mdinclude:: commands/reporterscreen.md From 620a85543d986702fdb9184b5a9c78d451360603 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 02:54:00 -0400 Subject: [PATCH 081/107] add data structure --- README.md | 6 +++--- 1 file changed, 3 insertions(+), 3 deletions(-) diff --git a/README.md b/README.md index dfe141c..ecf1a37 100755 --- a/README.md +++ b/README.md @@ -14,15 +14,15 @@ dag_bean_v2.svg -`bean` subcommands include the following: +`bean` subcommands include the following: Click on the [`links`]() to see the full documentation. 1. [`count`](https://pinellolab.github.io/crispr-bean/count.html), [`count-samples`](https://pinellolab.github.io/crispr-bean/count_samples.html): Base-editing-aware **mapping** of guide, optionally with reporter from `.fastq` files. * [`create-screen`](https://pinellolab.github.io/crispr-bean/create_screen.html) creates minimal ReporterScreen object from flat gRNA count file. Note that this way, allele counts are not included and many functionalities involving allele and edit counts are not supported. 2. [`profile`](https://pinellolab.github.io/crispr-bean/profile.html): Profile editing preferences of your editor. 3. [`qc`](https://pinellolab.github.io/crispr-bean/qc.html): Quality control report and filtering out / masking of aberrant sample and guides 4. [`filter`](https://pinellolab.github.io/crispr-bean/filter.html): Filter reporter alleles; essential for `tiling` mode that allows for all alleles generated from gRNA. 5. [`run`](https://pinellolab.github.io/crispr-bean/run.html): Quantify targeted variants' effect sizes from screen data. -* Screen data is saved as *ReporterScreen* object in the pipeline. -BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). See [Data Structure](#data-structure) section for more information. +* Screen data is saved as [`ReporterScreen` object](https://pinellolab.github.io/crispr-bean/reporterscreen.html) in the pipeline. +BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). ## Installation First install [PyTorch](https://pytorch.org/get-started/). Then download from PyPI: From 148957a4ae11dd124664f34f03c9fb8ad9e3f422 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 08:38:35 -0400 Subject: [PATCH 082/107] change images --- .github/workflows/documentation.yml | 3 +- README.md | 12 +- docs/commands/count.md | 2 +- docs/commands/filter.md | 2 +- docs/commands/profile.md | 2 +- imgs/cmd.rst | 5 + imgs/cmd_main.rst | 10 + imgs/library_design.svg | 1204 ++++++ imgs/summary.svg | 5452 +++++++++++++++++++++++++++ 9 files changed, 6681 insertions(+), 11 deletions(-) create mode 100755 imgs/cmd.rst create mode 100755 imgs/cmd_main.rst create mode 100644 imgs/library_design.svg create mode 100644 imgs/summary.svg diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index f36a502..e44207f 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -19,7 +19,8 @@ jobs: - name: Build Documentation working-directory: docs run: sphinx-build . _build - + - name: copy image files + run: cp -r imgs/ docs/_build/imgs - uses: actions/upload-pages-artifact@v3 with: name: github-pages diff --git a/README.md b/README.md index aeedb11..1d1d398 100755 --- a/README.md +++ b/README.md @@ -7,7 +7,7 @@ `bean` (**B**ase **E**diting reporter screens with guide **A**ctivity **N**ormalization) is an analysis toolkit for the pooled CRISPR reporter or sensor data. The reporter technique transfects cells with plasmid with not only sgRNA but with the **target sequence surrogate** which we call **reporter** or **sensor**. -Reporter construct +Reporter construct ## Overview `bean` supports end-to-end analysis of pooled sorting screens, with or without reporter. @@ -50,13 +50,11 @@ See the [documentation](https://pinellolab.github.io/crispr-bean/) for tutorials ### Library design: variant or tiling? The `bean filter` and `bean run` steps depend on the type of gRNA library design, where BEAN supports two modes of running. -1. `variant` library: Several gRNAs tile each of the targeted variants - Ex) - variant library design +variant library design -2. `tiling` library: gRNA densely tiles a long region (e.g. gene(s), exon(s), coding sequence(s)) - Ex) - tiling library design +1. `variant` library: Several gRNAs tile each of the targeted variants. Only the editing rate of the target variant is considered and the bystander effects are ignored. + +2. `tiling` library: gRNA densely tiles a long region (e.g. gene(s), exon(s), coding sequence(s)). Bystander edits are considered to obtain alleles with significant fractions. Edited alleles can be "translated" to output coding variants. ## Using BEAN as Python module ``` diff --git a/docs/commands/count.md b/docs/commands/count.md index 9dac49e..5f04b4c 100755 --- a/docs/commands/count.md +++ b/docs/commands/count.md @@ -18,7 +18,7 @@ bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r ``` By default, `bean count[-samples]` assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure. - Read structuren + Read structuren See full detail [below](#full-parameters). diff --git a/docs/commands/filter.md b/docs/commands/filter.md index 2809251..53d6ec1 100755 --- a/docs/commands/filter.md +++ b/docs/commands/filter.md @@ -16,7 +16,7 @@ You may want to adjust the flitering parameters to obtain optimal balance betwee # Translating alleles If you want to obtain **amino acid level variant** for coding sequence tiling screens, provide coding sequence positions which variants occuring within the coding sequence will be translated. *This is optional, but **highly recommended** to increase per-(coding)variant support.* -Allele translation +Allele translation ```bash diff --git a/docs/commands/profile.md b/docs/commands/profile.md index 7886ff5..5f1df8c 100755 --- a/docs/commands/profile.md +++ b/docs/commands/profile.md @@ -5,4 +5,4 @@ bean profile my_sorting_screen.h5ad -o output_prefix `# Prefix for editing profi # Output Above command produces `prefix_editing_preference.[html,ipynb]` as editing preferences ([see example](../notebooks/profile_editing_preference.ipynb)). -Allele translation \ No newline at end of file +Allele translation \ No newline at end of file diff --git a/imgs/cmd.rst b/imgs/cmd.rst new file mode 100755 index 0000000..ee52397 --- /dev/null +++ b/imgs/cmd.rst @@ -0,0 +1,5 @@ +.. toctree:: + :maxdepth: 2 + + cmd_main + \ No newline at end of file diff --git a/imgs/cmd_main.rst b/imgs/cmd_main.rst new file mode 100755 index 0000000..b7598db --- /dev/null +++ b/imgs/cmd_main.rst @@ -0,0 +1,10 @@ +Command line utilities +********************** +.. .. argparse:: +.. :filename: ../bean/cli/execute.py +.. :func: get_parser +.. :prog: execute + + subcommand + Here we add a reference to subcommand, to simplify navigation. + \ No newline at end of file diff --git a/imgs/library_design.svg b/imgs/library_design.svg new file mode 100644 index 0000000..1b00411 --- /dev/null +++ b/imgs/library_design.svg @@ -0,0 +1,1204 @@ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + diff --git a/imgs/summary.svg b/imgs/summary.svg new file mode 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+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + From 8ea81a2fae9e012e3fd7a1c72cb5b03b6f5dd749 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 10:56:27 -0400 Subject: [PATCH 083/107] update docs --- .github/workflows/documentation.yml | 2 +- README.md | 10 +- bean/model/survival_model.py | 18 +- bean/preprocessing/data_class.py | 14 + {imgs => docs/assets}/anbe.svg | 0 {imgs => docs/assets}/bean.gif | Bin {imgs => docs/assets}/bean_title2.svg | 0 {imgs => docs/assets}/cmd.rst | 0 {imgs => docs/assets}/cmd_main.rst | 0 {imgs => docs/assets}/dag_bean_v2.png | Bin docs/assets/dag_bean_v2.svg | 263 ++++ {imgs => docs/assets}/data_structure_v2.png | Bin docs/assets/library_design.png | Bin 0 -> 151586 bytes {imgs => docs/assets}/model_output.png | Bin {imgs => docs/assets}/output_20_2.png | Bin {imgs => docs/assets}/output_34_1.png | Bin {imgs => docs/assets}/profile_output.png | Bin {imgs => docs/assets}/qc_output.png | Bin {imgs => docs/assets}/reporter.jpg | Bin {imgs => docs/assets}/reporter_construct.png | Bin {imgs => docs/assets}/reporter_construct.svg | 0 {imgs => docs/assets}/reporter_screen.svg | 0 {imgs => docs/assets}/screendata.svg | 0 {imgs => docs/assets}/sequence_struct.png | Bin {imgs => docs/assets}/sorting_bins@8x.png | Bin {imgs => docs/assets}/summary.svg | 0 {imgs => docs/assets}/tiling.png | Bin {imgs => docs/assets}/translation.png | Bin {imgs => docs/assets}/variant.png | Bin docs/cds.rst | 2 +- docs/commands/count.md | 31 - docs/commands/create-screen.md | 9 - docs/commands/filter.md | 37 - docs/commands/input.md | 37 - docs/commands/profile.md | 8 - docs/commands/qc.md | 94 -- docs/commands/reporterscreen.md | 11 - docs/commands/run.md | 64 - docs/count.rst | 2 +- docs/create_screen.rst | 2 +- docs/filter.rst | 2 +- docs/gwas.rst | 2 +- docs/profile.rst | 2 +- docs/qc.rst | 2 +- docs/reporterscreen.rst | 2 +- docs/run.rst | 2 +- docs/tutorials/ldl_cds.md | 96 +- docs/tutorials/ldl_var.md | 43 +- imgs/bean_title.svg | 1 - imgs/dag_bean_v2.svg | 263 ---- imgs/library_design.svg | 1204 ------------------ notebooks/sample_quality_report.ipynb | 27 +- tests/test_qc.py | 13 + tests/test_run.py | 52 + 54 files changed, 448 insertions(+), 1867 deletions(-) rename {imgs => docs/assets}/anbe.svg (100%) rename {imgs => docs/assets}/bean.gif (100%) rename {imgs => docs/assets}/bean_title2.svg (100%) rename {imgs => docs/assets}/cmd.rst (100%) rename {imgs => docs/assets}/cmd_main.rst (100%) rename {imgs => docs/assets}/dag_bean_v2.png (100%) create mode 100644 docs/assets/dag_bean_v2.svg rename {imgs => docs/assets}/data_structure_v2.png (100%) create mode 100644 docs/assets/library_design.png rename {imgs => docs/assets}/model_output.png (100%) rename {imgs => docs/assets}/output_20_2.png (100%) rename {imgs => docs/assets}/output_34_1.png (100%) rename {imgs => docs/assets}/profile_output.png (100%) rename {imgs => docs/assets}/qc_output.png (100%) rename {imgs => docs/assets}/reporter.jpg (100%) rename {imgs => docs/assets}/reporter_construct.png (100%) rename {imgs => docs/assets}/reporter_construct.svg (100%) rename {imgs => docs/assets}/reporter_screen.svg (100%) rename {imgs => docs/assets}/screendata.svg (100%) rename {imgs => docs/assets}/sequence_struct.png (100%) rename {imgs => docs/assets}/sorting_bins@8x.png (100%) rename {imgs => docs/assets}/summary.svg (100%) rename {imgs => docs/assets}/tiling.png (100%) rename {imgs => docs/assets}/translation.png (100%) rename {imgs => docs/assets}/variant.png (100%) delete mode 100755 docs/commands/count.md delete mode 100755 docs/commands/create-screen.md delete mode 100755 docs/commands/filter.md delete mode 100755 docs/commands/input.md delete mode 100755 docs/commands/profile.md delete mode 100755 docs/commands/qc.md delete mode 100644 docs/commands/reporterscreen.md delete mode 100755 docs/commands/run.md delete mode 100755 imgs/bean_title.svg delete mode 100644 imgs/dag_bean_v2.svg delete mode 100644 imgs/library_design.svg diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index e44207f..53b8dfc 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -20,7 +20,7 @@ jobs: working-directory: docs run: sphinx-build . _build - name: copy image files - run: cp -r imgs/ docs/_build/imgs + run: cp -r docs/commands/imgs/ docs/_build/imgs - uses: actions/upload-pages-artifact@v3 with: name: github-pages diff --git a/README.md b/README.md index 1d1d398..376b851 100755 --- a/README.md +++ b/README.md @@ -7,12 +7,12 @@ `bean` (**B**ase **E**diting reporter screens with guide **A**ctivity **N**ormalization) is an analysis toolkit for the pooled CRISPR reporter or sensor data. The reporter technique transfects cells with plasmid with not only sgRNA but with the **target sequence surrogate** which we call **reporter** or **sensor**. -Reporter construct +Reporter construct ## Overview `bean` supports end-to-end analysis of pooled sorting screens, with or without reporter. -dag_bean_v2.svg +dag_bean_v2.svg `bean` subcommands include the following: Click on the [`links`]() to see the full documentation. 1. [`count`](https://pinellolab.github.io/crispr-bean/count.html), [`count-samples`](https://pinellolab.github.io/crispr-bean/count_samples.html): Base-editing-aware **mapping** of guide, optionally with reporter from `.fastq` files. @@ -42,15 +42,15 @@ See the [documentation](https://pinellolab.github.io/crispr-bean/) for tutorials ## Tutorials | [Library design](#pipeline-run-options-by-library-design) | Selection | Reporter | Tutorial link | |---------------| -------------- | --------- | -------- | -| GWAS variant library | FACS sorting | Yes/No | [GWAS variant screen](docs/tutorials/ldl_var.md) -| Coding sequence tiling libarary | FACS sorting | Yes/No | [Coding sequence tiling screen](docs/tutorials/ldl_cds.md) +| GWAS variant library | FACS sorting | Yes/No | [GWAS variant screen](https://pinellolab.github.io/crispr-bean/gwas) +| Coding sequence tiling libarary | FACS sorting | Yes/No | [Coding sequence tiling screen](https://pinellolab.github.io/crispr-bean/cds) | GWAS variant library | Survival / Proliferation | Yes/No | TKO simulated (Coming soon!) | Coding sequence tiling libarary | Survival / Proliferation | Yes/No | TKO simulated, tiling (Coming soon!) ### Library design: variant or tiling? The `bean filter` and `bean run` steps depend on the type of gRNA library design, where BEAN supports two modes of running. -variant library design +variant library design 1. `variant` library: Several gRNAs tile each of the targeted variants. Only the editing rate of the target variant is considered and the bystander effects are ignored. diff --git a/bean/model/survival_model.py b/bean/model/survival_model.py index ddfb362..88d7d10 100755 --- a/bean/model/survival_model.py +++ b/bean/model/survival_model.py @@ -151,15 +151,17 @@ def ControlNormalModel(data, mask_thres=10, use_bcmatch=True): with replicate_plate2: with pyro.plate("guide_plate3", data.n_guides, dim=-1): a = get_alpha(expected_guide_p, data.size_factor, data.sample_mask, data.a0) - - assert ( - data.X.shape - == data.X_bcmatch.shape - == ( + assert data.X.shape == ( + data.n_reps, + data.n_condits, + data.n_guides, + ), ( + data.X.shape, + ( data.n_reps, data.n_condits, data.n_guides, - ) + ), ) with poutine.mask( mask=torch.logical_and( @@ -307,7 +309,7 @@ def MixtureNormalModel( # assert a.shape == a_bcmatch.shape == (data.n_reps, data.n_guides, data.n_condits) assert ( data.X.shape - == data.X_bcmatch.shape + == data.X_bcmatch_masked.shape == ( data.n_reps, data.n_condits, @@ -563,7 +565,7 @@ def MultiMixtureNormalModel( # assert a.shape == a_bcmatch.shape == (data.n_reps, data.n_guides, data.n_condits) assert ( data.X.shape - == data.X_bcmatch.shape + == data.X_bcmatch_masked.shape == ( data.n_reps, data.n_condits, diff --git a/bean/preprocessing/data_class.py b/bean/preprocessing/data_class.py index 1418b39..0c37fb2 100755 --- a/bean/preprocessing/data_class.py +++ b/bean/preprocessing/data_class.py @@ -1296,6 +1296,20 @@ def __init__( screen, ) + def __getitem__(self, guide_idx): + ndata = super().__getitem__(guide_idx) + if hasattr(ndata, "X_bcmatch"): + ndata.X_bcmatch = ndata.X_bcmatch[:, :, guide_idx] + if hasattr(ndata, "X_bcmatch_masked"): + ndata.X_bcmatch_masked = ndata.X_bcmatch_masked[:, :, guide_idx] + if hasattr(ndata, "X_bcmatch_control"): + ndata.X_bcmatch_control = ndata.X_bcmatch_control[:, :, guide_idx] + if hasattr(ndata, "X_bcmatch_control_masked"): + ndata.X_bcmatch_control_masked = ndata.X_bcmatch_control_masked[ + :, :, guide_idx + ] + return ndata + def set_bcmatch(self, screen): screen.samples["size_factor_bcmatch"] = self.get_size_factor( screen.layers["X_bcmatch"] diff --git a/imgs/anbe.svg b/docs/assets/anbe.svg similarity index 100% rename from imgs/anbe.svg rename to docs/assets/anbe.svg diff --git a/imgs/bean.gif b/docs/assets/bean.gif similarity index 100% rename from imgs/bean.gif rename to docs/assets/bean.gif diff --git a/imgs/bean_title2.svg b/docs/assets/bean_title2.svg similarity index 100% rename from imgs/bean_title2.svg rename to docs/assets/bean_title2.svg diff --git a/imgs/cmd.rst b/docs/assets/cmd.rst similarity index 100% rename from imgs/cmd.rst rename to docs/assets/cmd.rst diff --git a/imgs/cmd_main.rst b/docs/assets/cmd_main.rst similarity index 100% rename from imgs/cmd_main.rst rename to docs/assets/cmd_main.rst diff --git a/imgs/dag_bean_v2.png b/docs/assets/dag_bean_v2.png similarity index 100% rename from imgs/dag_bean_v2.png rename to docs/assets/dag_bean_v2.png diff --git a/docs/assets/dag_bean_v2.svg b/docs/assets/dag_bean_v2.svg new file mode 100644 index 0000000..af61332 --- /dev/null +++ b/docs/assets/dag_bean_v2.svg @@ -0,0 +1,263 @@ + + + + + + + + + + + + + + + + + run + +qc +filter + +count-samples +R1.fastq + + + +R2.fastq +Coding regions oftargeted genes + + + + + + + + + + + Input + + + + Input(Optional) + +Legend + + +AnnData-based ReporterScreen object + + + + + + + + + + + + + + + + + guides + + samples + + + gRNA counts + + + + + + + + + + + gRNA + Allele + + samples + + + Allele counts + + + + + + + + + + + + + + gRNA1 + Allele1 + Allele2 + ... + + + + + + + + + + + + + + + + + + + + + + + + + +qc_report.[html,ipynb] + + + + + + + + + + +filter_log.txtfiltered_allele_stats.pdf + + + + + + + + + + +bean_element_result.csvbean_sgRNA_result.csv + + + + + Output + + + + + + + + + + + + +profile + + + + + + + + + + +editing_preferences.[html,ipynb] + +sample_list.csv + +gRNA_info.csv + + + + + + + + + + + + + diff --git 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b/docs/assets/tiling.png similarity index 100% rename from imgs/tiling.png rename to docs/assets/tiling.png diff --git a/imgs/translation.png b/docs/assets/translation.png similarity index 100% rename from imgs/translation.png rename to docs/assets/translation.png diff --git a/imgs/variant.png b/docs/assets/variant.png similarity index 100% rename from imgs/variant.png rename to docs/assets/variant.png diff --git a/docs/cds.rst b/docs/cds.rst index 5dabfde..2d15d26 100755 --- a/docs/cds.rst +++ b/docs/cds.rst @@ -1,5 +1,5 @@ Coding sequence tiling library *********************** -.. mdinclude:: tutorials/ldl_cds.md +.. mdinclude:: commands/ldl_cds.md See :ref:`subcommands` for the full details. diff --git a/docs/commands/count.md b/docs/commands/count.md deleted file mode 100755 index 5f04b4c..0000000 --- a/docs/commands/count.md +++ /dev/null @@ -1,31 +0,0 @@ -# `bean count[-samples]`: Count (reporter) screen data -`bean count-samples` (or `bean count` for a single sample) maps guide into guide counts, **allowing for base transition in spacer sequence**. When the matched reporter information is provided, it can count the **target site edits** and **alleles produced by each guide**. Mapping is efficiently done based on [CRISPResso2](https://github.com/pinellolab/CRISPResso2) modified for base-edit-aware mapping. - - - -```python -bean count-samples \ - --input sample_list.csv `# sample with lines 'R1_filepath,R2_filepath,sample_name\n'` \ - -b A `# base that is being edited (A/G)` \ - -f sgRNA_info_table.csv `# sgRNA information` \ - -o . `# output directory` \ - -r `# read edit/allele information from reporter` \ - -t 12 `# number of threads` \ - --name my_sorting_screen `# name of this sample run` \ -``` -```python -bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r -``` -By default, `bean count[-samples]` assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure. - - Read structuren - -See full detail [below](#full-parameters). - -# Input file format -See :ref:`input` for input file formats. - -# Output file format -`count` or `count-samples` produces `.h5ad` and `.xlsx` file with guide and per-guide allele counts. -* `.h5ad`: This output file follows annotated matrix format compatible with `AnnData` and is based on `Screen` object in [purturb_tools](https://github.com/pinellolab/perturb-tools). See [Data Structure](#data-structure) section for more information. -* `.xlsx`: This output file contains `.guides`, `.samples`, `.X[_bcmatch,_edits]`. (`allele_tables` are often too large to write into an Excel!) diff --git a/docs/commands/create-screen.md b/docs/commands/create-screen.md deleted file mode 100755 index d9200b7..0000000 --- a/docs/commands/create-screen.md +++ /dev/null @@ -1,9 +0,0 @@ -# `bean create-screen`: Create ReporterScreen object from flat files -```bash -bean create-screen gRNA_library.csv sample_list.csv gRNA_counts_table.csv -``` -## Input - * gRNA_library.csv - * sample_list.csv - * gRNA_counts_table.csv: Table with gRNA ID in the first column and sample IDs as the column names (first row) -`gRNA_library.csv` and `sample_list.csv` should be formatted as :ref:`input`. \ No newline at end of file diff --git a/docs/commands/filter.md b/docs/commands/filter.md deleted file mode 100755 index 53d6ec1..0000000 --- a/docs/commands/filter.md +++ /dev/null @@ -1,37 +0,0 @@ -# `filter`: Filtering (and optionally translating) alleles -As `tiling` mode of `bean run` accounts for any robustly observed alleles, `bean filter` filters for such alleles. -```bash -bean filter my_sorting_screen_masked.h5ad \ --o my_sorting_screen_filtered.h5ad `# Output file path` \ -``` - -# Output -Above command produces -* `my_sorting_screen_filtered.h5ad` with filtered alleles stored in `.uns`, -* `my_sorting_screen_filtered.filtered_allele_stats.pdf`, and `my_sorting_screen_filtered.filter_log.txt` that report allele count stats in each filtering step. - -You may want to adjust the flitering parameters to obtain optimal balance between # guides per variant & # variants that are scored. See example outputs of filtering step [here](docs/example_filtering_output/). - - -# Translating alleles -If you want to obtain **amino acid level variant** for coding sequence tiling screens, provide coding sequence positions which variants occuring within the coding sequence will be translated. *This is optional, but **highly recommended** to increase per-(coding)variant support.* - -Allele translation - - -```bash -bean filter my_sorting_screen.h5ad \ --o my_sorting_screen_masked.h5ad \ ---translate `# Translate coding variants` \ -[ --translate-gene-name GENE_SYMBOL OR - --translate-genes-list path_to_gene_names_file.txt OR - --translate-fasta gene_exon.fa, OR - --translate-fastas-csv gene_exon_fas.csv] -``` -* When library covers a single gene, do either of the following: - 1. Feed `--translate-gene-name GENE_SYMBOL` if your `genomic_pos` column of `sgRNA_info_tbl` is compatible with [MANE transcript](https://useast.ensembl.org/info/genome/genebuild/mane.html)'s reference genome. (Per 10/23/2023, GRCh38). This will automatically load the exon positions based on MANE transcript annotation. - 2. To use your custom coding sequence and exon positions, feed `--translate-fasta gene_exon.fa` argument where `gene_exon.fa` is the FASTA file with entries of exons. [See full details here](docs/exon_fa_format.md). -* When library covers multiple genes, do either of the following: - 1. Feed `--translate-genes-list path_to_gene_names_file.txt` where `path_to_gene_names_file.txt` is file with one gene symbol per line. - 2. Feed `--translate-fastas-csv gene_exon_fas.csv` where `gene_exon_fas.csv` is the csv file with lines `gene_id,gene_exon_fasta_path` without header. Each FASTA file in `gene_exon_fasta_path` is formatted [as the single-gene FASTA file](docs/exon_fa_format.md). -* Translation will keep the variants outside the coding sequence as nucleotide-level variants, while aggregating variants leading to the same coding sequence variants. diff --git a/docs/commands/input.md b/docs/commands/input.md deleted file mode 100755 index 9fa1de9..0000000 --- a/docs/commands/input.md +++ /dev/null @@ -1,37 +0,0 @@ -This document describes the input files of :ref:`count_samples`. -## sgRNA_info_table.csv -File should contain following columns. -* `name`: gRNA ID column -* `sequence`: gRNA sequence -* `barcode`: R2 barcode to help match reporter to gRNA, written in the sense direction (as in R1) -* In order to use accessibility in the [variant effect quantification](#bean-run-quantify-variant-effects), provide accessibility information in one of two options. (For non-targeting guides, provide NA values (empty cell).) - * Option 1: `chrom` & `genomic_pos`: Chromosome (ex. `chr19`) and genomic position of guide sequence. You will have to provide the path to the bigwig file with matching reference version in `bean run`. - * Option 2: `accessibility_signal`: ATAC-seq signal value of the target loci of each guide. -* For variant library (gRNAs are designed to target specific variants and ignores bystander edits) - * `target`: This column denotes which target variant/element of each gRNA. This is not used in `bean count[-samples]` but required to run `bean run` in later steps. - * `target_group`: If negative/positive control gRNA will be considered in `bean qc` and/or `bean run`, specify as "NegCtrl"/"PosCtrl" in this column. - * `target_pos`: If `--match_target_pos` flag is used, input file needs `target_pos` which specifies 0-based relative position of targeted base within Reporter sequence. -* For tiling library (gRNAs tile coding / noncoding sequences) - * `strand`: Specifies gRNA strand information relative to the reference genome. - * `chrom`: Chromosome of gRNA targeted locus. - * `start_pos`: gRNA starting position in the genome. Required when you provide `strand` column. Should specify the smaller coordinate value among start and end position regardless of gRNA strandedness. - -Also see examples for [variant library](tests/data/test_guide_info.csv) and [tiling library](tests/data/test_guide_info_tiling.csv). - -## sample_list.csv -File should contain following columns with header. -* `R1_filepath`: Path to read 1 `.fastq[.gz]` file -* `R2_filepath`: Path to read 1 `.fastq[.gz]` file -* `sample_id`: ID of sequencing sample -* `replicate`: Replicate # of this sample (Should NOT contain `.`) -* `condition`: Name of the sorting bin (ex. `top`, `bot`), or label of timepoint (ex. `D5`, `D18`) - -For FACS sorting screens: -* `upper_quantile`: FACS sorting upper quantile -* `lower_quantile`: FACS sorting lower quantile - -For proliferation / survival screens: -* `time`: Numeric time following the base editing of each sample. - - -Also see examples for [FACS sorting screen](tests/data/sample_list.csv). \ No newline at end of file diff --git a/docs/commands/profile.md b/docs/commands/profile.md deleted file mode 100755 index 5f1df8c..0000000 --- a/docs/commands/profile.md +++ /dev/null @@ -1,8 +0,0 @@ -# `bean profile`: Profile editing patterns -```bash -bean profile my_sorting_screen.h5ad -o output_prefix `# Prefix for editing profile report` -``` -# Output -Above command produces `prefix_editing_preference.[html,ipynb]` as editing preferences ([see example](../notebooks/profile_editing_preference.ipynb)). - -Allele translation \ No newline at end of file diff --git a/docs/commands/qc.md b/docs/commands/qc.md deleted file mode 100755 index d2ac1d5..0000000 --- a/docs/commands/qc.md +++ /dev/null @@ -1,94 +0,0 @@ -# `bean qc`: QC of reporter screen data -```bash -bean qc \ - my_sorting_screen.h5ad `# Input ReporterScreen .h5ad file path` \ - -o my_sorting_screen_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r qc_report_my_sorting_screen `# Prefix for QC report` \ - --ctrl-cond presort `# "condition" column in the control sample before selection. Mean gRNA editing rates in these samples are reported. ` \ -# Inspect the output qc_report_my_sorting_screen.html to tweak QC threshold - -bean qc \ - my_sorting_screen.h5ad \ - -o my_sorting_screen_masked.h5ad \ - -r qc_report_my_sorting_screen \ - #[--count-correlation-thres 0.7 ...]\ - -b -``` - -`bean qc` supports following quality control and masks samples with low quality. Specifically: - -Allele translation - -* Plots guide coverage and the uniformity of coverage -* Guide count correlation between samples -* Log fold change correlation when positive controls are provided -* Plots editing rate distribution -* Identify samples with low guide coverage/guide count correlation/editing rate and mask the sample in `bdata.samples.mask` -* Identify outlier guides to filter out - -# Output -Above command produces -* `my_sorting_screen_masked.h5ad` without problematic replicate and guides and with sample masks, and -* `qc_report_my_sorting_screen.[html,ipynb]` as QC report. -##### Optional arguments: -* `-o OUT_SCREEN_PATH`, `--out-screen-path OUT_SCREEN_PATH` - Path where quality-filtered ReporterScreen object to be written to -* `-r OUT_REPORT_PREFIX`, `--out-report-prefix OUT_REPORT_PREFIX` - Output prefix of qc report (prefix.html, prefix.ipynb) - -##### QC thresholds: -* `--count-correlation-thres COUNT_CORRELATION_THRES` - Correlation threshold to mask out. -* `--edit-rate-thres EDIT_RATE_THRES` - Mean editing rate threshold per sample to mask out. -* `--lfc-thres LFC_THRES` - Positive guides' correlation threshold to filter out. - -##### Run options: -* `-b`, `--remove-bad-replicates` - Remove replicates with at least two of its samples meet the QC threshold (bean run does not support having only one sorting bin sample for a replicate). -* `-i`, `--ignore-missing-samples` - If the flag is not provided, if the ReporterScreen object does not contain all condiitons for - each replicate, make fake empty samples. If the flag is provided, don't add dummy samples. -* `--no-editing` Ignore QC about editing. Can be used for QC of other editing modalities. -* `--dont-recalculate-edits` - When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from - ReporterScreen.uns['allele_count']. - -##### Input `.h5ad` formatting: -Note that these arguements will change the way the QC metrics are calculated for guides, samples, or replicates. -* `--tiling TILING` Specify that the guide library is tiling library without 'n guides per target' design -* `--replicate-label REPLICATE_LABEL` - Label of column in `bdata.samples` that describes replicate ID. -* `--sample-covariates SAMPLE_COVARIATES` - Comma-separated list of column names in `bdata.samples` that describes non-selective - experimental condition. (drug treatment, etc.) -* `--condition-label CONDITION_LABEL` - Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time, - etc.) -###### Editing rate calculation - * `--control-condition CTRL_COND` - Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate - to be calculated. Default is `None`, which considers all samples. - * `--rel-pos-is-reporter` - Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If - `False`, those are relative to spacer position. - Editing rate is calculated with following parameters in - * Variant screens: - * `--target-pos-col TARGET_POS_COL` - Target position column in `bdata.guides` specifying target edit position in reporter - * tiling screens: - * `--edit-start-pos EDIT_START_POS` - Edit start position to quantify editing rate on, 0-based inclusive. - * `--edit-end-pos EDIT_END_POS` - Edit end position to quantify editing rate on, 0-based exclusive. -###### LFC of positive controls - * `--posctrl-col POSCTRL_COL` - Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all - gRNAs, feed empty string ''. - * `--posctrl-val POSCTRL_VAL` - Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the - positive control in calculating log fold change. - * `--lfc-conds LFC_CONDS` - Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated - between, delimited by comma \ No newline at end of file diff --git a/docs/commands/reporterscreen.md b/docs/commands/reporterscreen.md deleted file mode 100644 index d939b5e..0000000 --- a/docs/commands/reporterscreen.md +++ /dev/null @@ -1,11 +0,0 @@ -BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). - -ReporterScreen object structure - - * `.guides`: guide information provided in input (`gRNA_library.csv` in above example) - * `.samples`: sample information provided in input (`sample_list.csv` in above example) - * `.X`: Main guide count matrix, where row corresponds to each guide in `.guides` and columns correspond to samples in `.samples`. -Following attributes are included if matched reporter is provided and you chose to read edit/allele information from the reporter using `-r` option. - * `.X_bcmatch [Optional]`: Contains information about number of barcode-matched reads. Information about R2 barcode should be specified as `barcode` column in your `gRNA_library.csv` file. - * `.X_edits [Optional]`: If target position of each guide is specified as `target_pos` in input `gRNA_library.csv` file and `--match-target-position` option is provided, the result has the matrix with the number of target edit at the specified positions. - * `.allele_tables [Optional]`: Dictionary with a single allele count table that counts per guide and allele combination, what is the count per sample. \ No newline at end of file diff --git a/docs/commands/run.md b/docs/commands/run.md deleted file mode 100755 index 3274276..0000000 --- a/docs/commands/run.md +++ /dev/null @@ -1,64 +0,0 @@ -# `bean run`: Quantify variant effects -BEAN uses Bayesian network to incorporate gRNA editing outcome to provide posterior estimate of variant phenotype. The Bayesian network reflects data generation process. Briefly, -1. Cellular phenotype (either for cells are sorted upon for sorting screen, or log(proliferation rate)) is modeled as the Gaussian mixture distribution of wild-type phenotype and variant phenotype. -2. The weight of the mixture components are inferred from the reporter editing outcome and the chromatin accessibility of the loci. -3. Cells with each gRNA, formulated as the mixture distribution, is sorted by the phenotypic quantile to produce the gRNA counts. - -For the full detail, see the method section of the [BEAN manuscript](https://www.medrxiv.org/content/10.1101/2023.09.08.23295253v1). - -model - -

    - -# Usage example -```bash -bean run sorting[survival] variant[tiling] my_sorting_screen_filtered.h5ad \ -[--uniform-edit, --scale-by-acc [--acc-bw-path accessibility_signal.bw, --acc-col accessibility]] \ --o output_prefix/ \ ---fit-negctrl -``` -See full list of parameters [below](#full-parameters). - - -# Input -`my_sorting_screen_filtered.h5ad` can be produced by one of the following: -1. [`bean count-samples`]((#bean-count-samples-count-reporter-screen-data)) when you have raw `.fastq` file -2. (Limited to `bean run variant` mode) `bean create-screen` when you have flat `.csv` tables of gRNA metadata table, sample metadata table, gRNA counts table (# guides x # samples), and optionally # edits table. - ```bash - bean create-screen gRNA_info_table.csv sample_info_table.csv gRNA_counts_table.csv \ - [--edits edit_counts_table.csv -o output.h5ad] - ``` - * `gRNA_info_table.csv` should have following columns. - * `name`: gRNA ID column - * `target`: This column denotes which target variant/element of each gRNA. - * `target_group [Optional]`: If negative control gRNA will be used, specify as "NegCtrl" in this column. - * `sample_info_table.csv` should have following columns. - * `sample_id`: ID of sequencing sample - * `replicate`: Replicate # of this sample - * `bin`: Name of the sorting bin - * `upper_quantile`: FACS sorting upper quantile - * `lower_quantile`: FACS sorting lower quantile - * `gRNA_counts_table.csv` should be formatted as follows. - * Columns include one of `sample_id` columns in `sample_info_table.csv` file. - * 1st row (row index) follows `name` (gRNA ID) in `gRNA_info_table.csv` file. -3. You can manually create the `AnnData` object with more annotations including allele counts: see [API tutorial](#using-bean-as-python-module) for full detail. - - -# Output -model - -Above command produces -* `output_prefix/bean_element_result.[model_type].csv` with following columns: - * Estimated variant effect sizes - * `mu` (Effect size): Mean of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. - * `mu_sd`: Mean of variant phenotype `mu` is modeled as normal distribution. The column shows fitted standard deviation of `mu` that quantify the uncertainty of the variant effect. - * `mu_z`: z-score of `mu` - * `sd`: Standard deviation of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. - * `CI[0.025`, `0.975]`: Credible interval of `mu` - * When negative control is provided, above columns with `_adj` suffix are provided, which are the corresponding values adjusted for negative control. - * Metrics on per-variant evidence provided in input (provided in `tiling` mode) - * `effective_edit_rate`: Sum of per-variant editing rates over all alleles observed in the input. Allele-level editing rate is divided by the number of variants observed in the allele prior to summing up. - * `n_guides`: # of guides covering the variant. - * `n_coocc`: # of cooccurring variants with a given variant in any alleles observed in the input. -* `output_prefix/bean_sgRNA_result.[model_type].csv`: - * `edit_rate`: Estimated editing rate at the target loci. diff --git a/docs/count.rst b/docs/count.rst index 206dbd2..fdbe236 100755 --- a/docs/count.rst +++ b/docs/count.rst @@ -1,6 +1,6 @@ `bean count` *********************** -.. mdinclude:: commands/count.md +.. mdinclude:: count.md Full parameters ================== diff --git a/docs/create_screen.rst b/docs/create_screen.rst index 6ffa9bf..ee36bfc 100755 --- a/docs/create_screen.rst +++ b/docs/create_screen.rst @@ -1,6 +1,6 @@ `bean create-screen` *********************** -.. mdinclude:: commands/create-screen.md +.. mdinclude:: create-screen.md Full parameters ================== diff --git a/docs/filter.rst b/docs/filter.rst index ce485ab..e0450cf 100755 --- a/docs/filter.rst +++ b/docs/filter.rst @@ -1,7 +1,7 @@ .. _filter: `bean filter` *********************** -.. mdinclude:: commands/filter.md +.. mdinclude:: filter.md Full parameters ================== diff --git a/docs/gwas.rst b/docs/gwas.rst index e407ad9..09d76fa 100755 --- a/docs/gwas.rst +++ b/docs/gwas.rst @@ -1,5 +1,5 @@ GWAS variant library *********************** -.. mdinclude:: tutorials/ldl_var.md +.. mdinclude:: ldl_var.md See :ref:`subcommands` for the full details. diff --git a/docs/profile.rst b/docs/profile.rst index 3ee525f..7316d9e 100755 --- a/docs/profile.rst +++ b/docs/profile.rst @@ -1,6 +1,6 @@ `bean profile` *********************** -.. mdinclude:: commands/profile.md +.. mdinclude:: profile.md Full parameters ================== diff --git a/docs/qc.rst b/docs/qc.rst index 604e77a..8cb0c7a 100755 --- a/docs/qc.rst +++ b/docs/qc.rst @@ -1,7 +1,7 @@ .. _qc: `bean qc` *********************** -.. mdinclude:: commands/qc.md +.. mdinclude:: qc.md Full parameters ================== diff --git a/docs/reporterscreen.rst b/docs/reporterscreen.rst index aa3c79d..dcd0a00 100644 --- a/docs/reporterscreen.rst +++ b/docs/reporterscreen.rst @@ -1,3 +1,3 @@ ReporterScreen object *********************** -.. mdinclude:: commands/reporterscreen.md +.. mdinclude:: reporterscreen.md diff --git a/docs/run.rst b/docs/run.rst index a6dc1fc..8378dbe 100755 --- a/docs/run.rst +++ b/docs/run.rst @@ -1,7 +1,7 @@ .. _run: `bean run` *********************** -.. mdinclude:: commands/run.md +.. mdinclude:: run.md Full parameters ================== diff --git a/docs/tutorials/ldl_cds.md b/docs/tutorials/ldl_cds.md index ba78a00..e2d5ce0 100755 --- a/docs/tutorials/ldl_cds.md +++ b/docs/tutorials/ldl_cds.md @@ -4,11 +4,11 @@ Tiling screen that tiles gRNA densely across locus or multiple loci, selected ba - + - +
    Library designTiling (gRNAs tile each locus densely)
    tiling library design     		Tiling (gRNAs tile each locus densely)
    tiling library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design    		Cells are sorted based on FACS signal quantiles
    variant library design
    @@ -17,35 +17,36 @@ Tiling screen that tiles gRNA densely across locus or multiple loci, selected ba ## Example workflow ```bash screen_id=my_sorting_tiling_screen +working_dir=my_workdir # 1. Count gRNA & reporter -bean-count-samples \ ---input tests/data/sample_list_tiling.csv `# Contains fastq file path; see test file for example.`\ +bean count-samples \ +--input ${working_dir}/sample_list_tiling.csv `# Contains fastq file path; see test file for example.`\ -b A `# Base A is edited (into G)` \ --f tests/data/test_guide_info_tiling_chrom.csv `# Contains gRNA metadata; see test file for example.`\ --o ./ `# Output directory` \ +-f ${working_dir}/test_guide_info_tiling_chrom.csv `# Contains gRNA metadata; see test file for example.`\ +-o $working_dir `# Output directory` \ -r `# Quantify reporter edits` \ -n ${screen_id} `# ID of the screen` \ --tiling # 2. QC samples & guides -bean-qc \ - bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ - -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r qc_report_${screen_id} `# Prefix for QC report` \ +bean qc \ + ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ # 3. Filter & translate alleles -bean-filter ./bean_count_${screen_id}_masked.h5ad \ --o ./bean_count_${screen_id}_alleleFiltered \ ---filter-target-basechange `# Filter based on intended base changes. If -b A was provided in bean-count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\ +bean filter ${working_dir}/bean_count_${screen_id}_masked.h5ad \ +-o ${working_dir}/bean_count_${screen_id}_alleleFiltered \ +--filter-target-basechange `# Filter based on intended base changes. If -b A was provided in bean count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\ --filter-window --edit-start-pos 0 --edit-end-pos 19 `# Filter based on editing window in spacer position within reporter.`\ --filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \ ---translate --translate-genes-list tests/data/gene_symbols.txt +--translate --translate-genes-list ${working_dir}/gene_symbols.txt # 4. Quantify variant effect -bean-run sorting tiling \ - ./bean_count_${screen_id}_alleleFiltered.h5ad \ - -o tests/test_res/var/ \ +bean run sorting tiling \ + ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ + -o $working_dir \ --fit-negctrl \ --scale-by-acc \ --accessibility-col accessibility @@ -55,12 +56,13 @@ See more details below. ## 1. Count gRNA & reporter (:ref:`count_samples`) ``` screen_id=my_sorting_tiling_screen +working_dir=my_workdir -bean-count-samples \ ---input tests/data/sample_list_tiling.csv `# Contains fastq file path; see test file for example.`\ +bean count-samples \ +--input ${working_dir}/sample_list_tiling.csv `# Contains fastq file path; see test file for example.`\ -b A `# Base A is edited (into G)` \ --f tests/data/test_guide_info_tiling_chrom.csv `# Contains gRNA metadata; see test file for example.`\ --o ./ `# Output directory` \ +-f ${working_dir}/test_guide_info_tiling_chrom.csv `# Contains gRNA metadata; see test file for example.`\ +-o $working_dir `# Output directory` \ -r `# Quantify reporter edits` \ -n ${screen_id} `# ID of the screen` \ --tiling @@ -68,12 +70,12 @@ bean-count-samples \ Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. ## 2. QC (:ref:`qc`) -Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean-qc`](../../README#bean-qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) +Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean qc`](../../README#bean qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) ``` -bean-qc \ - bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ - -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r qc_report_${screen_id} `# Prefix for QC report` \ +bean qc \ + ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ [--tiling] `# Not required if you have passed --tiling in counting step` ``` @@ -82,7 +84,7 @@ bean-qc \ If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. ## 3. Filter alleles (:ref:`filter`) -As tiling library doesn't have designated per-gRNA target variant, any base edit observed in reporter may be the candidate variant, while having too many variants with very low editing rate significantly decreases the power. Variants are filtered based on multiple criteria in `bean-fitler`. +As tiling library doesn't have designated per-gRNA target variant, any base edit observed in reporter may be the candidate variant, while having too many variants with very low editing rate significantly decreases the power. Variants are filtered based on multiple criteria in `bean fitler`. If the screen targets coding sequence, it's beneficial to translate edits into coding varaints whenever possible for better power. For translation, provide `--translate` and one of the following: ``` @@ -96,35 +98,35 @@ where `path_to_gene_names_file.txt` has one gene symbol per line, and gene symbo Example allele filtering given we're translating based on MANE transcript exons of multiple gene symbols: ```bash -bean-filter ./bean_count_${screen_id}_masked.h5ad \ --o ./bean_count_${screen_id}_alleleFiltered \ ---filter-target-basechange `# Filter based on intended base changes. If -b A was provided in bean-count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\ +bean filter ${working_dir}/bean_count_${screen_id}_masked.h5ad \ +-o ${working_dir}/bean_count_${screen_id}_alleleFiltered \ +--filter-target-basechange `# Filter based on intended base changes. If -b A was provided in bean count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\ --filter-window --edit-start-pos 0 --edit-end-pos 19 `# Filter based on editing window in spacer position within reporter.`\ --filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \ ---translate --translate-genes-list tests/data/gene_symbols.txt +--translate --translate-genes-list ${working_dir}/gene_symbols.txt ``` Ouptut file `` shows number of alleles per guide and number of guides per variant, where we want high enough values for the latter. See the typical output for dataset with good editing coverage & filtering result [here](../example_filtering_ouptut/). ## 4. Quantify variant effect (:ref:`run`) -By default, `bean-run [sorting,survival] tiling` uses most filtered allele counts table for variant identification and quantification of their effects. **Check [allele filtering output](../example_filtering_ouptut/)** and choose alternative filtered allele counts table if necessary. +By default, `bean run [sorting,survival] tiling` uses most filtered allele counts table for variant identification and quantification of their effects. **Check [allele filtering output](../example_filtering_ouptut/)** and choose alternative filtered allele counts table if necessary. -`bean-run` can take 3 run options to quantify editing rate: +`bean run` can take 3 run options to quantify editing rate: 1. From **reporter + accessibility** - 1-1. If your gRNA metadata table (`tests/data/test_guide_info.csv` above) included per-gRNA accessibility score, + 1-1. If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA accessibility score, ``` - bean-run sorting tiling \ - ./bean_count_${screen_id}_alleleFiltered.h5ad \ - -o tests/test_res/var/ \ + bean run sorting tiling \ + ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ + -o $working_dir \ --fit-negctrl \ --scale-by-acc \ --accessibility-col accessibility ``` - 1-2. If your gRNA metadata table (`tests/data/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, + 1-2. If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, ``` - bean-run sorting tiling \ - ./bean_count_${screen_id}_alleleFiltered.h5ad \ - -o tests/test_res/var/ \ + bean run sorting tiling \ + ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ + -o $working_dir \ --fit-negctrl \ --scale-by-acc \ --accessibility-bw accessibility.bw @@ -132,17 +134,17 @@ By default, `bean-run [sorting,survival] tiling` uses most filtered allele count 2. From **reporter** ``` - bean-run sorting tiling \ - ./bean_count_${screen_id}_alleleFiltered.h5ad \ - -o tests/test_res/var/ \ + bean run sorting tiling \ + ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ + -o $working_dir \ --fit-negctrl ``` 3. No reporter information, assume the same editing efficiency of all gRNAs. Use this option if your data don't have editing rate information. ``` - bean-run sorting tiling \ - ./bean_count_${screen_id}_alleleFiltered.h5ad \ - -o tests/test_res/var/ \ + bean run sorting tiling \ + ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ + -o $working_dir \ --fit-negctrl \ --uniform-edit ``` \ No newline at end of file diff --git a/docs/tutorials/ldl_var.md b/docs/tutorials/ldl_var.md index 1542536..cdbf869 100755 --- a/docs/tutorials/ldl_var.md +++ b/docs/tutorials/ldl_var.md @@ -4,11 +4,11 @@ GWAS variant screen with per-variant gRNA tiling design, selected based on FACS - + - +
    Library designVariant (gRNAs tile each target variant)
    variant library design    		Variant (gRNAs tile each target variant)
    variant library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design    		Cells are sorted based on FACS signal quantiles
    variant library design
    @@ -17,27 +17,28 @@ GWAS variant screen with per-variant gRNA tiling design, selected based on FACS ## Example workflow ```bash screen_id=my_sorting_tiling_screen +working_dir=my_workdir # 1. Count gRNA & reporter bean-count-samples \ ---input tests/data/sample_list.csv `# Contains fastq file path; see test file for example.`\ +--input ${working_dir}//sample_list.csv `# Contains fastq file path; see test file for example.`\ -b A `# Base A is edited (into G)` \ --f tests/data/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ +-f ${working_dir}/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ -o ./ `# Output directory` \ -r `# Quantify reporter edits` \ -n ${screen_id} `# ID of the screen to be counted` # 2. QC samples & guides bean-qc \ - bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ - -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r qc_report_${screen_id} `# Prefix for QC report` \ + ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ -b ` # Remove replicates with no good samples. # 3. Quantify variant effect bean-run sorting variant \ - tests/data/bean_count_${screen_id}_masked.h5ad \ - -o tests/test_res/var/ \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ --fit-negctrl \ --scale-by-acc \ --accessibility-col accessibility @@ -50,9 +51,9 @@ screen_id=my_sorting_tiling_screen # 1. Count gRNA & reporter bean-count-samples \ ---input tests/data/sample_list.csv `# Contains fastq file path; see test file for example.`\ +--input ${working_dir}/sample_list.csv `# Contains fastq file path; see test file for example.`\ -b A `# Base A is edited (into G)` \ --f tests/data/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ +-f ${working_dir}/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ -o ./ `# Output directory` \ -r `# Quantify reporter edits` \ -n ${screen_id} `# ID of the screen to be counted` @@ -77,20 +78,20 @@ If the data does not include reporter editing data, you can provide `--no-editin `bean-run` can take 3 run options to quantify editing rate: 1. From **reporter + accessibility** - If your gRNA metadata table (`tests/data/test_guide_info.csv` above) included per-gRNA accessibility score, + If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA accessibility score, ``` bean-run sorting variant \ - tests/data/bean_count_${screen_id}_masked.h5ad \ - -o tests/test_res/var/ \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ --fit-negctrl \ --scale-by-acc \ --accessibility-col accessibility ``` - If your gRNA metadata table (`tests/data/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, + If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, ``` bean-run sorting variant \ - tests/data/bean_count_${screen_id}_masked.h5ad \ - -o tests/test_res/var/ \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ --fit-negctrl \ --scale-by-acc \ --accessibility-bw accessibility.bw @@ -101,16 +102,16 @@ If the data does not include reporter editing data, you can provide `--no-editin This assumes the all target sites have the uniform chromatin accessibility. ``` bean-run sorting variant \ - tests/data/bean_count_${screen_id}_masked.h5ad \ - -o tests/test_res/var/ \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ --fit-negctrl ``` 3. No reporter information, assume the same editing efficiency of all gRNAs. Use this option if your data don't have editing outcome information. ``` bean-run sorting variant \ - tests/data/bean_count_${screen_id}_masked.h5ad \ - -o tests/test_res/var/ \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ --fit-negctrl \ --uniform-edit ``` \ No newline at end of file diff --git a/imgs/bean_title.svg b/imgs/bean_title.svg deleted file mode 100755 index 62f1e9c..0000000 --- a/imgs/bean_title.svg +++ /dev/null @@ -1 +0,0 @@ - \ No newline at end of file diff --git a/imgs/dag_bean_v2.svg b/imgs/dag_bean_v2.svg deleted file mode 100644 index 6f89f99..0000000 --- a/imgs/dag_bean_v2.svg +++ /dev/null @@ -1,263 +0,0 @@ - - - - - - - - - - - - - - - - run - -qc -filter - -count-samples -R1.fastq - - - -R2.fastq -Coding regions oftargeted genes - - - - - - - - - - - Input - - - - Input(Optional) - -Legend - - -AnnData-based ReporterScreen object - - - - - - - - - - - - - - - - - guides - - samples - - - gRNA counts - - - - - - - - - - - gRNA - Allele - - samples - - - Allele counts - - - - - - - - - - - - - - gRNA1 - Allele1 - Allele2 - ... - - - - - - - - - - - - - - - - - - - - - - - - - - -qc_report.[html,ipynb] - - - - - - - - - - -filter_log.txtfiltered_allele_stats.pdf - - - - - - - - - - -bean_element_result.csvbean_sgRNA_result.csv - - - - - Output - - - - - - - - - - - - -profile - - - - - - - - - - -editing_preferences.[html,ipynb] - -sample_list.csv - -gRNA_info.csv - - - - - - - - - - - - - diff --git a/imgs/library_design.svg b/imgs/library_design.svg deleted file mode 100644 index 1b00411..0000000 --- a/imgs/library_design.svg +++ /dev/null @@ -1,1204 +0,0 @@ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - diff --git a/notebooks/sample_quality_report.ipynb b/notebooks/sample_quality_report.ipynb index 3879bc7..2390ed7 100755 --- a/notebooks/sample_quality_report.ipynb +++ b/notebooks/sample_quality_report.ipynb @@ -402,17 +402,8 @@ "] = 0\n", "if \"mean_editing_rate\" in bdata.samples.columns.tolist():\n", " bdata.samples.loc[bdata.samples.mean_editing_rate < edit_rate_thres, \"mask\"] = 0\n", - "if (\n", - " isinstance(replicate_label, str)\n", - " and len(bdata.samples[replicate_label].unique()) > 1\n", - " or isinstance(replicate_label, list)\n", - " and len(bdata.samples[replicate_label].drop_duplicates()) > 1\n", - "):\n", - " bdata_filtered = bdata[\n", - " :, bdata.samples[f\"median_lfc_corr.{comp_cond1}_{comp_cond2}\"] > lfc_thres\n", - " ]\n", - "else:\n", - " bdata_filtered = bdata" + "if f\"median_lfc_corr.{comp_cond1}_{comp_cond2}\" in bdata.samples.columns.tolist():\n", + " bdata.samples.loc[bdata.samples[f\"median_lfc_corr.{comp_cond1}_{comp_cond2}\"] < lfc_thres, \"mask\"] = 0" ] }, { @@ -423,23 +414,25 @@ "source": [ "# leave replicate with more than 1 sorting bin data\n", "if remove_bad_replciates:\n", - " rep_n_samples = bdata_filtered.samples.groupby(replicate_label)[\"mask\"].sum()\n", + " rep_n_samples = bdata.samples.groupby(replicate_label)[\"mask\"].sum()\n", " print(rep_n_samples)\n", " rep_has_too_small_sample = rep_n_samples.loc[rep_n_samples < 2].index.tolist()\n", " print(\n", " f\"Excluding reps {rep_has_too_small_sample} that has less than 2 samples per replicate.\"\n", " )\n", " if isinstance(replicate_label, str):\n", - " samples_include = ~bdata_filtered.samples[replicate_label].isin(\n", + " samples_include = ~bdata.samples[replicate_label].isin(\n", " rep_has_too_small_sample\n", " )\n", " else:\n", - " bdata_filtered.samples[\"_rc\"] = bdata_filtered.samples[\n", + " bdata.samples[\"_rc\"] = bdata.samples[\n", " replicate_label\n", " ].values.tolist()\n", - " samples_include = ~bdata_filtered.samples[\"_rc\"].isin(rep_has_too_small_sample)\n", - " bdata_filtered.samples.pop(\"_rc\")\n", - " bdata_filtered = bdata_filtered[:, samples_include]" + " samples_include = ~bdata.samples[\"_rc\"].isin(rep_has_too_small_sample)\n", + " bdata.samples.pop(\"_rc\")\n", + " bdata_filtered = bdata[:, samples_include]\n", + "else:\n", + " bdata_filtered = bdata" ] }, { diff --git a/tests/test_qc.py b/tests/test_qc.py index b2c0c05..5f9f752 100755 --- a/tests/test_qc.py +++ b/tests/test_qc.py @@ -28,6 +28,19 @@ def test_qc_tiling(): raise exc +@pytest.mark.order(7) +def test_qc_survival(): + cmd = "bean qc tests/data/survival_var_mini_screen.h5ad -o tests/data/survival_var_mini_screen_masked.h5ad -r tests/test_res/qc_report_survival_var_mini_screen --count-correlation-thres 0.6 --lfc-conds D0,D14" + try: + subprocess.check_output( + cmd, + shell=True, + universal_newlines=True, + ) + except subprocess.CalledProcessError as exc: + raise exc + + @pytest.mark.order(9) def test_dummy_insertion_varscreen(): cmd = "bean qc tests/data/var_mini_screen_missing.h5ad -o tests/data/var_mini_screen_missing_masked.h5ad -r tests/test_res/qc_report_var_mini_screen_missing --count-correlation-thres 0.6" diff --git a/tests/test_run.py b/tests/test_run.py index 699d1c3..9c0534d 100755 --- a/tests/test_run.py +++ b/tests/test_run.py @@ -159,3 +159,55 @@ def test_run_tiling_with_negctrl_uniform(): # Add fit_negctrl examples + + +@pytest.mark.order(17) +def test_survival_run_variant_noacc(): + cmd = "bean run survival variant tests/data/survival_var_mini_screen_masked.h5ad -o tests/test_res/var/ --n-iter 10 --control-condition=D7" + try: + subprocess.check_output( + cmd, + shell=True, + universal_newlines=True, + ) + except subprocess.CalledProcessError as exc: + raise exc + + +@pytest.mark.order(18) +def test_survival_run_variant_wo_negctrl_uniform(): + cmd = "bean run survival variant tests/data/survival_var_mini_screen_masked.h5ad -o tests/test_res/var/ --uniform-edit --n-iter 10 --control-condition=D7" + try: + subprocess.check_output( + cmd, + shell=True, + universal_newlines=True, + ) + except subprocess.CalledProcessError as exc: + raise exc + + +@pytest.mark.order(20) +def test_survival_run_variant_noacc_negctrl(): + cmd = "bean run survival variant tests/data/survival_var_mini_screen_masked.h5ad -o tests/test_res/var/ --fit-negctrl --n-iter 10 --control-condition=D7" + try: + subprocess.check_output( + cmd, + shell=True, + universal_newlines=True, + ) + except subprocess.CalledProcessError as exc: + raise exc + + +@pytest.mark.order(21) +def test_survival_run_variant_uniform_negctrl(): + cmd = "bean run survival variant tests/data/survival_var_mini_screen_masked.h5ad -o tests/test_res/var/ --uniform-edit --fit-negctrl --n-iter 10 --control-condition=D7" + try: + subprocess.check_output( + cmd, + shell=True, + universal_newlines=True, + ) + except subprocess.CalledProcessError as exc: + raise exc From 16e3b5d2be238a963a6e6c908c991a9d27310484 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 11:00:40 -0400 Subject: [PATCH 084/107] add tutorial for survival screen --- docs/_index.md | 4 ++ docs/count.md | 31 +++++++++ docs/create-screen.md | 9 +++ docs/filter.md | 37 ++++++++++ docs/index.rst | 1 + docs/input.md | 37 ++++++++++ docs/ldl_cds.md | 150 +++++++++++++++++++++++++++++++++++++++++ docs/ldl_var.md | 117 ++++++++++++++++++++++++++++++++ docs/profile.md | 8 +++ docs/prolif_gwas.md | 118 ++++++++++++++++++++++++++++++++ docs/prolif_gwas.rst | 5 ++ docs/qc.md | 94 ++++++++++++++++++++++++++ docs/reporterscreen.md | 11 +++ docs/run.md | 64 ++++++++++++++++++ 14 files changed, 686 insertions(+) create mode 100755 docs/_index.md create mode 100755 docs/count.md create mode 100755 docs/create-screen.md create mode 100755 docs/filter.md create mode 100755 docs/input.md create mode 100755 docs/ldl_cds.md create mode 100755 docs/ldl_var.md create mode 100755 docs/profile.md create mode 100644 docs/prolif_gwas.md create mode 100644 docs/prolif_gwas.rst create mode 100755 docs/qc.md create mode 100644 docs/reporterscreen.md create mode 100755 docs/run.md diff --git a/docs/_index.md b/docs/_index.md new file mode 100755 index 0000000..45e2414 --- /dev/null +++ b/docs/_index.md @@ -0,0 +1,4 @@ +--- +layout: default +title: CRISPR-BEAN +--- diff --git a/docs/count.md b/docs/count.md new file mode 100755 index 0000000..68c9ef7 --- /dev/null +++ b/docs/count.md @@ -0,0 +1,31 @@ +# `bean count[-samples]`: Count (reporter) screen data +`bean count-samples` (or `bean count` for a single sample) maps guide into guide counts, **allowing for base transition in spacer sequence**. When the matched reporter information is provided, it can count the **target site edits** and **alleles produced by each guide**. Mapping is efficiently done based on [CRISPResso2](https://github.com/pinellolab/CRISPResso2) modified for base-edit-aware mapping. + + + +```python +bean count-samples \ + --input sample_list.csv `# sample with lines 'R1_filepath,R2_filepath,sample_name\n'` \ + -b A `# base that is being edited (A/G)` \ + -f sgRNA_info_table.csv `# sgRNA information` \ + -o . `# output directory` \ + -r `# read edit/allele information from reporter` \ + -t 12 `# number of threads` \ + --name my_sorting_screen `# name of this sample run` \ +``` +```python +bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r +``` +By default, `bean count[-samples]` assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure. + + Read structuren + +See full detail [below](#full-parameters). + +# Input file format +See :ref:`input` for input file formats. + +# Output file format +`count` or `count-samples` produces `.h5ad` and `.xlsx` file with guide and per-guide allele counts. +* `.h5ad`: This output file follows annotated matrix format compatible with `AnnData` and is based on `Screen` object in [purturb_tools](https://github.com/pinellolab/perturb-tools). See [Data Structure](#data-structure) section for more information. +* `.xlsx`: This output file contains `.guides`, `.samples`, `.X[_bcmatch,_edits]`. (`allele_tables` are often too large to write into an Excel!) diff --git a/docs/create-screen.md b/docs/create-screen.md new file mode 100755 index 0000000..d9200b7 --- /dev/null +++ b/docs/create-screen.md @@ -0,0 +1,9 @@ +# `bean create-screen`: Create ReporterScreen object from flat files +```bash +bean create-screen gRNA_library.csv sample_list.csv gRNA_counts_table.csv +``` +## Input + * gRNA_library.csv + * sample_list.csv + * gRNA_counts_table.csv: Table with gRNA ID in the first column and sample IDs as the column names (first row) +`gRNA_library.csv` and `sample_list.csv` should be formatted as :ref:`input`. \ No newline at end of file diff --git a/docs/filter.md b/docs/filter.md new file mode 100755 index 0000000..4a04745 --- /dev/null +++ b/docs/filter.md @@ -0,0 +1,37 @@ +# `filter`: Filtering (and optionally translating) alleles +As `tiling` mode of `bean run` accounts for any robustly observed alleles, `bean filter` filters for such alleles. +```bash +bean filter my_sorting_screen_masked.h5ad \ +-o my_sorting_screen_filtered.h5ad `# Output file path` \ +``` + +# Output +Above command produces +* `my_sorting_screen_filtered.h5ad` with filtered alleles stored in `.uns`, +* `my_sorting_screen_filtered.filtered_allele_stats.pdf`, and `my_sorting_screen_filtered.filter_log.txt` that report allele count stats in each filtering step. + +You may want to adjust the flitering parameters to obtain optimal balance between # guides per variant & # variants that are scored. See example outputs of filtering step [here](docs/example_filtering_output/). + + +# Translating alleles +If you want to obtain **amino acid level variant** for coding sequence tiling screens, provide coding sequence positions which variants occuring within the coding sequence will be translated. *This is optional, but **highly recommended** to increase per-(coding)variant support.* + +Allele translation + + +```bash +bean filter my_sorting_screen.h5ad \ +-o my_sorting_screen_masked.h5ad \ +--translate `# Translate coding variants` \ +[ --translate-gene-name GENE_SYMBOL OR + --translate-genes-list path_to_gene_names_file.txt OR + --translate-fasta gene_exon.fa, OR + --translate-fastas-csv gene_exon_fas.csv] +``` +* When library covers a single gene, do either of the following: + 1. Feed `--translate-gene-name GENE_SYMBOL` if your `genomic_pos` column of `sgRNA_info_tbl` is compatible with [MANE transcript](https://useast.ensembl.org/info/genome/genebuild/mane.html)'s reference genome. (Per 10/23/2023, GRCh38). This will automatically load the exon positions based on MANE transcript annotation. + 2. To use your custom coding sequence and exon positions, feed `--translate-fasta gene_exon.fa` argument where `gene_exon.fa` is the FASTA file with entries of exons. [See full details here](docs/exon_fa_format.md). +* When library covers multiple genes, do either of the following: + 1. Feed `--translate-genes-list path_to_gene_names_file.txt` where `path_to_gene_names_file.txt` is file with one gene symbol per line. + 2. Feed `--translate-fastas-csv gene_exon_fas.csv` where `gene_exon_fas.csv` is the csv file with lines `gene_id,gene_exon_fasta_path` without header. Each FASTA file in `gene_exon_fasta_path` is formatted [as the single-gene FASTA file](docs/exon_fa_format.md). +* Translation will keep the variants outside the coding sequence as nucleotide-level variants, while aggregating variants leading to the same coding sequence variants. diff --git a/docs/index.rst b/docs/index.rst index 9f04032..567b619 100755 --- a/docs/index.rst +++ b/docs/index.rst @@ -13,6 +13,7 @@ Workflows gwas cds + prolif_gwas input =================== diff --git a/docs/input.md b/docs/input.md new file mode 100755 index 0000000..21368b8 --- /dev/null +++ b/docs/input.md @@ -0,0 +1,37 @@ +This document describes the input files of :ref:`count_samples`. +## sgRNA_info_table.csv +File should contain following columns. +* `name`: gRNA ID column +* `sequence`: gRNA sequence +* `barcode`: R2 barcode to help match reporter to gRNA, written in the sense direction (as in R1) +* In order to use accessibility in the [variant effect quantification](#bean-run-quantify-variant-effects), provide accessibility information in one of two options. (For non-targeting guides, provide NA values (empty cell).) + * Option 1: `chrom` & `genomic_pos`: Chromosome (ex. `chr19`) and genomic position of guide sequence. You will have to provide the path to the bigwig file with matching reference version in `bean run`. + * Option 2: `accessibility_signal`: ATAC-seq signal value of the target loci of each guide. +* For variant library (gRNAs are designed to target specific variants and ignores bystander edits) + * `target`: This column denotes which target variant/element of each gRNA. This is not used in `bean count[-samples]` but required to run `bean run` in later steps. + * `target_group`: If negative/positive control gRNA will be considered in `bean qc` and/or `bean run`, specify as "NegCtrl"/"PosCtrl" in this column. + * `target_pos`: If `--match_target_pos` flag is used, input file needs `target_pos` which specifies 0-based relative position of targeted base within Reporter sequence. +* For tiling library (gRNAs tile coding / noncoding sequences) + * `strand`: Specifies gRNA strand information relative to the reference genome. + * `chrom`: Chromosome of gRNA targeted locus. + * `start_pos`: gRNA starting position in the genome. Required when you provide `strand` column. Should specify the smaller coordinate value among start and end position regardless of gRNA strandedness. + +Also see examples for [variant library](tests/data/test_guide_info.csv) and [tiling library](tests/data/test_guide_info_tiling.csv). + +## sample_list.csv +File should contain following columns with header. +* `R1_filepath`: Path to read 1 `.fastq[.gz]` file +* `R2_filepath`: Path to read 1 `.fastq[.gz]` file +* `sample_id`: ID of sequencing sample +* `replicate`: Replicate # of this sample (Should NOT contain `.`) +* `condition`: Name of the sorting bin (ex. `top`, `bot`), or label of timepoint (ex. `D5`, `D18`) + +For FACS sorting screens: +* `upper_quantile`: FACS sorting upper quantile +* `lower_quantile`: FACS sorting lower quantile + +For proliferation / survival screens: +* `time`: Numeric time following the base editing of each sample. + + +Also see examples for [FACS sorting screen](tests/data/sample_list.csv) and [proliferation / survival screen](tests/data/sample_list_survival.csv). \ No newline at end of file diff --git a/docs/ldl_cds.md b/docs/ldl_cds.md new file mode 100755 index 0000000..e2d5ce0 --- /dev/null +++ b/docs/ldl_cds.md @@ -0,0 +1,150 @@ +# Tiling sorting screen tutorial +Tiling screen that tiles gRNA densely across locus or multiple loci, selected based on FACS signal quantiles. + + + + + + + + + + +
    Library designTiling (gRNAs tile each locus densely)
    tiling library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design
    + +

    + +## Example workflow +```bash +screen_id=my_sorting_tiling_screen +working_dir=my_workdir + +# 1. Count gRNA & reporter +bean count-samples \ +--input ${working_dir}/sample_list_tiling.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f ${working_dir}/test_guide_info_tiling_chrom.csv `# Contains gRNA metadata; see test file for example.`\ +-o $working_dir `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen` \ +--tiling + +# 2. QC samples & guides +bean qc \ + ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ + +# 3. Filter & translate alleles +bean filter ${working_dir}/bean_count_${screen_id}_masked.h5ad \ +-o ${working_dir}/bean_count_${screen_id}_alleleFiltered \ +--filter-target-basechange `# Filter based on intended base changes. If -b A was provided in bean count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\ +--filter-window --edit-start-pos 0 --edit-end-pos 19 `# Filter based on editing window in spacer position within reporter.`\ +--filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \ +--translate --translate-genes-list ${working_dir}/gene_symbols.txt + +# 4. Quantify variant effect +bean run sorting tiling \ + ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ + -o $working_dir \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility +``` +See more details below. + +## 1. Count gRNA & reporter (:ref:`count_samples`) +``` +screen_id=my_sorting_tiling_screen +working_dir=my_workdir + +bean count-samples \ +--input ${working_dir}/sample_list_tiling.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f ${working_dir}/test_guide_info_tiling_chrom.csv `# Contains gRNA metadata; see test file for example.`\ +-o $working_dir `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen` \ +--tiling +``` +Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. + +## 2. QC (:ref:`qc`) +Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean qc`](../../README#bean qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) +``` +bean qc \ + ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ + [--tiling] `# Not required if you have passed --tiling in counting step` +``` + + + +If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. + +## 3. Filter alleles (:ref:`filter`) +As tiling library doesn't have designated per-gRNA target variant, any base edit observed in reporter may be the candidate variant, while having too many variants with very low editing rate significantly decreases the power. Variants are filtered based on multiple criteria in `bean fitler`. + +If the screen targets coding sequence, it's beneficial to translate edits into coding varaints whenever possible for better power. For translation, provide `--translate` and one of the following: +``` +[ --translate-gene-name GENE_SYMBOL OR + --translate-genes-list path_to_gene_names_file.txt OR + --translate-fasta gene_exon.fa, OR + --translate-fastas-csv gene_exon_fas.csv] +``` +where `path_to_gene_names_file.txt` has one gene symbol per line, and gene symbol uses its MANE transcript (hg38) coordinates of exons. In order to use other reference versions or transcript ID, you'll need to feed in fasta file. See detailed formatting of fasta file [here](../../README#translating-alleles). + +Example allele filtering given we're translating based on MANE transcript exons of multiple gene symbols: + +```bash +bean filter ${working_dir}/bean_count_${screen_id}_masked.h5ad \ +-o ${working_dir}/bean_count_${screen_id}_alleleFiltered \ +--filter-target-basechange `# Filter based on intended base changes. If -b A was provided in bean count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\ +--filter-window --edit-start-pos 0 --edit-end-pos 19 `# Filter based on editing window in spacer position within reporter.`\ +--filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \ +--translate --translate-genes-list ${working_dir}/gene_symbols.txt +``` + +Ouptut file `` shows number of alleles per guide and number of guides per variant, where we want high enough values for the latter. See the typical output for dataset with good editing coverage & filtering result [here](../example_filtering_ouptut/). + +## 4. Quantify variant effect (:ref:`run`) +By default, `bean run [sorting,survival] tiling` uses most filtered allele counts table for variant identification and quantification of their effects. **Check [allele filtering output](../example_filtering_ouptut/)** and choose alternative filtered allele counts table if necessary. + +`bean run` can take 3 run options to quantify editing rate: +1. From **reporter + accessibility** + 1-1. If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA accessibility score, + ``` + bean run sorting tiling \ + ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ + -o $working_dir \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility + ``` + 1-2. If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, + ``` + bean run sorting tiling \ + ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ + -o $working_dir \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-bw accessibility.bw + ``` + +2. From **reporter** + ``` + bean run sorting tiling \ + ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ + -o $working_dir \ + --fit-negctrl + ``` +3. No reporter information, assume the same editing efficiency of all gRNAs. + Use this option if your data don't have editing rate information. + ``` + bean run sorting tiling \ + ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ + -o $working_dir \ + --fit-negctrl \ + --uniform-edit + ``` \ No newline at end of file diff --git a/docs/ldl_var.md b/docs/ldl_var.md new file mode 100755 index 0000000..cdbf869 --- /dev/null +++ b/docs/ldl_var.md @@ -0,0 +1,117 @@ +# Variant sorting screen tutorial +GWAS variant screen with per-variant gRNA tiling design, selected based on FACS signal quantiles. + + + + + + + + + + +
    Library designVariant (gRNAs tile each target variant)
    variant library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design
    + +

    + +## Example workflow +```bash +screen_id=my_sorting_tiling_screen +working_dir=my_workdir + +# 1. Count gRNA & reporter +bean-count-samples \ +--input ${working_dir}//sample_list.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f ${working_dir}/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ +-o ./ `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen to be counted` + +# 2. QC samples & guides +bean-qc \ + ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ + -b ` # Remove replicates with no good samples. + +# 3. Quantify variant effect +bean-run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility +``` +See more details below. + +## 1. Count gRNA & reporter (:ref:`count_samples`) +```bash +screen_id=my_sorting_tiling_screen + +# 1. Count gRNA & reporter +bean-count-samples \ +--input ${working_dir}/sample_list.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f ${working_dir}/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ +-o ./ `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen to be counted` +``` +Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. + +## 2. QC samples & guides (:ref:`qc`) +Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean-qc`](../../README#bean-qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) +``` +bean-qc \ + bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r qc_report_${screen_id} `# Prefix for QC report` +``` + + + +If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. + + +## 3. Quantify variant effect (:ref:`run`) + +`bean-run` can take 3 run options to quantify editing rate: +1. From **reporter + accessibility** + If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA accessibility score, + ``` + bean-run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility + ``` + If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, + ``` + bean-run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-bw accessibility.bw + ``` + +2. From **reporter**, without accessibility + + This assumes the all target sites have the uniform chromatin accessibility. + ``` + bean-run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ + --fit-negctrl + ``` +3. No reporter information, assume the same editing efficiency of all gRNAs. + Use this option if your data don't have editing outcome information. + ``` + bean-run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ + --fit-negctrl \ + --uniform-edit + ``` \ No newline at end of file diff --git a/docs/profile.md b/docs/profile.md new file mode 100755 index 0000000..0300c16 --- /dev/null +++ b/docs/profile.md @@ -0,0 +1,8 @@ +# `bean profile`: Profile editing patterns +```bash +bean profile my_sorting_screen.h5ad -o output_prefix `# Prefix for editing profile report` +``` +# Output +Above command produces `prefix_editing_preference.[html,ipynb]` as editing preferences ([see example](../notebooks/profile_editing_preference.ipynb)). + +Allele translation \ No newline at end of file diff --git a/docs/prolif_gwas.md b/docs/prolif_gwas.md new file mode 100644 index 0000000..3cac063 --- /dev/null +++ b/docs/prolif_gwas.md @@ -0,0 +1,118 @@ +# Variant sorting screen tutorial +GWAS variant screen with per-variant gRNA tiling design, selected based on FACS signal quantiles. + + + + + + + + + + +
    Library designVariant (gRNAs tile each target variant)
    variant library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design
    + +

    + +## Example workflow +```bash +screen_id=my_sorting_tiling_screen +working_dir=my_workdir + +# 1. Count gRNA & reporter +bean count-samples \ +--input ${working_dir}/sample_list.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f ${working_dir}/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ +-o ${working_dir} `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen to be counted` + +# 2. QC samples & guides +bean qc \ + ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ + --lfc-conds D0,D14 `# Conditions to calculate LFC of positive controls` \ + -b ` # Remove replicates with no good samples. + +# 3. Quantify variant effect +bean run survival variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility +``` +See more details below. + +## 1. Count gRNA & reporter (:ref:`count_samples`) +```bash +bean count-samples \ +--input ${working_dir}/sample_list.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f ${working_dir}/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ +-o ${working_dir} `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen to be counted` +``` +Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. + +## 2. QC samples & guides (:ref:`qc`) +Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean qc`](../../README#bean qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) +``` +bean qc \ + ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ + --lfc-conds D0,D14 `# Conditions to calculate LFC of positive controls` \ + -b ` # Remove replicates with no good samples. + +``` + + + +If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. + + +## 3. Quantify variant effect (:ref:`run`) + +`bean run` can take 3 run options to quantify editing rate: +1. From **reporter + accessibility** + If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA accessibility score, + ``` + bean run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o $working_dir \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility + ``` + If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, + ``` + bean run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o $working_dir \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-bw accessibility.bw + ``` + +2. From **reporter**, without accessibility + + This assumes the all target sites have the uniform chromatin accessibility. + ``` + bean run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o $working_dir \ + --fit-negctrl + ``` +3. No reporter information, assume the same editing efficiency of all gRNAs. + Use this option if your data don't have editing outcome information. + ``` + bean run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o $working_dir \ + --fit-negctrl \ + --uniform-edit + ``` \ No newline at end of file diff --git a/docs/prolif_gwas.rst b/docs/prolif_gwas.rst new file mode 100644 index 0000000..35d4175 --- /dev/null +++ b/docs/prolif_gwas.rst @@ -0,0 +1,5 @@ +GWAS variant library +*********************** +.. mdinclude:: profile_gwas.md + +See :ref:`subcommands` for the full details. diff --git a/docs/qc.md b/docs/qc.md new file mode 100755 index 0000000..ce85c44 --- /dev/null +++ b/docs/qc.md @@ -0,0 +1,94 @@ +# `bean qc`: QC of reporter screen data +```bash +bean qc \ + my_sorting_screen.h5ad `# Input ReporterScreen .h5ad file path` \ + -o my_sorting_screen_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r qc_report_my_sorting_screen `# Prefix for QC report` \ + --ctrl-cond presort `# "condition" column in the control sample before selection. Mean gRNA editing rates in these samples are reported. ` \ +# Inspect the output qc_report_my_sorting_screen.html to tweak QC threshold + +bean qc \ + my_sorting_screen.h5ad \ + -o my_sorting_screen_masked.h5ad \ + -r qc_report_my_sorting_screen \ + #[--count-correlation-thres 0.7 ...]\ + -b +``` + +`bean qc` supports following quality control and masks samples with low quality. Specifically: + +Allele translation + +* Plots guide coverage and the uniformity of coverage +* Guide count correlation between samples +* Log fold change correlation when positive controls are provided +* Plots editing rate distribution +* Identify samples with low guide coverage/guide count correlation/editing rate and mask the sample in `bdata.samples.mask` +* Identify outlier guides to filter out + +# Output +Above command produces +* `my_sorting_screen_masked.h5ad` without problematic replicate and guides and with sample masks, and +* `qc_report_my_sorting_screen.[html,ipynb]` as QC report. +##### Optional arguments: +* `-o OUT_SCREEN_PATH`, `--out-screen-path OUT_SCREEN_PATH` + Path where quality-filtered ReporterScreen object to be written to +* `-r OUT_REPORT_PREFIX`, `--out-report-prefix OUT_REPORT_PREFIX` + Output prefix of qc report (prefix.html, prefix.ipynb) + +##### QC thresholds: +* `--count-correlation-thres COUNT_CORRELATION_THRES` + Correlation threshold to mask out. +* `--edit-rate-thres EDIT_RATE_THRES` + Mean editing rate threshold per sample to mask out. +* `--lfc-thres LFC_THRES` + Positive guides' correlation threshold to filter out. + +##### Run options: +* `-b`, `--remove-bad-replicates` + Remove replicates with at least two of its samples meet the QC threshold (bean run does not support having only one sorting bin sample for a replicate). +* `-i`, `--ignore-missing-samples` + If the flag is not provided, if the ReporterScreen object does not contain all condiitons for + each replicate, make fake empty samples. If the flag is provided, don't add dummy samples. +* `--no-editing` Ignore QC about editing. Can be used for QC of other editing modalities. +* `--dont-recalculate-edits` + When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from + ReporterScreen.uns['allele_count']. + +##### Input `.h5ad` formatting: +Note that these arguements will change the way the QC metrics are calculated for guides, samples, or replicates. +* `--tiling TILING` Specify that the guide library is tiling library without 'n guides per target' design +* `--replicate-label REPLICATE_LABEL` + Label of column in `bdata.samples` that describes replicate ID. +* `--sample-covariates SAMPLE_COVARIATES` + Comma-separated list of column names in `bdata.samples` that describes non-selective + experimental condition. (drug treatment, etc.) +* `--condition-label CONDITION_LABEL` + Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time, + etc.) +###### Editing rate calculation + * `--control-condition CTRL_COND` + Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate + to be calculated. Default is `None`, which considers all samples. + * `--rel-pos-is-reporter` + Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If + `False`, those are relative to spacer position. + Editing rate is calculated with following parameters in + * Variant screens: + * `--target-pos-col TARGET_POS_COL` + Target position column in `bdata.guides` specifying target edit position in reporter + * tiling screens: + * `--edit-start-pos EDIT_START_POS` + Edit start position to quantify editing rate on, 0-based inclusive. + * `--edit-end-pos EDIT_END_POS` + Edit end position to quantify editing rate on, 0-based exclusive. +###### LFC of positive controls + * `--posctrl-col POSCTRL_COL` + Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all + gRNAs, feed empty string ''. + * `--posctrl-val POSCTRL_VAL` + Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the + positive control in calculating log fold change. + * `--lfc-conds LFC_CONDS` + Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated + between, delimited by comma \ No newline at end of file diff --git a/docs/reporterscreen.md b/docs/reporterscreen.md new file mode 100644 index 0000000..0450470 --- /dev/null +++ b/docs/reporterscreen.md @@ -0,0 +1,11 @@ +BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). + +ReporterScreen object structure + + * `.guides`: guide information provided in input (`gRNA_library.csv` in above example) + * `.samples`: sample information provided in input (`sample_list.csv` in above example) + * `.X`: Main guide count matrix, where row corresponds to each guide in `.guides` and columns correspond to samples in `.samples`. +Following attributes are included if matched reporter is provided and you chose to read edit/allele information from the reporter using `-r` option. + * `.X_bcmatch [Optional]`: Contains information about number of barcode-matched reads. Information about R2 barcode should be specified as `barcode` column in your `gRNA_library.csv` file. + * `.X_edits [Optional]`: If target position of each guide is specified as `target_pos` in input `gRNA_library.csv` file and `--match-target-position` option is provided, the result has the matrix with the number of target edit at the specified positions. + * `.allele_tables [Optional]`: Dictionary with a single allele count table that counts per guide and allele combination, what is the count per sample. \ No newline at end of file diff --git a/docs/run.md b/docs/run.md new file mode 100755 index 0000000..c1de5bf --- /dev/null +++ b/docs/run.md @@ -0,0 +1,64 @@ +# `bean run`: Quantify variant effects +BEAN uses Bayesian network to incorporate gRNA editing outcome to provide posterior estimate of variant phenotype. The Bayesian network reflects data generation process. Briefly, +1. Cellular phenotype (either for cells are sorted upon for sorting screen, or log(proliferation rate)) is modeled as the Gaussian mixture distribution of wild-type phenotype and variant phenotype. +2. The weight of the mixture components are inferred from the reporter editing outcome and the chromatin accessibility of the loci. +3. Cells with each gRNA, formulated as the mixture distribution, is sorted by the phenotypic quantile to produce the gRNA counts. + +For the full detail, see the method section of the [BEAN manuscript](https://www.medrxiv.org/content/10.1101/2023.09.08.23295253v1). + +model + +

    + +# Usage example +```bash +bean run sorting[survival] variant[tiling] my_sorting_screen_filtered.h5ad \ +[--uniform-edit, --scale-by-acc [--acc-bw-path accessibility_signal.bw, --acc-col accessibility]] \ +-o output_prefix/ \ +--fit-negctrl +``` +See full list of parameters [below](#full-parameters). + + +# Input +`my_sorting_screen_filtered.h5ad` can be produced by one of the following: +1. [`bean count-samples`]((#bean-count-samples-count-reporter-screen-data)) when you have raw `.fastq` file +2. (Limited to `bean run variant` mode) `bean create-screen` when you have flat `.csv` tables of gRNA metadata table, sample metadata table, gRNA counts table (# guides x # samples), and optionally # edits table. + ```bash + bean create-screen gRNA_info_table.csv sample_info_table.csv gRNA_counts_table.csv \ + [--edits edit_counts_table.csv -o output.h5ad] + ``` + * `gRNA_info_table.csv` should have following columns. + * `name`: gRNA ID column + * `target`: This column denotes which target variant/element of each gRNA. + * `target_group [Optional]`: If negative control gRNA will be used, specify as "NegCtrl" in this column. + * `sample_info_table.csv` should have following columns. + * `sample_id`: ID of sequencing sample + * `replicate`: Replicate # of this sample + * `bin`: Name of the sorting bin + * `upper_quantile`: FACS sorting upper quantile + * `lower_quantile`: FACS sorting lower quantile + * `gRNA_counts_table.csv` should be formatted as follows. + * Columns include one of `sample_id` columns in `sample_info_table.csv` file. + * 1st row (row index) follows `name` (gRNA ID) in `gRNA_info_table.csv` file. +3. You can manually create the `AnnData` object with more annotations including allele counts: see [API tutorial](#using-bean-as-python-module) for full detail. + + +# Output +model + +Above command produces +* `output_prefix/bean_element_result.[model_type].csv` with following columns: + * Estimated variant effect sizes + * `mu` (Effect size): Mean of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. + * `mu_sd`: Mean of variant phenotype `mu` is modeled as normal distribution. The column shows fitted standard deviation of `mu` that quantify the uncertainty of the variant effect. + * `mu_z`: z-score of `mu` + * `sd`: Standard deviation of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. + * `CI[0.025`, `0.975]`: Credible interval of `mu` + * When negative control is provided, above columns with `_adj` suffix are provided, which are the corresponding values adjusted for negative control. + * Metrics on per-variant evidence provided in input (provided in `tiling` mode) + * `effective_edit_rate`: Sum of per-variant editing rates over all alleles observed in the input. Allele-level editing rate is divided by the number of variants observed in the allele prior to summing up. + * `n_guides`: # of guides covering the variant. + * `n_coocc`: # of cooccurring variants with a given variant in any alleles observed in the input. +* `output_prefix/bean_sgRNA_result.[model_type].csv`: + * `edit_rate`: Estimated editing rate at the target loci. From 1174c70df7512b2fd5566b6edd896f9248312f32 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 11:01:54 -0400 Subject: [PATCH 085/107] remove copying img directory --- .github/workflows/documentation.yml | 1 - README.md | 10 +++++----- 2 files changed, 5 insertions(+), 6 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 53b8dfc..8d947d4 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -20,7 +20,6 @@ jobs: working-directory: docs run: sphinx-build . _build - name: copy image files - run: cp -r docs/commands/imgs/ docs/_build/imgs - uses: actions/upload-pages-artifact@v3 with: name: github-pages diff --git a/README.md b/README.md index 376b851..a451910 100755 --- a/README.md +++ b/README.md @@ -7,12 +7,12 @@ `bean` (**B**ase **E**diting reporter screens with guide **A**ctivity **N**ormalization) is an analysis toolkit for the pooled CRISPR reporter or sensor data. The reporter technique transfects cells with plasmid with not only sgRNA but with the **target sequence surrogate** which we call **reporter** or **sensor**. -Reporter construct +Reporter construct ## Overview `bean` supports end-to-end analysis of pooled sorting screens, with or without reporter. -dag_bean_v2.svg +dag_bean_v2.svg `bean` subcommands include the following: Click on the [`links`]() to see the full documentation. 1. [`count`](https://pinellolab.github.io/crispr-bean/count.html), [`count-samples`](https://pinellolab.github.io/crispr-bean/count_samples.html): Base-editing-aware **mapping** of guide, optionally with reporter from `.fastq` files. @@ -44,13 +44,13 @@ See the [documentation](https://pinellolab.github.io/crispr-bean/) for tutorials |---------------| -------------- | --------- | -------- | | GWAS variant library | FACS sorting | Yes/No | [GWAS variant screen](https://pinellolab.github.io/crispr-bean/gwas) | Coding sequence tiling libarary | FACS sorting | Yes/No | [Coding sequence tiling screen](https://pinellolab.github.io/crispr-bean/cds) -| GWAS variant library | Survival / Proliferation | Yes/No | TKO simulated (Coming soon!) -| Coding sequence tiling libarary | Survival / Proliferation | Yes/No | TKO simulated, tiling (Coming soon!) +| GWAS variant library | Survival / Proliferation | Yes/No | [GWAS variant screen](https://pinellolab.github.io/crispr-bean/prolif_gwas) +| Coding sequence tiling libarary | Survival / Proliferation | Yes/No | Coming soon! ### Library design: variant or tiling? The `bean filter` and `bean run` steps depend on the type of gRNA library design, where BEAN supports two modes of running. -variant library design +variant library design 1. `variant` library: Several gRNAs tile each of the targeted variants. Only the editing rate of the target variant is considered and the bystander effects are ignored. From 217a3170f13eb683ce3b8851b6312efac0254010 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 11:03:46 -0400 Subject: [PATCH 086/107] remove copying img directory --- .github/workflows/documentation.yml | 1 - 1 file changed, 1 deletion(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 8d947d4..c7000b6 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -19,7 +19,6 @@ jobs: - name: Build Documentation working-directory: docs run: sphinx-build . _build - - name: copy image files - uses: actions/upload-pages-artifact@v3 with: name: github-pages From 78193f76d3141f686f932480493298c4ff24907d Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 11:14:40 -0400 Subject: [PATCH 087/107] add copy again --- .github/workflows/documentation.yml | 2 ++ docs/count.md | 2 +- docs/filter.md | 2 +- docs/ldl_cds.md | 4 ++-- docs/ldl_var.md | 6 +++--- docs/profile.md | 2 +- docs/prolif_gwas.md | 6 +++--- docs/prolif_gwas.rst | 2 +- docs/qc.md | 2 +- docs/reporterscreen.md | 2 +- docs/run.md | 4 ++-- docs/tutorials/ldl_cds.md | 4 ++-- docs/tutorials/ldl_var.md | 6 +++--- 13 files changed, 23 insertions(+), 21 deletions(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index c7000b6..44e9f15 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -19,6 +19,8 @@ jobs: - name: Build Documentation working-directory: docs run: sphinx-build . _build + - name: copy image files + run: cp -r docs/assets docs/_build/assets/ - uses: actions/upload-pages-artifact@v3 with: name: github-pages diff --git a/docs/count.md b/docs/count.md index 68c9ef7..7400cba 100755 --- a/docs/count.md +++ b/docs/count.md @@ -18,7 +18,7 @@ bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r ``` By default, `bean count[-samples]` assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure. - Read structuren + Read structuren See full detail [below](#full-parameters). diff --git a/docs/filter.md b/docs/filter.md index 4a04745..e05373d 100755 --- a/docs/filter.md +++ b/docs/filter.md @@ -16,7 +16,7 @@ You may want to adjust the flitering parameters to obtain optimal balance betwee # Translating alleles If you want to obtain **amino acid level variant** for coding sequence tiling screens, provide coding sequence positions which variants occuring within the coding sequence will be translated. *This is optional, but **highly recommended** to increase per-(coding)variant support.* -Allele translation +Allele translation ```bash diff --git a/docs/ldl_cds.md b/docs/ldl_cds.md index e2d5ce0..8b0379f 100755 --- a/docs/ldl_cds.md +++ b/docs/ldl_cds.md @@ -4,11 +4,11 @@ Tiling screen that tiles gRNA densely across locus or multiple loci, selected ba - + - +
    Library designTiling (gRNAs tile each locus densely)
    tiling library design     		Tiling (gRNAs tile each locus densely)
    tiling library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design    		Cells are sorted based on FACS signal quantiles
    variant library design
    diff --git a/docs/ldl_var.md b/docs/ldl_var.md index cdbf869..7eb1af7 100755 --- a/docs/ldl_var.md +++ b/docs/ldl_var.md @@ -1,14 +1,14 @@ -# Variant sorting screen tutorial +## Variant sorting screen tutorial GWAS variant screen with per-variant gRNA tiling design, selected based on FACS signal quantiles. - + - +
    Library designVariant (gRNAs tile each target variant)
    variant library design    		Variant (gRNAs tile each target variant)
    variant library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design    		Cells are sorted based on FACS signal quantiles
    variant library design
    diff --git a/docs/profile.md b/docs/profile.md index 0300c16..cd40cb4 100755 --- a/docs/profile.md +++ b/docs/profile.md @@ -5,4 +5,4 @@ bean profile my_sorting_screen.h5ad -o output_prefix `# Prefix for editing profi # Output Above command produces `prefix_editing_preference.[html,ipynb]` as editing preferences ([see example](../notebooks/profile_editing_preference.ipynb)). -Allele translation \ No newline at end of file +Allele translation \ No newline at end of file diff --git a/docs/prolif_gwas.md b/docs/prolif_gwas.md index 3cac063..9af45ee 100644 --- a/docs/prolif_gwas.md +++ b/docs/prolif_gwas.md @@ -1,14 +1,14 @@ -# Variant sorting screen tutorial +## Variant survival screen tutorial GWAS variant screen with per-variant gRNA tiling design, selected based on FACS signal quantiles. - + - +
    Library designVariant (gRNAs tile each target variant)
    variant library design    		Variant (gRNAs tile each target variant)
    variant library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design    		Cells are sorted based on FACS signal quantiles
    variant library design
    diff --git a/docs/prolif_gwas.rst b/docs/prolif_gwas.rst index 35d4175..2bdfa6b 100644 --- a/docs/prolif_gwas.rst +++ b/docs/prolif_gwas.rst @@ -1,4 +1,4 @@ -GWAS variant library +Proliferation screen with GWAS library *********************** .. mdinclude:: profile_gwas.md diff --git a/docs/qc.md b/docs/qc.md index ce85c44..ee89c3e 100755 --- a/docs/qc.md +++ b/docs/qc.md @@ -17,7 +17,7 @@ bean qc \ `bean qc` supports following quality control and masks samples with low quality. Specifically: -Allele translation +Allele translation * Plots guide coverage and the uniformity of coverage * Guide count correlation between samples diff --git a/docs/reporterscreen.md b/docs/reporterscreen.md index 0450470..03ce056 100644 --- a/docs/reporterscreen.md +++ b/docs/reporterscreen.md @@ -1,6 +1,6 @@ BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). -ReporterScreen object structure +ReporterScreen object structure * `.guides`: guide information provided in input (`gRNA_library.csv` in above example) * `.samples`: sample information provided in input (`sample_list.csv` in above example) diff --git a/docs/run.md b/docs/run.md index c1de5bf..b0a478c 100755 --- a/docs/run.md +++ b/docs/run.md @@ -6,7 +6,7 @@ BEAN uses Bayesian network to incorporate gRNA editing outcome to provide poster For the full detail, see the method section of the [BEAN manuscript](https://www.medrxiv.org/content/10.1101/2023.09.08.23295253v1). -model +model

    @@ -45,7 +45,7 @@ See full list of parameters [below](#full-parameters). # Output -model +model Above command produces * `output_prefix/bean_element_result.[model_type].csv` with following columns: diff --git a/docs/tutorials/ldl_cds.md b/docs/tutorials/ldl_cds.md index e2d5ce0..8b0379f 100755 --- a/docs/tutorials/ldl_cds.md +++ b/docs/tutorials/ldl_cds.md @@ -4,11 +4,11 @@ Tiling screen that tiles gRNA densely across locus or multiple loci, selected ba - + - +
    Library designTiling (gRNAs tile each locus densely)
    tiling library design     		Tiling (gRNAs tile each locus densely)
    tiling library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design    		Cells are sorted based on FACS signal quantiles
    variant library design
    diff --git a/docs/tutorials/ldl_var.md b/docs/tutorials/ldl_var.md index cdbf869..7eb1af7 100755 --- a/docs/tutorials/ldl_var.md +++ b/docs/tutorials/ldl_var.md @@ -1,14 +1,14 @@ -# Variant sorting screen tutorial +## Variant sorting screen tutorial GWAS variant screen with per-variant gRNA tiling design, selected based on FACS signal quantiles. - + - +
    Library designVariant (gRNAs tile each target variant)
    variant library design    		Variant (gRNAs tile each target variant)
    variant library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design    		Cells are sorted based on FACS signal quantiles
    variant library design
    From d9445abc145e85d6710a720a6d7d419cb32ec666 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 11:17:06 -0400 Subject: [PATCH 088/107] change copy dir --- .github/workflows/documentation.yml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/.github/workflows/documentation.yml b/.github/workflows/documentation.yml index 44e9f15..d8b6316 100755 --- a/.github/workflows/documentation.yml +++ b/.github/workflows/documentation.yml @@ -20,7 +20,7 @@ jobs: working-directory: docs run: sphinx-build . _build - name: copy image files - run: cp -r docs/assets docs/_build/assets/ + run: cp -r docs/assets docs/_build/ - uses: actions/upload-pages-artifact@v3 with: name: github-pages From 2ac37dc2ff9510d86185463dab952afc52c66351 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 11:21:45 -0400 Subject: [PATCH 089/107] add missing files --- docs/cds.rst | 2 +- docs/index.rst | 2 +- docs/input.rst | 2 +- docs/prolif_gwas.rst | 2 +- 4 files changed, 4 insertions(+), 4 deletions(-) diff --git a/docs/cds.rst b/docs/cds.rst index 2d15d26..c566387 100755 --- a/docs/cds.rst +++ b/docs/cds.rst @@ -1,5 +1,5 @@ Coding sequence tiling library *********************** -.. mdinclude:: commands/ldl_cds.md +.. mdinclude:: ldl_cds.md See :ref:`subcommands` for the full details. diff --git a/docs/index.rst b/docs/index.rst index 567b619..8ef251d 100755 --- a/docs/index.rst +++ b/docs/index.rst @@ -9,7 +9,7 @@ Welcome to `bean`'s documentation! Workflows =================== .. toctree:: - :maxdepth: 2 + :maxdepth: 1 gwas cds diff --git a/docs/input.rst b/docs/input.rst index d63417e..d018e97 100755 --- a/docs/input.rst +++ b/docs/input.rst @@ -1,4 +1,4 @@ .. _input: Input file format *********************** -.. mdinclude:: commands/input.md \ No newline at end of file +.. mdinclude:: input.md \ No newline at end of file diff --git a/docs/prolif_gwas.rst b/docs/prolif_gwas.rst index 2bdfa6b..34adb83 100644 --- a/docs/prolif_gwas.rst +++ b/docs/prolif_gwas.rst @@ -1,5 +1,5 @@ Proliferation screen with GWAS library -*********************** +********************************************** .. mdinclude:: profile_gwas.md See :ref:`subcommands` for the full details. From 03d093ef6fe8e504ea8ed643e00530cf7f294297 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 11:32:26 -0400 Subject: [PATCH 090/107] prioritize rst --- docs/conf.py | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/docs/conf.py b/docs/conf.py index 0a8e58c..6b99fd5 100755 --- a/docs/conf.py +++ b/docs/conf.py @@ -21,7 +21,7 @@ root_doc = "index" numpydoc_show_class_members = False - +source_suffix = [".rst", ".md"] # -- Options for HTML output ------------------------------------------------- # https://www.sphinx-doc.org/en/master/usage/configuration.html#options-for-html-output From a0993419e0ca1295855fbb711e5609d4568294ce Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 11:37:35 -0400 Subject: [PATCH 091/107] disamgibuagte names --- docs/_count.md | 31 +++++++++ docs/_create-screen.md | 9 +++ docs/_filter.md | 37 ++++++++++ docs/_input.md | 37 ++++++++++ docs/_ldl_cds.md | 150 ++++++++++++++++++++++++++++++++++++++++ docs/_ldl_var.md | 117 +++++++++++++++++++++++++++++++ docs/_profile.md | 8 +++ docs/_prolif_gwas.md | 118 +++++++++++++++++++++++++++++++ docs/_qc.md | 94 +++++++++++++++++++++++++ docs/_reporterscreen.md | 11 +++ docs/_run.md | 64 +++++++++++++++++ 11 files changed, 676 insertions(+) create mode 100755 docs/_count.md create mode 100755 docs/_create-screen.md create mode 100755 docs/_filter.md create mode 100755 docs/_input.md create mode 100755 docs/_ldl_cds.md create mode 100755 docs/_ldl_var.md create mode 100755 docs/_profile.md create mode 100644 docs/_prolif_gwas.md create mode 100755 docs/_qc.md create mode 100644 docs/_reporterscreen.md create mode 100755 docs/_run.md diff --git a/docs/_count.md b/docs/_count.md new file mode 100755 index 0000000..7400cba --- /dev/null +++ b/docs/_count.md @@ -0,0 +1,31 @@ +# `bean count[-samples]`: Count (reporter) screen data +`bean count-samples` (or `bean count` for a single sample) maps guide into guide counts, **allowing for base transition in spacer sequence**. When the matched reporter information is provided, it can count the **target site edits** and **alleles produced by each guide**. Mapping is efficiently done based on [CRISPResso2](https://github.com/pinellolab/CRISPResso2) modified for base-edit-aware mapping. + + + +```python +bean count-samples \ + --input sample_list.csv `# sample with lines 'R1_filepath,R2_filepath,sample_name\n'` \ + -b A `# base that is being edited (A/G)` \ + -f sgRNA_info_table.csv `# sgRNA information` \ + -o . `# output directory` \ + -r `# read edit/allele information from reporter` \ + -t 12 `# number of threads` \ + --name my_sorting_screen `# name of this sample run` \ +``` +```python +bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r +``` +By default, `bean count[-samples]` assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure. + + Read structuren + +See full detail [below](#full-parameters). + +# Input file format +See :ref:`input` for input file formats. + +# Output file format +`count` or `count-samples` produces `.h5ad` and `.xlsx` file with guide and per-guide allele counts. +* `.h5ad`: This output file follows annotated matrix format compatible with `AnnData` and is based on `Screen` object in [purturb_tools](https://github.com/pinellolab/perturb-tools). See [Data Structure](#data-structure) section for more information. +* `.xlsx`: This output file contains `.guides`, `.samples`, `.X[_bcmatch,_edits]`. (`allele_tables` are often too large to write into an Excel!) diff --git a/docs/_create-screen.md b/docs/_create-screen.md new file mode 100755 index 0000000..d9200b7 --- /dev/null +++ b/docs/_create-screen.md @@ -0,0 +1,9 @@ +# `bean create-screen`: Create ReporterScreen object from flat files +```bash +bean create-screen gRNA_library.csv sample_list.csv gRNA_counts_table.csv +``` +## Input + * gRNA_library.csv + * sample_list.csv + * gRNA_counts_table.csv: Table with gRNA ID in the first column and sample IDs as the column names (first row) +`gRNA_library.csv` and `sample_list.csv` should be formatted as :ref:`input`. \ No newline at end of file diff --git a/docs/_filter.md b/docs/_filter.md new file mode 100755 index 0000000..e05373d --- /dev/null +++ b/docs/_filter.md @@ -0,0 +1,37 @@ +# `filter`: Filtering (and optionally translating) alleles +As `tiling` mode of `bean run` accounts for any robustly observed alleles, `bean filter` filters for such alleles. +```bash +bean filter my_sorting_screen_masked.h5ad \ +-o my_sorting_screen_filtered.h5ad `# Output file path` \ +``` + +# Output +Above command produces +* `my_sorting_screen_filtered.h5ad` with filtered alleles stored in `.uns`, +* `my_sorting_screen_filtered.filtered_allele_stats.pdf`, and `my_sorting_screen_filtered.filter_log.txt` that report allele count stats in each filtering step. + +You may want to adjust the flitering parameters to obtain optimal balance between # guides per variant & # variants that are scored. See example outputs of filtering step [here](docs/example_filtering_output/). + + +# Translating alleles +If you want to obtain **amino acid level variant** for coding sequence tiling screens, provide coding sequence positions which variants occuring within the coding sequence will be translated. *This is optional, but **highly recommended** to increase per-(coding)variant support.* + +Allele translation + + +```bash +bean filter my_sorting_screen.h5ad \ +-o my_sorting_screen_masked.h5ad \ +--translate `# Translate coding variants` \ +[ --translate-gene-name GENE_SYMBOL OR + --translate-genes-list path_to_gene_names_file.txt OR + --translate-fasta gene_exon.fa, OR + --translate-fastas-csv gene_exon_fas.csv] +``` +* When library covers a single gene, do either of the following: + 1. Feed `--translate-gene-name GENE_SYMBOL` if your `genomic_pos` column of `sgRNA_info_tbl` is compatible with [MANE transcript](https://useast.ensembl.org/info/genome/genebuild/mane.html)'s reference genome. (Per 10/23/2023, GRCh38). This will automatically load the exon positions based on MANE transcript annotation. + 2. To use your custom coding sequence and exon positions, feed `--translate-fasta gene_exon.fa` argument where `gene_exon.fa` is the FASTA file with entries of exons. [See full details here](docs/exon_fa_format.md). +* When library covers multiple genes, do either of the following: + 1. Feed `--translate-genes-list path_to_gene_names_file.txt` where `path_to_gene_names_file.txt` is file with one gene symbol per line. + 2. Feed `--translate-fastas-csv gene_exon_fas.csv` where `gene_exon_fas.csv` is the csv file with lines `gene_id,gene_exon_fasta_path` without header. Each FASTA file in `gene_exon_fasta_path` is formatted [as the single-gene FASTA file](docs/exon_fa_format.md). +* Translation will keep the variants outside the coding sequence as nucleotide-level variants, while aggregating variants leading to the same coding sequence variants. diff --git a/docs/_input.md b/docs/_input.md new file mode 100755 index 0000000..21368b8 --- /dev/null +++ b/docs/_input.md @@ -0,0 +1,37 @@ +This document describes the input files of :ref:`count_samples`. +## sgRNA_info_table.csv +File should contain following columns. +* `name`: gRNA ID column +* `sequence`: gRNA sequence +* `barcode`: R2 barcode to help match reporter to gRNA, written in the sense direction (as in R1) +* In order to use accessibility in the [variant effect quantification](#bean-run-quantify-variant-effects), provide accessibility information in one of two options. (For non-targeting guides, provide NA values (empty cell).) + * Option 1: `chrom` & `genomic_pos`: Chromosome (ex. `chr19`) and genomic position of guide sequence. You will have to provide the path to the bigwig file with matching reference version in `bean run`. + * Option 2: `accessibility_signal`: ATAC-seq signal value of the target loci of each guide. +* For variant library (gRNAs are designed to target specific variants and ignores bystander edits) + * `target`: This column denotes which target variant/element of each gRNA. This is not used in `bean count[-samples]` but required to run `bean run` in later steps. + * `target_group`: If negative/positive control gRNA will be considered in `bean qc` and/or `bean run`, specify as "NegCtrl"/"PosCtrl" in this column. + * `target_pos`: If `--match_target_pos` flag is used, input file needs `target_pos` which specifies 0-based relative position of targeted base within Reporter sequence. +* For tiling library (gRNAs tile coding / noncoding sequences) + * `strand`: Specifies gRNA strand information relative to the reference genome. + * `chrom`: Chromosome of gRNA targeted locus. + * `start_pos`: gRNA starting position in the genome. Required when you provide `strand` column. Should specify the smaller coordinate value among start and end position regardless of gRNA strandedness. + +Also see examples for [variant library](tests/data/test_guide_info.csv) and [tiling library](tests/data/test_guide_info_tiling.csv). + +## sample_list.csv +File should contain following columns with header. +* `R1_filepath`: Path to read 1 `.fastq[.gz]` file +* `R2_filepath`: Path to read 1 `.fastq[.gz]` file +* `sample_id`: ID of sequencing sample +* `replicate`: Replicate # of this sample (Should NOT contain `.`) +* `condition`: Name of the sorting bin (ex. `top`, `bot`), or label of timepoint (ex. `D5`, `D18`) + +For FACS sorting screens: +* `upper_quantile`: FACS sorting upper quantile +* `lower_quantile`: FACS sorting lower quantile + +For proliferation / survival screens: +* `time`: Numeric time following the base editing of each sample. + + +Also see examples for [FACS sorting screen](tests/data/sample_list.csv) and [proliferation / survival screen](tests/data/sample_list_survival.csv). \ No newline at end of file diff --git a/docs/_ldl_cds.md b/docs/_ldl_cds.md new file mode 100755 index 0000000..8b0379f --- /dev/null +++ b/docs/_ldl_cds.md @@ -0,0 +1,150 @@ +# Tiling sorting screen tutorial +Tiling screen that tiles gRNA densely across locus or multiple loci, selected based on FACS signal quantiles. + + + + + + + + + + +
    Library designTiling (gRNAs tile each locus densely)
    tiling library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design
    + +

    + +## Example workflow +```bash +screen_id=my_sorting_tiling_screen +working_dir=my_workdir + +# 1. Count gRNA & reporter +bean count-samples \ +--input ${working_dir}/sample_list_tiling.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f ${working_dir}/test_guide_info_tiling_chrom.csv `# Contains gRNA metadata; see test file for example.`\ +-o $working_dir `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen` \ +--tiling + +# 2. QC samples & guides +bean qc \ + ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ + +# 3. Filter & translate alleles +bean filter ${working_dir}/bean_count_${screen_id}_masked.h5ad \ +-o ${working_dir}/bean_count_${screen_id}_alleleFiltered \ +--filter-target-basechange `# Filter based on intended base changes. If -b A was provided in bean count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\ +--filter-window --edit-start-pos 0 --edit-end-pos 19 `# Filter based on editing window in spacer position within reporter.`\ +--filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \ +--translate --translate-genes-list ${working_dir}/gene_symbols.txt + +# 4. Quantify variant effect +bean run sorting tiling \ + ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ + -o $working_dir \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility +``` +See more details below. + +## 1. Count gRNA & reporter (:ref:`count_samples`) +``` +screen_id=my_sorting_tiling_screen +working_dir=my_workdir + +bean count-samples \ +--input ${working_dir}/sample_list_tiling.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f ${working_dir}/test_guide_info_tiling_chrom.csv `# Contains gRNA metadata; see test file for example.`\ +-o $working_dir `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen` \ +--tiling +``` +Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. + +## 2. QC (:ref:`qc`) +Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean qc`](../../README#bean qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) +``` +bean qc \ + ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ + [--tiling] `# Not required if you have passed --tiling in counting step` +``` + + + +If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. + +## 3. Filter alleles (:ref:`filter`) +As tiling library doesn't have designated per-gRNA target variant, any base edit observed in reporter may be the candidate variant, while having too many variants with very low editing rate significantly decreases the power. Variants are filtered based on multiple criteria in `bean fitler`. + +If the screen targets coding sequence, it's beneficial to translate edits into coding varaints whenever possible for better power. For translation, provide `--translate` and one of the following: +``` +[ --translate-gene-name GENE_SYMBOL OR + --translate-genes-list path_to_gene_names_file.txt OR + --translate-fasta gene_exon.fa, OR + --translate-fastas-csv gene_exon_fas.csv] +``` +where `path_to_gene_names_file.txt` has one gene symbol per line, and gene symbol uses its MANE transcript (hg38) coordinates of exons. In order to use other reference versions or transcript ID, you'll need to feed in fasta file. See detailed formatting of fasta file [here](../../README#translating-alleles). + +Example allele filtering given we're translating based on MANE transcript exons of multiple gene symbols: + +```bash +bean filter ${working_dir}/bean_count_${screen_id}_masked.h5ad \ +-o ${working_dir}/bean_count_${screen_id}_alleleFiltered \ +--filter-target-basechange `# Filter based on intended base changes. If -b A was provided in bean count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\ +--filter-window --edit-start-pos 0 --edit-end-pos 19 `# Filter based on editing window in spacer position within reporter.`\ +--filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \ +--translate --translate-genes-list ${working_dir}/gene_symbols.txt +``` + +Ouptut file `` shows number of alleles per guide and number of guides per variant, where we want high enough values for the latter. See the typical output for dataset with good editing coverage & filtering result [here](../example_filtering_ouptut/). + +## 4. Quantify variant effect (:ref:`run`) +By default, `bean run [sorting,survival] tiling` uses most filtered allele counts table for variant identification and quantification of their effects. **Check [allele filtering output](../example_filtering_ouptut/)** and choose alternative filtered allele counts table if necessary. + +`bean run` can take 3 run options to quantify editing rate: +1. From **reporter + accessibility** + 1-1. If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA accessibility score, + ``` + bean run sorting tiling \ + ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ + -o $working_dir \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility + ``` + 1-2. If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, + ``` + bean run sorting tiling \ + ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ + -o $working_dir \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-bw accessibility.bw + ``` + +2. From **reporter** + ``` + bean run sorting tiling \ + ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ + -o $working_dir \ + --fit-negctrl + ``` +3. No reporter information, assume the same editing efficiency of all gRNAs. + Use this option if your data don't have editing rate information. + ``` + bean run sorting tiling \ + ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ + -o $working_dir \ + --fit-negctrl \ + --uniform-edit + ``` \ No newline at end of file diff --git a/docs/_ldl_var.md b/docs/_ldl_var.md new file mode 100755 index 0000000..7eb1af7 --- /dev/null +++ b/docs/_ldl_var.md @@ -0,0 +1,117 @@ +## Variant sorting screen tutorial +GWAS variant screen with per-variant gRNA tiling design, selected based on FACS signal quantiles. + + + + + + + + + + +
    Library designVariant (gRNAs tile each target variant)
    variant library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design
    + +

    + +## Example workflow +```bash +screen_id=my_sorting_tiling_screen +working_dir=my_workdir + +# 1. Count gRNA & reporter +bean-count-samples \ +--input ${working_dir}//sample_list.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f ${working_dir}/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ +-o ./ `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen to be counted` + +# 2. QC samples & guides +bean-qc \ + ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ + -b ` # Remove replicates with no good samples. + +# 3. Quantify variant effect +bean-run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility +``` +See more details below. + +## 1. Count gRNA & reporter (:ref:`count_samples`) +```bash +screen_id=my_sorting_tiling_screen + +# 1. Count gRNA & reporter +bean-count-samples \ +--input ${working_dir}/sample_list.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f ${working_dir}/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ +-o ./ `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen to be counted` +``` +Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. + +## 2. QC samples & guides (:ref:`qc`) +Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean-qc`](../../README#bean-qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) +``` +bean-qc \ + bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r qc_report_${screen_id} `# Prefix for QC report` +``` + + + +If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. + + +## 3. Quantify variant effect (:ref:`run`) + +`bean-run` can take 3 run options to quantify editing rate: +1. From **reporter + accessibility** + If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA accessibility score, + ``` + bean-run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility + ``` + If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, + ``` + bean-run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-bw accessibility.bw + ``` + +2. From **reporter**, without accessibility + + This assumes the all target sites have the uniform chromatin accessibility. + ``` + bean-run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ + --fit-negctrl + ``` +3. No reporter information, assume the same editing efficiency of all gRNAs. + Use this option if your data don't have editing outcome information. + ``` + bean-run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ + --fit-negctrl \ + --uniform-edit + ``` \ No newline at end of file diff --git a/docs/_profile.md b/docs/_profile.md new file mode 100755 index 0000000..cd40cb4 --- /dev/null +++ b/docs/_profile.md @@ -0,0 +1,8 @@ +# `bean profile`: Profile editing patterns +```bash +bean profile my_sorting_screen.h5ad -o output_prefix `# Prefix for editing profile report` +``` +# Output +Above command produces `prefix_editing_preference.[html,ipynb]` as editing preferences ([see example](../notebooks/profile_editing_preference.ipynb)). + +Allele translation \ No newline at end of file diff --git a/docs/_prolif_gwas.md b/docs/_prolif_gwas.md new file mode 100644 index 0000000..9af45ee --- /dev/null +++ b/docs/_prolif_gwas.md @@ -0,0 +1,118 @@ +## Variant survival screen tutorial +GWAS variant screen with per-variant gRNA tiling design, selected based on FACS signal quantiles. + + + + + + + + + + +
    Library designVariant (gRNAs tile each target variant)
    variant library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design
    + +

    + +## Example workflow +```bash +screen_id=my_sorting_tiling_screen +working_dir=my_workdir + +# 1. Count gRNA & reporter +bean count-samples \ +--input ${working_dir}/sample_list.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f ${working_dir}/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ +-o ${working_dir} `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen to be counted` + +# 2. QC samples & guides +bean qc \ + ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ + --lfc-conds D0,D14 `# Conditions to calculate LFC of positive controls` \ + -b ` # Remove replicates with no good samples. + +# 3. Quantify variant effect +bean run survival variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o ${working_dir}/ \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility +``` +See more details below. + +## 1. Count gRNA & reporter (:ref:`count_samples`) +```bash +bean count-samples \ +--input ${working_dir}/sample_list.csv `# Contains fastq file path; see test file for example.`\ +-b A `# Base A is edited (into G)` \ +-f ${working_dir}/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ +-o ${working_dir} `# Output directory` \ +-r `# Quantify reporter edits` \ +-n ${screen_id} `# ID of the screen to be counted` +``` +Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. + +## 2. QC samples & guides (:ref:`qc`) +Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean qc`](../../README#bean qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) +``` +bean qc \ + ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ + -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ + --lfc-conds D0,D14 `# Conditions to calculate LFC of positive controls` \ + -b ` # Remove replicates with no good samples. + +``` + + + +If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. + + +## 3. Quantify variant effect (:ref:`run`) + +`bean run` can take 3 run options to quantify editing rate: +1. From **reporter + accessibility** + If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA accessibility score, + ``` + bean run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o $working_dir \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-col accessibility + ``` + If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, + ``` + bean run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o $working_dir \ + --fit-negctrl \ + --scale-by-acc \ + --accessibility-bw accessibility.bw + ``` + +2. From **reporter**, without accessibility + + This assumes the all target sites have the uniform chromatin accessibility. + ``` + bean run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o $working_dir \ + --fit-negctrl + ``` +3. No reporter information, assume the same editing efficiency of all gRNAs. + Use this option if your data don't have editing outcome information. + ``` + bean run sorting variant \ + ${working_dir}/bean_count_${screen_id}_masked.h5ad \ + -o $working_dir \ + --fit-negctrl \ + --uniform-edit + ``` \ No newline at end of file diff --git a/docs/_qc.md b/docs/_qc.md new file mode 100755 index 0000000..ee89c3e --- /dev/null +++ b/docs/_qc.md @@ -0,0 +1,94 @@ +# `bean qc`: QC of reporter screen data +```bash +bean qc \ + my_sorting_screen.h5ad `# Input ReporterScreen .h5ad file path` \ + -o my_sorting_screen_masked.h5ad `# Output ReporterScreen .h5ad file path` \ + -r qc_report_my_sorting_screen `# Prefix for QC report` \ + --ctrl-cond presort `# "condition" column in the control sample before selection. Mean gRNA editing rates in these samples are reported. ` \ +# Inspect the output qc_report_my_sorting_screen.html to tweak QC threshold + +bean qc \ + my_sorting_screen.h5ad \ + -o my_sorting_screen_masked.h5ad \ + -r qc_report_my_sorting_screen \ + #[--count-correlation-thres 0.7 ...]\ + -b +``` + +`bean qc` supports following quality control and masks samples with low quality. Specifically: + +Allele translation + +* Plots guide coverage and the uniformity of coverage +* Guide count correlation between samples +* Log fold change correlation when positive controls are provided +* Plots editing rate distribution +* Identify samples with low guide coverage/guide count correlation/editing rate and mask the sample in `bdata.samples.mask` +* Identify outlier guides to filter out + +# Output +Above command produces +* `my_sorting_screen_masked.h5ad` without problematic replicate and guides and with sample masks, and +* `qc_report_my_sorting_screen.[html,ipynb]` as QC report. +##### Optional arguments: +* `-o OUT_SCREEN_PATH`, `--out-screen-path OUT_SCREEN_PATH` + Path where quality-filtered ReporterScreen object to be written to +* `-r OUT_REPORT_PREFIX`, `--out-report-prefix OUT_REPORT_PREFIX` + Output prefix of qc report (prefix.html, prefix.ipynb) + +##### QC thresholds: +* `--count-correlation-thres COUNT_CORRELATION_THRES` + Correlation threshold to mask out. +* `--edit-rate-thres EDIT_RATE_THRES` + Mean editing rate threshold per sample to mask out. +* `--lfc-thres LFC_THRES` + Positive guides' correlation threshold to filter out. + +##### Run options: +* `-b`, `--remove-bad-replicates` + Remove replicates with at least two of its samples meet the QC threshold (bean run does not support having only one sorting bin sample for a replicate). +* `-i`, `--ignore-missing-samples` + If the flag is not provided, if the ReporterScreen object does not contain all condiitons for + each replicate, make fake empty samples. If the flag is provided, don't add dummy samples. +* `--no-editing` Ignore QC about editing. Can be used for QC of other editing modalities. +* `--dont-recalculate-edits` + When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from + ReporterScreen.uns['allele_count']. + +##### Input `.h5ad` formatting: +Note that these arguements will change the way the QC metrics are calculated for guides, samples, or replicates. +* `--tiling TILING` Specify that the guide library is tiling library without 'n guides per target' design +* `--replicate-label REPLICATE_LABEL` + Label of column in `bdata.samples` that describes replicate ID. +* `--sample-covariates SAMPLE_COVARIATES` + Comma-separated list of column names in `bdata.samples` that describes non-selective + experimental condition. (drug treatment, etc.) +* `--condition-label CONDITION_LABEL` + Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time, + etc.) +###### Editing rate calculation + * `--control-condition CTRL_COND` + Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate + to be calculated. Default is `None`, which considers all samples. + * `--rel-pos-is-reporter` + Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If + `False`, those are relative to spacer position. + Editing rate is calculated with following parameters in + * Variant screens: + * `--target-pos-col TARGET_POS_COL` + Target position column in `bdata.guides` specifying target edit position in reporter + * tiling screens: + * `--edit-start-pos EDIT_START_POS` + Edit start position to quantify editing rate on, 0-based inclusive. + * `--edit-end-pos EDIT_END_POS` + Edit end position to quantify editing rate on, 0-based exclusive. +###### LFC of positive controls + * `--posctrl-col POSCTRL_COL` + Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all + gRNAs, feed empty string ''. + * `--posctrl-val POSCTRL_VAL` + Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the + positive control in calculating log fold change. + * `--lfc-conds LFC_CONDS` + Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated + between, delimited by comma \ No newline at end of file diff --git a/docs/_reporterscreen.md b/docs/_reporterscreen.md new file mode 100644 index 0000000..03ce056 --- /dev/null +++ b/docs/_reporterscreen.md @@ -0,0 +1,11 @@ +BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). + +ReporterScreen object structure + + * `.guides`: guide information provided in input (`gRNA_library.csv` in above example) + * `.samples`: sample information provided in input (`sample_list.csv` in above example) + * `.X`: Main guide count matrix, where row corresponds to each guide in `.guides` and columns correspond to samples in `.samples`. +Following attributes are included if matched reporter is provided and you chose to read edit/allele information from the reporter using `-r` option. + * `.X_bcmatch [Optional]`: Contains information about number of barcode-matched reads. Information about R2 barcode should be specified as `barcode` column in your `gRNA_library.csv` file. + * `.X_edits [Optional]`: If target position of each guide is specified as `target_pos` in input `gRNA_library.csv` file and `--match-target-position` option is provided, the result has the matrix with the number of target edit at the specified positions. + * `.allele_tables [Optional]`: Dictionary with a single allele count table that counts per guide and allele combination, what is the count per sample. \ No newline at end of file diff --git a/docs/_run.md b/docs/_run.md new file mode 100755 index 0000000..b0a478c --- /dev/null +++ b/docs/_run.md @@ -0,0 +1,64 @@ +# `bean run`: Quantify variant effects +BEAN uses Bayesian network to incorporate gRNA editing outcome to provide posterior estimate of variant phenotype. The Bayesian network reflects data generation process. Briefly, +1. Cellular phenotype (either for cells are sorted upon for sorting screen, or log(proliferation rate)) is modeled as the Gaussian mixture distribution of wild-type phenotype and variant phenotype. +2. The weight of the mixture components are inferred from the reporter editing outcome and the chromatin accessibility of the loci. +3. Cells with each gRNA, formulated as the mixture distribution, is sorted by the phenotypic quantile to produce the gRNA counts. + +For the full detail, see the method section of the [BEAN manuscript](https://www.medrxiv.org/content/10.1101/2023.09.08.23295253v1). + +model + +

    + +# Usage example +```bash +bean run sorting[survival] variant[tiling] my_sorting_screen_filtered.h5ad \ +[--uniform-edit, --scale-by-acc [--acc-bw-path accessibility_signal.bw, --acc-col accessibility]] \ +-o output_prefix/ \ +--fit-negctrl +``` +See full list of parameters [below](#full-parameters). + + +# Input +`my_sorting_screen_filtered.h5ad` can be produced by one of the following: +1. [`bean count-samples`]((#bean-count-samples-count-reporter-screen-data)) when you have raw `.fastq` file +2. (Limited to `bean run variant` mode) `bean create-screen` when you have flat `.csv` tables of gRNA metadata table, sample metadata table, gRNA counts table (# guides x # samples), and optionally # edits table. + ```bash + bean create-screen gRNA_info_table.csv sample_info_table.csv gRNA_counts_table.csv \ + [--edits edit_counts_table.csv -o output.h5ad] + ``` + * `gRNA_info_table.csv` should have following columns. + * `name`: gRNA ID column + * `target`: This column denotes which target variant/element of each gRNA. + * `target_group [Optional]`: If negative control gRNA will be used, specify as "NegCtrl" in this column. + * `sample_info_table.csv` should have following columns. + * `sample_id`: ID of sequencing sample + * `replicate`: Replicate # of this sample + * `bin`: Name of the sorting bin + * `upper_quantile`: FACS sorting upper quantile + * `lower_quantile`: FACS sorting lower quantile + * `gRNA_counts_table.csv` should be formatted as follows. + * Columns include one of `sample_id` columns in `sample_info_table.csv` file. + * 1st row (row index) follows `name` (gRNA ID) in `gRNA_info_table.csv` file. +3. You can manually create the `AnnData` object with more annotations including allele counts: see [API tutorial](#using-bean-as-python-module) for full detail. + + +# Output +model + +Above command produces +* `output_prefix/bean_element_result.[model_type].csv` with following columns: + * Estimated variant effect sizes + * `mu` (Effect size): Mean of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. + * `mu_sd`: Mean of variant phenotype `mu` is modeled as normal distribution. The column shows fitted standard deviation of `mu` that quantify the uncertainty of the variant effect. + * `mu_z`: z-score of `mu` + * `sd`: Standard deviation of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. + * `CI[0.025`, `0.975]`: Credible interval of `mu` + * When negative control is provided, above columns with `_adj` suffix are provided, which are the corresponding values adjusted for negative control. + * Metrics on per-variant evidence provided in input (provided in `tiling` mode) + * `effective_edit_rate`: Sum of per-variant editing rates over all alleles observed in the input. Allele-level editing rate is divided by the number of variants observed in the allele prior to summing up. + * `n_guides`: # of guides covering the variant. + * `n_coocc`: # of cooccurring variants with a given variant in any alleles observed in the input. +* `output_prefix/bean_sgRNA_result.[model_type].csv`: + * `edit_rate`: Estimated editing rate at the target loci. From d29e73db337589161bfc604996931d67b04440ed Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 11:42:00 -0400 Subject: [PATCH 092/107] remove old files --- docs/count.md | 31 --------- docs/create-screen.md | 9 --- docs/filter.md | 37 ---------- docs/input.md | 37 ---------- docs/ldl_cds.md | 150 ----------------------------------------- docs/ldl_var.md | 117 -------------------------------- docs/profile.md | 8 --- docs/prolif_gwas.md | 118 -------------------------------- docs/qc.md | 94 -------------------------- docs/reporterscreen.md | 11 --- docs/run.md | 64 ------------------ 11 files changed, 676 deletions(-) delete mode 100755 docs/count.md delete mode 100755 docs/create-screen.md delete mode 100755 docs/filter.md delete mode 100755 docs/input.md delete mode 100755 docs/ldl_cds.md delete mode 100755 docs/ldl_var.md delete mode 100755 docs/profile.md delete mode 100644 docs/prolif_gwas.md delete mode 100755 docs/qc.md delete mode 100644 docs/reporterscreen.md delete mode 100755 docs/run.md diff --git a/docs/count.md b/docs/count.md deleted file mode 100755 index 7400cba..0000000 --- a/docs/count.md +++ /dev/null @@ -1,31 +0,0 @@ -# `bean count[-samples]`: Count (reporter) screen data -`bean count-samples` (or `bean count` for a single sample) maps guide into guide counts, **allowing for base transition in spacer sequence**. When the matched reporter information is provided, it can count the **target site edits** and **alleles produced by each guide**. Mapping is efficiently done based on [CRISPResso2](https://github.com/pinellolab/CRISPResso2) modified for base-edit-aware mapping. - - - -```python -bean count-samples \ - --input sample_list.csv `# sample with lines 'R1_filepath,R2_filepath,sample_name\n'` \ - -b A `# base that is being edited (A/G)` \ - -f sgRNA_info_table.csv `# sgRNA information` \ - -o . `# output directory` \ - -r `# read edit/allele information from reporter` \ - -t 12 `# number of threads` \ - --name my_sorting_screen `# name of this sample run` \ -``` -```python -bean count --R1 R1.fq --R2 R2.fq -b A -f sgRNA_info_table.csv -r -``` -By default, `bean count[-samples]` assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure. - - Read structuren - -See full detail [below](#full-parameters). - -# Input file format -See :ref:`input` for input file formats. - -# Output file format -`count` or `count-samples` produces `.h5ad` and `.xlsx` file with guide and per-guide allele counts. -* `.h5ad`: This output file follows annotated matrix format compatible with `AnnData` and is based on `Screen` object in [purturb_tools](https://github.com/pinellolab/perturb-tools). See [Data Structure](#data-structure) section for more information. -* `.xlsx`: This output file contains `.guides`, `.samples`, `.X[_bcmatch,_edits]`. (`allele_tables` are often too large to write into an Excel!) diff --git a/docs/create-screen.md b/docs/create-screen.md deleted file mode 100755 index d9200b7..0000000 --- a/docs/create-screen.md +++ /dev/null @@ -1,9 +0,0 @@ -# `bean create-screen`: Create ReporterScreen object from flat files -```bash -bean create-screen gRNA_library.csv sample_list.csv gRNA_counts_table.csv -``` -## Input - * gRNA_library.csv - * sample_list.csv - * gRNA_counts_table.csv: Table with gRNA ID in the first column and sample IDs as the column names (first row) -`gRNA_library.csv` and `sample_list.csv` should be formatted as :ref:`input`. \ No newline at end of file diff --git a/docs/filter.md b/docs/filter.md deleted file mode 100755 index e05373d..0000000 --- a/docs/filter.md +++ /dev/null @@ -1,37 +0,0 @@ -# `filter`: Filtering (and optionally translating) alleles -As `tiling` mode of `bean run` accounts for any robustly observed alleles, `bean filter` filters for such alleles. -```bash -bean filter my_sorting_screen_masked.h5ad \ --o my_sorting_screen_filtered.h5ad `# Output file path` \ -``` - -# Output -Above command produces -* `my_sorting_screen_filtered.h5ad` with filtered alleles stored in `.uns`, -* `my_sorting_screen_filtered.filtered_allele_stats.pdf`, and `my_sorting_screen_filtered.filter_log.txt` that report allele count stats in each filtering step. - -You may want to adjust the flitering parameters to obtain optimal balance between # guides per variant & # variants that are scored. See example outputs of filtering step [here](docs/example_filtering_output/). - - -# Translating alleles -If you want to obtain **amino acid level variant** for coding sequence tiling screens, provide coding sequence positions which variants occuring within the coding sequence will be translated. *This is optional, but **highly recommended** to increase per-(coding)variant support.* - -Allele translation - - -```bash -bean filter my_sorting_screen.h5ad \ --o my_sorting_screen_masked.h5ad \ ---translate `# Translate coding variants` \ -[ --translate-gene-name GENE_SYMBOL OR - --translate-genes-list path_to_gene_names_file.txt OR - --translate-fasta gene_exon.fa, OR - --translate-fastas-csv gene_exon_fas.csv] -``` -* When library covers a single gene, do either of the following: - 1. Feed `--translate-gene-name GENE_SYMBOL` if your `genomic_pos` column of `sgRNA_info_tbl` is compatible with [MANE transcript](https://useast.ensembl.org/info/genome/genebuild/mane.html)'s reference genome. (Per 10/23/2023, GRCh38). This will automatically load the exon positions based on MANE transcript annotation. - 2. To use your custom coding sequence and exon positions, feed `--translate-fasta gene_exon.fa` argument where `gene_exon.fa` is the FASTA file with entries of exons. [See full details here](docs/exon_fa_format.md). -* When library covers multiple genes, do either of the following: - 1. Feed `--translate-genes-list path_to_gene_names_file.txt` where `path_to_gene_names_file.txt` is file with one gene symbol per line. - 2. Feed `--translate-fastas-csv gene_exon_fas.csv` where `gene_exon_fas.csv` is the csv file with lines `gene_id,gene_exon_fasta_path` without header. Each FASTA file in `gene_exon_fasta_path` is formatted [as the single-gene FASTA file](docs/exon_fa_format.md). -* Translation will keep the variants outside the coding sequence as nucleotide-level variants, while aggregating variants leading to the same coding sequence variants. diff --git a/docs/input.md b/docs/input.md deleted file mode 100755 index 21368b8..0000000 --- a/docs/input.md +++ /dev/null @@ -1,37 +0,0 @@ -This document describes the input files of :ref:`count_samples`. -## sgRNA_info_table.csv -File should contain following columns. -* `name`: gRNA ID column -* `sequence`: gRNA sequence -* `barcode`: R2 barcode to help match reporter to gRNA, written in the sense direction (as in R1) -* In order to use accessibility in the [variant effect quantification](#bean-run-quantify-variant-effects), provide accessibility information in one of two options. (For non-targeting guides, provide NA values (empty cell).) - * Option 1: `chrom` & `genomic_pos`: Chromosome (ex. `chr19`) and genomic position of guide sequence. You will have to provide the path to the bigwig file with matching reference version in `bean run`. - * Option 2: `accessibility_signal`: ATAC-seq signal value of the target loci of each guide. -* For variant library (gRNAs are designed to target specific variants and ignores bystander edits) - * `target`: This column denotes which target variant/element of each gRNA. This is not used in `bean count[-samples]` but required to run `bean run` in later steps. - * `target_group`: If negative/positive control gRNA will be considered in `bean qc` and/or `bean run`, specify as "NegCtrl"/"PosCtrl" in this column. - * `target_pos`: If `--match_target_pos` flag is used, input file needs `target_pos` which specifies 0-based relative position of targeted base within Reporter sequence. -* For tiling library (gRNAs tile coding / noncoding sequences) - * `strand`: Specifies gRNA strand information relative to the reference genome. - * `chrom`: Chromosome of gRNA targeted locus. - * `start_pos`: gRNA starting position in the genome. Required when you provide `strand` column. Should specify the smaller coordinate value among start and end position regardless of gRNA strandedness. - -Also see examples for [variant library](tests/data/test_guide_info.csv) and [tiling library](tests/data/test_guide_info_tiling.csv). - -## sample_list.csv -File should contain following columns with header. -* `R1_filepath`: Path to read 1 `.fastq[.gz]` file -* `R2_filepath`: Path to read 1 `.fastq[.gz]` file -* `sample_id`: ID of sequencing sample -* `replicate`: Replicate # of this sample (Should NOT contain `.`) -* `condition`: Name of the sorting bin (ex. `top`, `bot`), or label of timepoint (ex. `D5`, `D18`) - -For FACS sorting screens: -* `upper_quantile`: FACS sorting upper quantile -* `lower_quantile`: FACS sorting lower quantile - -For proliferation / survival screens: -* `time`: Numeric time following the base editing of each sample. - - -Also see examples for [FACS sorting screen](tests/data/sample_list.csv) and [proliferation / survival screen](tests/data/sample_list_survival.csv). \ No newline at end of file diff --git a/docs/ldl_cds.md b/docs/ldl_cds.md deleted file mode 100755 index 8b0379f..0000000 --- a/docs/ldl_cds.md +++ /dev/null @@ -1,150 +0,0 @@ -# Tiling sorting screen tutorial -Tiling screen that tiles gRNA densely across locus or multiple loci, selected based on FACS signal quantiles. - - - - - - - - - - -
    Library designTiling (gRNAs tile each locus densely)
    tiling library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design
    - -

    - -## Example workflow -```bash -screen_id=my_sorting_tiling_screen -working_dir=my_workdir - -# 1. Count gRNA & reporter -bean count-samples \ ---input ${working_dir}/sample_list_tiling.csv `# Contains fastq file path; see test file for example.`\ --b A `# Base A is edited (into G)` \ --f ${working_dir}/test_guide_info_tiling_chrom.csv `# Contains gRNA metadata; see test file for example.`\ --o $working_dir `# Output directory` \ --r `# Quantify reporter edits` \ --n ${screen_id} `# ID of the screen` \ ---tiling - -# 2. QC samples & guides -bean qc \ - ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ - -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ - -# 3. Filter & translate alleles -bean filter ${working_dir}/bean_count_${screen_id}_masked.h5ad \ --o ${working_dir}/bean_count_${screen_id}_alleleFiltered \ ---filter-target-basechange `# Filter based on intended base changes. If -b A was provided in bean count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\ ---filter-window --edit-start-pos 0 --edit-end-pos 19 `# Filter based on editing window in spacer position within reporter.`\ ---filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \ ---translate --translate-genes-list ${working_dir}/gene_symbols.txt - -# 4. Quantify variant effect -bean run sorting tiling \ - ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ - -o $working_dir \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-col accessibility -``` -See more details below. - -## 1. Count gRNA & reporter (:ref:`count_samples`) -``` -screen_id=my_sorting_tiling_screen -working_dir=my_workdir - -bean count-samples \ ---input ${working_dir}/sample_list_tiling.csv `# Contains fastq file path; see test file for example.`\ --b A `# Base A is edited (into G)` \ --f ${working_dir}/test_guide_info_tiling_chrom.csv `# Contains gRNA metadata; see test file for example.`\ --o $working_dir `# Output directory` \ --r `# Quantify reporter edits` \ --n ${screen_id} `# ID of the screen` \ ---tiling -``` -Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. - -## 2. QC (:ref:`qc`) -Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean qc`](../../README#bean qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) -``` -bean qc \ - ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ - -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ - [--tiling] `# Not required if you have passed --tiling in counting step` -``` - - - -If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. - -## 3. Filter alleles (:ref:`filter`) -As tiling library doesn't have designated per-gRNA target variant, any base edit observed in reporter may be the candidate variant, while having too many variants with very low editing rate significantly decreases the power. Variants are filtered based on multiple criteria in `bean fitler`. - -If the screen targets coding sequence, it's beneficial to translate edits into coding varaints whenever possible for better power. For translation, provide `--translate` and one of the following: -``` -[ --translate-gene-name GENE_SYMBOL OR - --translate-genes-list path_to_gene_names_file.txt OR - --translate-fasta gene_exon.fa, OR - --translate-fastas-csv gene_exon_fas.csv] -``` -where `path_to_gene_names_file.txt` has one gene symbol per line, and gene symbol uses its MANE transcript (hg38) coordinates of exons. In order to use other reference versions or transcript ID, you'll need to feed in fasta file. See detailed formatting of fasta file [here](../../README#translating-alleles). - -Example allele filtering given we're translating based on MANE transcript exons of multiple gene symbols: - -```bash -bean filter ${working_dir}/bean_count_${screen_id}_masked.h5ad \ --o ${working_dir}/bean_count_${screen_id}_alleleFiltered \ ---filter-target-basechange `# Filter based on intended base changes. If -b A was provided in bean count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\ ---filter-window --edit-start-pos 0 --edit-end-pos 19 `# Filter based on editing window in spacer position within reporter.`\ ---filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \ ---translate --translate-genes-list ${working_dir}/gene_symbols.txt -``` - -Ouptut file `` shows number of alleles per guide and number of guides per variant, where we want high enough values for the latter. See the typical output for dataset with good editing coverage & filtering result [here](../example_filtering_ouptut/). - -## 4. Quantify variant effect (:ref:`run`) -By default, `bean run [sorting,survival] tiling` uses most filtered allele counts table for variant identification and quantification of their effects. **Check [allele filtering output](../example_filtering_ouptut/)** and choose alternative filtered allele counts table if necessary. - -`bean run` can take 3 run options to quantify editing rate: -1. From **reporter + accessibility** - 1-1. If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA accessibility score, - ``` - bean run sorting tiling \ - ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ - -o $working_dir \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-col accessibility - ``` - 1-2. If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, - ``` - bean run sorting tiling \ - ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ - -o $working_dir \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-bw accessibility.bw - ``` - -2. From **reporter** - ``` - bean run sorting tiling \ - ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ - -o $working_dir \ - --fit-negctrl - ``` -3. No reporter information, assume the same editing efficiency of all gRNAs. - Use this option if your data don't have editing rate information. - ``` - bean run sorting tiling \ - ${working_dir}/bean_count_${screen_id}_alleleFiltered.h5ad \ - -o $working_dir \ - --fit-negctrl \ - --uniform-edit - ``` \ No newline at end of file diff --git a/docs/ldl_var.md b/docs/ldl_var.md deleted file mode 100755 index 7eb1af7..0000000 --- a/docs/ldl_var.md +++ /dev/null @@ -1,117 +0,0 @@ -## Variant sorting screen tutorial -GWAS variant screen with per-variant gRNA tiling design, selected based on FACS signal quantiles. - - - - - - - - - - -
    Library designVariant (gRNAs tile each target variant)
    variant library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design
    - -

    - -## Example workflow -```bash -screen_id=my_sorting_tiling_screen -working_dir=my_workdir - -# 1. Count gRNA & reporter -bean-count-samples \ ---input ${working_dir}//sample_list.csv `# Contains fastq file path; see test file for example.`\ --b A `# Base A is edited (into G)` \ --f ${working_dir}/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ --o ./ `# Output directory` \ --r `# Quantify reporter edits` \ --n ${screen_id} `# ID of the screen to be counted` - -# 2. QC samples & guides -bean-qc \ - ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ - -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ - -b ` # Remove replicates with no good samples. - -# 3. Quantify variant effect -bean-run sorting variant \ - ${working_dir}/bean_count_${screen_id}_masked.h5ad \ - -o ${working_dir}/ \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-col accessibility -``` -See more details below. - -## 1. Count gRNA & reporter (:ref:`count_samples`) -```bash -screen_id=my_sorting_tiling_screen - -# 1. Count gRNA & reporter -bean-count-samples \ ---input ${working_dir}/sample_list.csv `# Contains fastq file path; see test file for example.`\ --b A `# Base A is edited (into G)` \ --f ${working_dir}/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ --o ./ `# Output directory` \ --r `# Quantify reporter edits` \ --n ${screen_id} `# ID of the screen to be counted` -``` -Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. - -## 2. QC samples & guides (:ref:`qc`) -Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean-qc`](../../README#bean-qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) -``` -bean-qc \ - bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ - -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r qc_report_${screen_id} `# Prefix for QC report` -``` - - - -If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. - - -## 3. Quantify variant effect (:ref:`run`) - -`bean-run` can take 3 run options to quantify editing rate: -1. From **reporter + accessibility** - If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA accessibility score, - ``` - bean-run sorting variant \ - ${working_dir}/bean_count_${screen_id}_masked.h5ad \ - -o ${working_dir}/ \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-col accessibility - ``` - If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, - ``` - bean-run sorting variant \ - ${working_dir}/bean_count_${screen_id}_masked.h5ad \ - -o ${working_dir}/ \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-bw accessibility.bw - ``` - -2. From **reporter**, without accessibility - - This assumes the all target sites have the uniform chromatin accessibility. - ``` - bean-run sorting variant \ - ${working_dir}/bean_count_${screen_id}_masked.h5ad \ - -o ${working_dir}/ \ - --fit-negctrl - ``` -3. No reporter information, assume the same editing efficiency of all gRNAs. - Use this option if your data don't have editing outcome information. - ``` - bean-run sorting variant \ - ${working_dir}/bean_count_${screen_id}_masked.h5ad \ - -o ${working_dir}/ \ - --fit-negctrl \ - --uniform-edit - ``` \ No newline at end of file diff --git a/docs/profile.md b/docs/profile.md deleted file mode 100755 index cd40cb4..0000000 --- a/docs/profile.md +++ /dev/null @@ -1,8 +0,0 @@ -# `bean profile`: Profile editing patterns -```bash -bean profile my_sorting_screen.h5ad -o output_prefix `# Prefix for editing profile report` -``` -# Output -Above command produces `prefix_editing_preference.[html,ipynb]` as editing preferences ([see example](../notebooks/profile_editing_preference.ipynb)). - -Allele translation \ No newline at end of file diff --git a/docs/prolif_gwas.md b/docs/prolif_gwas.md deleted file mode 100644 index 9af45ee..0000000 --- a/docs/prolif_gwas.md +++ /dev/null @@ -1,118 +0,0 @@ -## Variant survival screen tutorial -GWAS variant screen with per-variant gRNA tiling design, selected based on FACS signal quantiles. - - - - - - - - - - -
    Library designVariant (gRNAs tile each target variant)
    variant library design
    SelectionCells are sorted based on FACS signal quantiles
    variant library design
    - -

    - -## Example workflow -```bash -screen_id=my_sorting_tiling_screen -working_dir=my_workdir - -# 1. Count gRNA & reporter -bean count-samples \ ---input ${working_dir}/sample_list.csv `# Contains fastq file path; see test file for example.`\ --b A `# Base A is edited (into G)` \ --f ${working_dir}/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ --o ${working_dir} `# Output directory` \ --r `# Quantify reporter edits` \ --n ${screen_id} `# ID of the screen to be counted` - -# 2. QC samples & guides -bean qc \ - ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ - -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ - --lfc-conds D0,D14 `# Conditions to calculate LFC of positive controls` \ - -b ` # Remove replicates with no good samples. - -# 3. Quantify variant effect -bean run survival variant \ - ${working_dir}/bean_count_${screen_id}_masked.h5ad \ - -o ${working_dir}/ \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-col accessibility -``` -See more details below. - -## 1. Count gRNA & reporter (:ref:`count_samples`) -```bash -bean count-samples \ ---input ${working_dir}/sample_list.csv `# Contains fastq file path; see test file for example.`\ --b A `# Base A is edited (into G)` \ --f ${working_dir}/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ --o ${working_dir} `# Output directory` \ --r `# Quantify reporter edits` \ --n ${screen_id} `# ID of the screen to be counted` -``` -Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. - -## 2. QC samples & guides (:ref:`qc`) -Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean qc`](../../README#bean qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) -``` -bean qc \ - ${working_dir}/bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ - -o ${working_dir}/bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r ${working_dir}/qc_report_${screen_id} `# Prefix for QC report` \ - --lfc-conds D0,D14 `# Conditions to calculate LFC of positive controls` \ - -b ` # Remove replicates with no good samples. - -``` - - - -If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. - - -## 3. Quantify variant effect (:ref:`run`) - -`bean run` can take 3 run options to quantify editing rate: -1. From **reporter + accessibility** - If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA accessibility score, - ``` - bean run sorting variant \ - ${working_dir}/bean_count_${screen_id}_masked.h5ad \ - -o $working_dir \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-col accessibility - ``` - If your gRNA metadata table (`${working_dir}/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, - ``` - bean run sorting variant \ - ${working_dir}/bean_count_${screen_id}_masked.h5ad \ - -o $working_dir \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-bw accessibility.bw - ``` - -2. From **reporter**, without accessibility - - This assumes the all target sites have the uniform chromatin accessibility. - ``` - bean run sorting variant \ - ${working_dir}/bean_count_${screen_id}_masked.h5ad \ - -o $working_dir \ - --fit-negctrl - ``` -3. No reporter information, assume the same editing efficiency of all gRNAs. - Use this option if your data don't have editing outcome information. - ``` - bean run sorting variant \ - ${working_dir}/bean_count_${screen_id}_masked.h5ad \ - -o $working_dir \ - --fit-negctrl \ - --uniform-edit - ``` \ No newline at end of file diff --git a/docs/qc.md b/docs/qc.md deleted file mode 100755 index ee89c3e..0000000 --- a/docs/qc.md +++ /dev/null @@ -1,94 +0,0 @@ -# `bean qc`: QC of reporter screen data -```bash -bean qc \ - my_sorting_screen.h5ad `# Input ReporterScreen .h5ad file path` \ - -o my_sorting_screen_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r qc_report_my_sorting_screen `# Prefix for QC report` \ - --ctrl-cond presort `# "condition" column in the control sample before selection. Mean gRNA editing rates in these samples are reported. ` \ -# Inspect the output qc_report_my_sorting_screen.html to tweak QC threshold - -bean qc \ - my_sorting_screen.h5ad \ - -o my_sorting_screen_masked.h5ad \ - -r qc_report_my_sorting_screen \ - #[--count-correlation-thres 0.7 ...]\ - -b -``` - -`bean qc` supports following quality control and masks samples with low quality. Specifically: - -Allele translation - -* Plots guide coverage and the uniformity of coverage -* Guide count correlation between samples -* Log fold change correlation when positive controls are provided -* Plots editing rate distribution -* Identify samples with low guide coverage/guide count correlation/editing rate and mask the sample in `bdata.samples.mask` -* Identify outlier guides to filter out - -# Output -Above command produces -* `my_sorting_screen_masked.h5ad` without problematic replicate and guides and with sample masks, and -* `qc_report_my_sorting_screen.[html,ipynb]` as QC report. -##### Optional arguments: -* `-o OUT_SCREEN_PATH`, `--out-screen-path OUT_SCREEN_PATH` - Path where quality-filtered ReporterScreen object to be written to -* `-r OUT_REPORT_PREFIX`, `--out-report-prefix OUT_REPORT_PREFIX` - Output prefix of qc report (prefix.html, prefix.ipynb) - -##### QC thresholds: -* `--count-correlation-thres COUNT_CORRELATION_THRES` - Correlation threshold to mask out. -* `--edit-rate-thres EDIT_RATE_THRES` - Mean editing rate threshold per sample to mask out. -* `--lfc-thres LFC_THRES` - Positive guides' correlation threshold to filter out. - -##### Run options: -* `-b`, `--remove-bad-replicates` - Remove replicates with at least two of its samples meet the QC threshold (bean run does not support having only one sorting bin sample for a replicate). -* `-i`, `--ignore-missing-samples` - If the flag is not provided, if the ReporterScreen object does not contain all condiitons for - each replicate, make fake empty samples. If the flag is provided, don't add dummy samples. -* `--no-editing` Ignore QC about editing. Can be used for QC of other editing modalities. -* `--dont-recalculate-edits` - When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from - ReporterScreen.uns['allele_count']. - -##### Input `.h5ad` formatting: -Note that these arguements will change the way the QC metrics are calculated for guides, samples, or replicates. -* `--tiling TILING` Specify that the guide library is tiling library without 'n guides per target' design -* `--replicate-label REPLICATE_LABEL` - Label of column in `bdata.samples` that describes replicate ID. -* `--sample-covariates SAMPLE_COVARIATES` - Comma-separated list of column names in `bdata.samples` that describes non-selective - experimental condition. (drug treatment, etc.) -* `--condition-label CONDITION_LABEL` - Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time, - etc.) -###### Editing rate calculation - * `--control-condition CTRL_COND` - Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate - to be calculated. Default is `None`, which considers all samples. - * `--rel-pos-is-reporter` - Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If - `False`, those are relative to spacer position. - Editing rate is calculated with following parameters in - * Variant screens: - * `--target-pos-col TARGET_POS_COL` - Target position column in `bdata.guides` specifying target edit position in reporter - * tiling screens: - * `--edit-start-pos EDIT_START_POS` - Edit start position to quantify editing rate on, 0-based inclusive. - * `--edit-end-pos EDIT_END_POS` - Edit end position to quantify editing rate on, 0-based exclusive. -###### LFC of positive controls - * `--posctrl-col POSCTRL_COL` - Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all - gRNAs, feed empty string ''. - * `--posctrl-val POSCTRL_VAL` - Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the - positive control in calculating log fold change. - * `--lfc-conds LFC_CONDS` - Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated - between, delimited by comma \ No newline at end of file diff --git a/docs/reporterscreen.md b/docs/reporterscreen.md deleted file mode 100644 index 03ce056..0000000 --- a/docs/reporterscreen.md +++ /dev/null @@ -1,11 +0,0 @@ -BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). - -ReporterScreen object structure - - * `.guides`: guide information provided in input (`gRNA_library.csv` in above example) - * `.samples`: sample information provided in input (`sample_list.csv` in above example) - * `.X`: Main guide count matrix, where row corresponds to each guide in `.guides` and columns correspond to samples in `.samples`. -Following attributes are included if matched reporter is provided and you chose to read edit/allele information from the reporter using `-r` option. - * `.X_bcmatch [Optional]`: Contains information about number of barcode-matched reads. Information about R2 barcode should be specified as `barcode` column in your `gRNA_library.csv` file. - * `.X_edits [Optional]`: If target position of each guide is specified as `target_pos` in input `gRNA_library.csv` file and `--match-target-position` option is provided, the result has the matrix with the number of target edit at the specified positions. - * `.allele_tables [Optional]`: Dictionary with a single allele count table that counts per guide and allele combination, what is the count per sample. \ No newline at end of file diff --git a/docs/run.md b/docs/run.md deleted file mode 100755 index b0a478c..0000000 --- a/docs/run.md +++ /dev/null @@ -1,64 +0,0 @@ -# `bean run`: Quantify variant effects -BEAN uses Bayesian network to incorporate gRNA editing outcome to provide posterior estimate of variant phenotype. The Bayesian network reflects data generation process. Briefly, -1. Cellular phenotype (either for cells are sorted upon for sorting screen, or log(proliferation rate)) is modeled as the Gaussian mixture distribution of wild-type phenotype and variant phenotype. -2. The weight of the mixture components are inferred from the reporter editing outcome and the chromatin accessibility of the loci. -3. Cells with each gRNA, formulated as the mixture distribution, is sorted by the phenotypic quantile to produce the gRNA counts. - -For the full detail, see the method section of the [BEAN manuscript](https://www.medrxiv.org/content/10.1101/2023.09.08.23295253v1). - -model - -

    - -# Usage example -```bash -bean run sorting[survival] variant[tiling] my_sorting_screen_filtered.h5ad \ -[--uniform-edit, --scale-by-acc [--acc-bw-path accessibility_signal.bw, --acc-col accessibility]] \ --o output_prefix/ \ ---fit-negctrl -``` -See full list of parameters [below](#full-parameters). - - -# Input -`my_sorting_screen_filtered.h5ad` can be produced by one of the following: -1. [`bean count-samples`]((#bean-count-samples-count-reporter-screen-data)) when you have raw `.fastq` file -2. (Limited to `bean run variant` mode) `bean create-screen` when you have flat `.csv` tables of gRNA metadata table, sample metadata table, gRNA counts table (# guides x # samples), and optionally # edits table. - ```bash - bean create-screen gRNA_info_table.csv sample_info_table.csv gRNA_counts_table.csv \ - [--edits edit_counts_table.csv -o output.h5ad] - ``` - * `gRNA_info_table.csv` should have following columns. - * `name`: gRNA ID column - * `target`: This column denotes which target variant/element of each gRNA. - * `target_group [Optional]`: If negative control gRNA will be used, specify as "NegCtrl" in this column. - * `sample_info_table.csv` should have following columns. - * `sample_id`: ID of sequencing sample - * `replicate`: Replicate # of this sample - * `bin`: Name of the sorting bin - * `upper_quantile`: FACS sorting upper quantile - * `lower_quantile`: FACS sorting lower quantile - * `gRNA_counts_table.csv` should be formatted as follows. - * Columns include one of `sample_id` columns in `sample_info_table.csv` file. - * 1st row (row index) follows `name` (gRNA ID) in `gRNA_info_table.csv` file. -3. You can manually create the `AnnData` object with more annotations including allele counts: see [API tutorial](#using-bean-as-python-module) for full detail. - - -# Output -model - -Above command produces -* `output_prefix/bean_element_result.[model_type].csv` with following columns: - * Estimated variant effect sizes - * `mu` (Effect size): Mean of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. - * `mu_sd`: Mean of variant phenotype `mu` is modeled as normal distribution. The column shows fitted standard deviation of `mu` that quantify the uncertainty of the variant effect. - * `mu_z`: z-score of `mu` - * `sd`: Standard deviation of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. - * `CI[0.025`, `0.975]`: Credible interval of `mu` - * When negative control is provided, above columns with `_adj` suffix are provided, which are the corresponding values adjusted for negative control. - * Metrics on per-variant evidence provided in input (provided in `tiling` mode) - * `effective_edit_rate`: Sum of per-variant editing rates over all alleles observed in the input. Allele-level editing rate is divided by the number of variants observed in the allele prior to summing up. - * `n_guides`: # of guides covering the variant. - * `n_coocc`: # of cooccurring variants with a given variant in any alleles observed in the input. -* `output_prefix/bean_sgRNA_result.[model_type].csv`: - * `edit_rate`: Estimated editing rate at the target loci. From c3cee14b17e439f37c08f2113192cd7660d8ee3f Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 11:58:11 -0400 Subject: [PATCH 093/107] remove old files --- docs/assets/anbe.svg | 1 - docs/assets/cmd.rst | 5 ----- docs/assets/cmd_main.rst | 10 ---------- docs/assets/dag_bean_v2.png | Bin 605372 -> 0 bytes docs/assets/reporter.jpg | Bin 66628 -> 0 bytes docs/assets/reporter_construct.png | Bin 171933 -> 0 bytes docs/assets/reporter_construct.svg | 1 - docs/assets/reporter_screen.svg | 1 - 8 files changed, 18 deletions(-) delete mode 100755 docs/assets/anbe.svg delete mode 100755 docs/assets/cmd.rst delete mode 100755 docs/assets/cmd_main.rst delete mode 100755 docs/assets/dag_bean_v2.png delete mode 100755 docs/assets/reporter.jpg delete mode 100755 docs/assets/reporter_construct.png delete mode 100755 docs/assets/reporter_construct.svg delete mode 100755 docs/assets/reporter_screen.svg diff --git a/docs/assets/anbe.svg b/docs/assets/anbe.svg deleted file mode 100755 index b39b191..0000000 --- a/docs/assets/anbe.svg +++ /dev/null @@ -1 +0,0 @@ -gRNA spacerreporterendogenous target site \ No newline at end of file diff --git a/docs/assets/cmd.rst b/docs/assets/cmd.rst deleted file mode 100755 index ee52397..0000000 --- a/docs/assets/cmd.rst +++ /dev/null @@ -1,5 +0,0 @@ -.. toctree:: - :maxdepth: 2 - - cmd_main - \ No newline at end of file diff --git a/docs/assets/cmd_main.rst b/docs/assets/cmd_main.rst deleted file mode 100755 index b7598db..0000000 --- a/docs/assets/cmd_main.rst +++ /dev/null @@ -1,10 +0,0 @@ -Command line utilities -********************** -.. .. argparse:: -.. :filename: ../bean/cli/execute.py -.. :func: get_parser -.. :prog: execute - - subcommand - Here we add a reference to subcommand, to simplify navigation. - \ No newline at end of file diff --git a/docs/assets/dag_bean_v2.png b/docs/assets/dag_bean_v2.png deleted file mode 100755 index a48822c5d8e2c5e6d726260436e289d9c24f0c85..0000000000000000000000000000000000000000 GIT binary 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The reporter technique transfects cells with plasmid with not only sgRNA but with the **target sequence surrogate** which we call **reporter** or **sensor**. +`bean` unconfounds variant effect of CRISPR reporter screens by considering genotypic outcome from *reporter* sequence. -Reporter construct +Reporter construct ## Overview `bean` supports end-to-end analysis of pooled sorting screens, with or without reporter. diff --git a/docs/cds.rst b/docs/cds.rst index c566387..dc4ea75 100755 --- a/docs/cds.rst +++ b/docs/cds.rst @@ -1,5 +1,5 @@ Coding sequence tiling library *********************** -.. mdinclude:: ldl_cds.md +.. mdinclude:: _ldl_cds.md See :ref:`subcommands` for the full details. diff --git a/docs/count.rst b/docs/count.rst index fdbe236..9a186dc 100755 --- a/docs/count.rst +++ b/docs/count.rst @@ -1,6 +1,6 @@ `bean count` *********************** -.. mdinclude:: count.md +.. mdinclude:: _count.md Full parameters ================== diff --git a/docs/count_samples.rst b/docs/count_samples.rst index fbbf8f1..db89732 100755 --- a/docs/count_samples.rst +++ b/docs/count_samples.rst @@ -1,7 +1,7 @@ .. _count_samples: `bean count-samples` *********************** -.. mdinclude:: commands/count.md +.. mdinclude:: _count.md Full parameters ================== diff --git a/docs/create_screen.rst b/docs/create_screen.rst index ee36bfc..485ee04 100755 --- a/docs/create_screen.rst +++ b/docs/create_screen.rst @@ -1,6 +1,6 @@ `bean create-screen` *********************** -.. mdinclude:: create-screen.md +.. mdinclude:: _create-screen.md Full parameters ================== diff --git a/docs/filter.rst b/docs/filter.rst index e0450cf..3b10bc4 100755 --- a/docs/filter.rst +++ b/docs/filter.rst @@ -1,7 +1,7 @@ .. _filter: `bean filter` *********************** -.. mdinclude:: filter.md +.. mdinclude:: _filter.md Full parameters ================== diff --git a/docs/gwas.rst b/docs/gwas.rst index 09d76fa..31e8c68 100755 --- a/docs/gwas.rst +++ b/docs/gwas.rst @@ -1,5 +1,5 @@ GWAS variant library *********************** -.. mdinclude:: ldl_var.md +.. mdinclude:: _ldl_var.md See :ref:`subcommands` for the full details. diff --git a/docs/index.rst b/docs/index.rst index 8ef251d..14287f9 100755 --- a/docs/index.rst +++ b/docs/index.rst @@ -5,6 +5,12 @@ Welcome to `bean`'s documentation! ================================ +`bean` unconfounds variant effect of CRISPR reporter screens by considering genotypic outcome from *reporter* sequence. + +.. image:: assets/summary.png + :width: 700 + :alt: BEAN schematic + =================== Workflows =================== diff --git a/docs/input.rst b/docs/input.rst index d018e97..79285ff 100755 --- a/docs/input.rst +++ b/docs/input.rst @@ -1,4 +1,4 @@ .. _input: Input file format *********************** -.. mdinclude:: input.md \ No newline at end of file +.. mdinclude:: _input.md \ No newline at end of file diff --git a/docs/profile.rst b/docs/profile.rst index 7316d9e..c25a422 100755 --- a/docs/profile.rst +++ b/docs/profile.rst @@ -1,6 +1,6 @@ `bean profile` *********************** -.. mdinclude:: profile.md +.. mdinclude:: _profile.md Full parameters ================== diff --git a/docs/prolif_gwas.rst b/docs/prolif_gwas.rst index 34adb83..26bdbe6 100644 --- a/docs/prolif_gwas.rst +++ b/docs/prolif_gwas.rst @@ -1,5 +1,5 @@ Proliferation screen with GWAS library ********************************************** -.. mdinclude:: profile_gwas.md +.. mdinclude:: _profile_gwas.md See :ref:`subcommands` for the full details. diff --git a/docs/qc.rst b/docs/qc.rst index 8cb0c7a..bd233cf 100755 --- a/docs/qc.rst +++ b/docs/qc.rst @@ -1,7 +1,7 @@ .. _qc: `bean qc` *********************** -.. mdinclude:: qc.md +.. mdinclude:: _qc.md Full parameters ================== diff --git a/docs/reporterscreen.rst b/docs/reporterscreen.rst index dcd0a00..687a31b 100644 --- a/docs/reporterscreen.rst +++ b/docs/reporterscreen.rst @@ -1,3 +1,3 @@ ReporterScreen object *********************** -.. mdinclude:: reporterscreen.md +.. mdinclude:: _reporterscreen.md diff --git a/docs/run.rst b/docs/run.rst index 8378dbe..db09224 100755 --- a/docs/run.rst +++ b/docs/run.rst @@ -1,7 +1,7 @@ .. _run: `bean run` *********************** -.. mdinclude:: run.md +.. mdinclude:: _run.md Full parameters ================== From 749df5f23c7a2d17fe6de77bf5982d77c917f3ed Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 12:13:06 -0400 Subject: [PATCH 095/107] add image --- docs/assets/summary.png | Bin 0 -> 416940 bytes 1 file changed, 0 insertions(+), 0 deletions(-) create mode 100644 docs/assets/summary.png diff --git a/docs/assets/summary.png b/docs/assets/summary.png new file mode 100644 index 0000000000000000000000000000000000000000..397a37c48f40faa0ab1254c845747e1b806d2617 GIT binary patch literal 416940 zcmZsD1yodfwD%~8gb0FybT`u79g?C*H%NCk0t!gCv~+h#hcwbi4KOG%z|b&syfb+3 z_wHNkJ8LZBn zoIxP($A~`+xsbgIyxvE)ya=j*ehPtLE z$!z6dH4yHRfh}o5uH3YS8H#P9Yrq$R-S5T@H581DkbL(2cg-J=7B^Z$5MNgqCIjEf 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zW6zItd3`Knd(M})_tvuy48?X}DB?xxm5;ac()HrJJu{9s$HB4b#+D!HvT{Bs7stKr z*yl0FksimJ%3ivtC!`OCABy!*FYc{}|IbpC3mRJv_JeW##_6Cxh7XE3{9vr-IJmLO z=@z8}KNNmu#64v{7|Pof#nB$2*uIG2gRVjyB_&sJ7`fH z>ziA#e@K_hgLrc*^5Oi8^1*iS6)}93QsiHxoPUwOq3b#Q=3 Date: Sat, 30 Mar 2024 12:27:48 -0400 Subject: [PATCH 097/107] fix filtering qc --- tests/test_qc.py | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/tests/test_qc.py b/tests/test_qc.py index 5f9f752..3910432 100755 --- a/tests/test_qc.py +++ b/tests/test_qc.py @@ -43,7 +43,7 @@ def test_qc_survival(): @pytest.mark.order(9) def test_dummy_insertion_varscreen(): - cmd = "bean qc tests/data/var_mini_screen_missing.h5ad -o tests/data/var_mini_screen_missing_masked.h5ad -r tests/test_res/qc_report_var_mini_screen_missing --count-correlation-thres 0.6" + cmd = "bean qc tests/data/var_mini_screen_missing.h5ad -o tests/data/var_mini_screen_missing_masked.h5ad -r tests/test_res/qc_report_var_mini_screen_missing --count-correlation-thres 0.6 -b" try: subprocess.check_output( cmd, shell=True, universal_newlines=True, stderr=subprocess.STDOUT @@ -56,7 +56,7 @@ def test_dummy_insertion_varscreen(): @pytest.mark.order(10) def test_dummy_insertion_tilingscreen(): - cmd = "bean qc tests/data/tiling_mini_screen_missing.h5ad -o tests/data/tiling_mini_screen_missing_masked.h5ad -r tests/test_res/qc_report_tiling_mini_screen_missing --count-correlation-thres 0.6 --posctrl-col ''" + cmd = "bean qc tests/data/tiling_mini_screen_missing.h5ad -o tests/data/tiling_mini_screen_missing_masked.h5ad -r tests/test_res/qc_report_tiling_mini_screen_missing --count-correlation-thres 0.6 --posctrl-col -b''" try: subprocess.check_output( cmd, shell=True, universal_newlines=True, stderr=subprocess.STDOUT From f52937ccd9942dd021da47897231a758522da53f Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 14:29:16 -0400 Subject: [PATCH 098/107] Fix QC notebook --- notebooks/sample_quality_report.ipynb | 42 +++++++++++++++++---------- tests/test_qc.py | 7 ++--- 2 files changed, 29 insertions(+), 20 deletions(-) diff --git a/notebooks/sample_quality_report.ipynb b/notebooks/sample_quality_report.ipynb index 2390ed7..23f49ec 100755 --- a/notebooks/sample_quality_report.ipynb +++ b/notebooks/sample_quality_report.ipynb @@ -60,7 +60,7 @@ "recalculate_edits = True\n", "tiling = None\n", "base_edit_data = True\n", - "remove_bad_replciates = False" + "remove_bad_replicates = False" ] }, { @@ -128,7 +128,7 @@ "metadata": {}, "outputs": [], "source": [ - "bdata.X" + "bdata.guides" ] }, { @@ -397,13 +397,31 @@ "source": [ "bdata.samples[\"mask\"] = 1\n", "bdata.samples.loc[\n", - " bdata.samples.median_corr_X.isnull() | (bdata.samples.median_corr_X < count_correlation_thres),\n", + " (\"PosCtrl\" in bdata.guides.target_group) & bdata.samples.median_corr_X.isnull() | (bdata.samples.median_corr_X < count_correlation_thres),\n", " \"mask\",\n", "] = 0\n", "if \"mean_editing_rate\" in bdata.samples.columns.tolist():\n", " bdata.samples.loc[bdata.samples.mean_editing_rate < edit_rate_thres, \"mask\"] = 0\n", - "if f\"median_lfc_corr.{comp_cond1}_{comp_cond2}\" in bdata.samples.columns.tolist():\n", - " bdata.samples.loc[bdata.samples[f\"median_lfc_corr.{comp_cond1}_{comp_cond2}\"] < lfc_thres, \"mask\"] = 0" + "\n", + "bdata.samples.loc[\n", + " bdata.samples[f\"median_lfc_corr.{comp_cond1}_{comp_cond2}\"] < lfc_thres,\n", + " \"mask\",\n", + "] = 0\n", + "if posctrl_val in bdata.guides[posctrl_col].values:\n", + " print(\"filter with posctrl LFC\")\n", + " bdata.samples.loc[\n", + " bdata.samples[f\"median_lfc_corr.{comp_cond1}_{comp_cond2}\"].isnull(),\n", + " \"mask\",\n", + " ] = 0" + ] + }, + { + "cell_type": "code", + "execution_count": null, + "metadata": {}, + "outputs": [], + "source": [ + "bdata.samples.style.background_gradient(cmap=\"coolwarm_r\")" ] }, { @@ -413,7 +431,7 @@ "outputs": [], "source": [ "# leave replicate with more than 1 sorting bin data\n", - "if remove_bad_replciates:\n", + "if remove_bad_replicates:\n", " rep_n_samples = bdata.samples.groupby(replicate_label)[\"mask\"].sum()\n", " print(rep_n_samples)\n", " rep_has_too_small_sample = rep_n_samples.loc[rep_n_samples < 2].index.tolist()\n", @@ -431,20 +449,12 @@ " samples_include = ~bdata.samples[\"_rc\"].isin(rep_has_too_small_sample)\n", " bdata.samples.pop(\"_rc\")\n", " bdata_filtered = bdata[:, samples_include]\n", + " if isinstance(replicate_label, str) and len(bdata_filtered.samples[replicate_label].unique()) <= 1 or isinstance(replicate_label, list) and len(bdata_filtered.samples[replicate_label].drop_duplicates() <= 1): \n", + " raise ValueError(\"Too small number of replicate left after QC. Check the input data or adjust the QC metric thresholds.\")\n", "else:\n", " bdata_filtered = bdata" ] }, - { - "cell_type": "code", - "execution_count": null, - "metadata": {}, - "outputs": [], - "source": [ - "if isinstance(replicate_label, str) and len(bdata_filtered.samples[replicate_label].unique()) <= 1 or isinstance(replicate_label, list) and len(bdata_filtered.samples[replicate_label].drop_duplicates() <= 1): \n", - " raise ValueError(\"Too small number of replicate left after QC. Check the input data or adjust the QC metric thresholds.\")" - ] - }, { "cell_type": "code", "execution_count": null, diff --git a/tests/test_qc.py b/tests/test_qc.py index 3910432..241ad01 100755 --- a/tests/test_qc.py +++ b/tests/test_qc.py @@ -17,7 +17,7 @@ def test_qc(): @pytest.mark.order(8) def test_qc_tiling(): - cmd = "bean qc tests/data/tiling_mini_screen.h5ad -o tests/data/tiling_mini_screen_masked.h5ad -r tests/test_res/qc_report_tiling_mini_screen --count-correlation-thres 0.6 --posctrl-col ''" + cmd = "bean qc tests/data/tiling_mini_screen.h5ad -o tests/data/tiling_mini_screen_masked.h5ad -r tests/test_res/qc_report_tiling_mini_screen --count-correlation-thres 0.6 " try: subprocess.check_output( cmd, @@ -56,12 +56,11 @@ def test_dummy_insertion_varscreen(): @pytest.mark.order(10) def test_dummy_insertion_tilingscreen(): - cmd = "bean qc tests/data/tiling_mini_screen_missing.h5ad -o tests/data/tiling_mini_screen_missing_masked.h5ad -r tests/test_res/qc_report_tiling_mini_screen_missing --count-correlation-thres 0.6 --posctrl-col -b''" + cmd = "bean qc tests/data/tiling_mini_screen_missing.h5ad -o tests/data/tiling_mini_screen_missing_masked.h5ad -r tests/test_res/qc_report_tiling_mini_screen_missing --count-correlation-thres 0.6 -b" try: subprocess.check_output( cmd, shell=True, universal_newlines=True, stderr=subprocess.STDOUT ) raise ValueError("Filtering should fail with too small number of replicates.") except subprocess.CalledProcessError as exc: - if "Too small number of replicate left after QC" not in exc.output: - raise exc + raise exc From 4d4791d4c585f135ff8d66dc6ccc0d2df5178125 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 14:33:33 -0400 Subject: [PATCH 099/107] change intro message --- README.md | 2 +- docs/index.rst | 2 +- 2 files changed, 2 insertions(+), 2 deletions(-) diff --git a/README.md b/README.md index 5ad9a83..54337f3 100755 --- a/README.md +++ b/README.md @@ -4,7 +4,7 @@ [![PyPI version](https://img.shields.io/pypi/v/crispr-bean)](https://pypi.org/project/crispr-bean/) [![Code style](https://img.shields.io/badge/code%20style-black-black)](https://github.com/psf/black) -`bean` unconfounds variant effect of CRISPR reporter screens by considering genotypic outcome from *reporter* sequence. +`bean` unconfounds variant effect from variable editing outcome of CRISPR screens by considering genotypic outcome from *reporter* sequence. Reporter construct diff --git a/docs/index.rst b/docs/index.rst index 14287f9..8e312d9 100755 --- a/docs/index.rst +++ b/docs/index.rst @@ -5,7 +5,7 @@ Welcome to `bean`'s documentation! ================================ -`bean` unconfounds variant effect of CRISPR reporter screens by considering genotypic outcome from *reporter* sequence. +`bean` unconfounds variant effect from variable editing outcome of CRISPR screens by considering genotypic outcome from *reporter* sequence. .. image:: assets/summary.png :width: 700 From 6efb8d3ee1aebdd04ebc1f34e66724fe912277c3 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 14:45:10 -0400 Subject: [PATCH 100/107] fix qc error message --- tests/test_qc.py | 3 ++- 1 file changed, 2 insertions(+), 1 deletion(-) diff --git a/tests/test_qc.py b/tests/test_qc.py index 241ad01..3cffec6 100755 --- a/tests/test_qc.py +++ b/tests/test_qc.py @@ -61,6 +61,7 @@ def test_dummy_insertion_tilingscreen(): subprocess.check_output( cmd, shell=True, universal_newlines=True, stderr=subprocess.STDOUT ) - raise ValueError("Filtering should fail with too small number of replicates.") except subprocess.CalledProcessError as exc: raise exc + #if "Too small number of replicate left after QC" not in exc.output: + raise exc From 2ecc9b5ac072ed0d42df18e53d094ee264a37477 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 14:49:54 -0400 Subject: [PATCH 101/107] fix typo --- tests/test_qc.py | 2 -- 1 file changed, 2 deletions(-) diff --git a/tests/test_qc.py b/tests/test_qc.py index 3cffec6..1499f96 100755 --- a/tests/test_qc.py +++ b/tests/test_qc.py @@ -63,5 +63,3 @@ def test_dummy_insertion_tilingscreen(): ) except subprocess.CalledProcessError as exc: raise exc - #if "Too small number of replicate left after QC" not in exc.output: - raise exc From 51e2351b3c60aeea64f16dbb7d9f7a74afaa03eb Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 15:11:22 -0400 Subject: [PATCH 102/107] commit before merge --- 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---------------------------- 2 files changed, 196 deletions(-) delete mode 100644 docs/tutorials/ldl_cds.md delete mode 100644 docs/tutorials/ldl_var.md diff --git a/docs/tutorials/ldl_cds.md b/docs/tutorials/ldl_cds.md deleted file mode 100644 index c3de47a..0000000 --- a/docs/tutorials/ldl_cds.md +++ /dev/null @@ -1,112 +0,0 @@ -# Tiling sorting screen tutorial -Tiling screen that tiles gRNA densely across locus or multiple loci, selected based on FACS signal quantiles. - - - - - - - - - - -
      Library designTiling (gRNAs tile each locus densely)
      tiling library design
      SelectionCells are sorted based on FACS signal quantiles
      variant library design
      - -

      - -## 1. Count gRNA & reporter ([`bean-count-samples`](../../README#bean-count-samples-count-reporter-screen-data)) -``` -screen_id=my_sorting_tiling_screen - -bean-count-samples \ ---input tests/data/sample_list_tiling.csv `# Contains fastq file path; see test file for example.`\ --b A `# Base A is edited (into G)` \ --f tests/data/test_guide_info_tiling_chrom.csv `# Contains gRNA metadata; see test file for example.`\ --o ./ `# Output directory` \ --r `# Quantify reporter edits` \ --n ${screen_id} `# ID of the screen` \ ---tiling -``` -Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. - -## 2. QC ([`bean-qc`](../../README#bean-qc-qc-of-reporter-screen-data)) -Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean-qc`](../../README#bean-qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) -``` -bean-qc \ - bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ - -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r qc_report_${screen_id} `# Prefix for QC report` \ - [--tiling] `# Not required if you have passed --tiling in counting step` -``` - - - -If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. - -## 3. Filter alleles ([`bean-filter`](../../README#bean-filter-filtering-and-optionally-translating-alleles)) -As tiling library doesn't have designated per-gRNA target variant, any base edit observed in reporter may be the candidate variant, while having too many variants with very low editing rate significantly decreases the power. Variants are filtered based on multiple criteria in `bean-fitler`. - -If the screen targets coding sequence, it's beneficial to translate edits into coding varaints whenever possible for better power. For translation, provide `--translate` and one of the following: -``` -[ --translate-gene-name GENE_SYMBOL OR - --translate-genes-list path_to_gene_names_file.txt OR - --translate-fasta gene_exon.fa, OR - --translate-fastas-csv gene_exon_fas.csv] -``` -where `path_to_gene_names_file.txt` has one gene symbol per line, and gene symbol uses its MANE transcript (hg38) coordinates of exons. In order to use other reference versions or transcript ID, you'll need to feed in fasta file. See detailed formatting of fasta file [here](../../README#translating-alleles). - -Example allele filtering given we're translating based on MANE transcript exons of multiple gene symbols: - -```bash -bean-filter ./bean_count_${screen_id}_masked.h5ad \ --o ./bean_count_${screen_id}_alleleFiltered \ ---filter-target-basechange `# Filter based on intended base changes. If -b A was provided in bean-count, filters for A>G edit. If -b C was provided, filters for C>T edit.`\ ---filter-window --edit-start-pos 0 --edit-end-pos 19 `# Filter based on editing window in spacer position within reporter.`\ ---filter-allele-proportion 0.1 --filter-sample-proportion 0.3 `#Filter based on allele proportion larger than 0.1 in at least 0.3 (30%) of the control samples.` \ ---translate --translate-genes-list tests/data/gene_symbols.txt -``` - -Ouptut file `` shows number of alleles per guide and number of guides per variant, where we want high enough values for the latter. See the typical output for dataset with good editing coverage & filtering result [here](../example_filtering_ouptut/). - -## 4. Quantify variant effect ([`bean-run`](../../README#bean-run-quantify-variant-effects)) -By default, `bean-run [sorting,survival] tiling` uses most filtered allele counts table for variant identification and quantification of their effects. **Check [allele filtering output](../example_filtering_ouptut/)** and choose alternative filtered allele counts table if necessary. - -`bean-run` can take 3 run options to quantify editing rate: -1. From **reporter + accessibility** - 1-1. If your gRNA metadata table (`tests/data/test_guide_info.csv` above) included per-gRNA accessibility score, - ``` - bean-run sorting tiling \ - ./bean_count_${screen_id}_alleleFiltered.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-col accessibility - ``` - 1-2. If your gRNA metadata table (`tests/data/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, - ``` - bean-run sorting tiling \ - ./bean_count_${screen_id}_alleleFiltered.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-bw accessibility.bw - ``` - -2. From **reporter** - ``` - bean-run sorting tiling \ - ./bean_count_${screen_id}_alleleFiltered.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl - ``` -3. No reporter information, assume the same editing efficiency of all gRNAs. - Use this option if your data don't have editing rate information. - ``` - bean-run sorting tiling \ - ./bean_count_${screen_id}_alleleFiltered.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl \ - --uniform-edit - ``` - -See [full argument list](../../README#optional-parameters) to accommodate different input sample & guide metadata columns/values and run options. \ No newline at end of file diff --git a/docs/tutorials/ldl_var.md b/docs/tutorials/ldl_var.md deleted file mode 100644 index 263899a..0000000 --- a/docs/tutorials/ldl_var.md +++ /dev/null @@ -1,84 +0,0 @@ -# Variant sorting screen tutorial -GWAS variant screen with per-variant gRNA tiling design, selected based on FACS signal quantiles. - - - - - - - - - - -
      Library designVariant (gRNAs tile each target variant)
      variant library design
      SelectionCells are sorted based on FACS signal quantiles
      variant library design
      - -

      - -## 1. Count gRNA & reporter ([`bean-count-samples`](../../README#bean-count-samples-count-reporter-screen-data)) -``` -screen_id=my_sorting_tiling_screen - -bean-count-samples \ ---input tests/data/sample_list.csv `# Contains fastq file path; see test file for example.`\ --b A `# Base A is edited (into G)` \ --f tests/data/test_guide_info.csv `# Contains gRNA metadata; see test file for example.`\ --o ./ `# Output directory` \ --r `# Quantify reporter edits` \ --n ${screen_id} `# ID of the screen to be counted` -``` -Make sure you follow the [input file format](../../README#input-file-format) for seamless downstream steps. This will produce `./bean_count_${screen_id}.h5ad`. - -## 2. QC ([`bean-qc`](../../README#bean-qc-qc-of-reporter-screen-data)) -Base editing data will include QC about editing efficiency. As QC uses predefined column names and values, beware to follow the [input file guideline](../../README#input-file-format), but you can change the parameters with the full argument list of [`bean-qc`](../../README#bean-qc-qc-of-reporter-screen-data). (Common factors you may want to tweak is `--ctrl-cond=bulk` and `--lfc-conds=top,bot` if you have different sample condition labels.) -``` -bean-qc \ - bean_count_${screen_id}.h5ad `# Input ReporterScreen .h5ad file path` \ - -o bean_count_${screen_id}_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r qc_report_${screen_id} `# Prefix for QC report` -``` - - - -If the data does not include reporter editing data, you can provide `--no-editing` flag to omit the editing rate QC. - - -## 3. Quantify variant effect ([`bean-run`](../../README#bean-run-quantify-variant-effects)) - -`bean-run` can take 3 run options to quantify editing rate: -1. From **reporter + accessibility** - If your gRNA metadata table (`tests/data/test_guide_info.csv` above) included per-gRNA accessibility score, - ``` - bean-run sorting variant \ - tests/data/bean_count_${screen_id}_masked.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-col accessibility - ``` - If your gRNA metadata table (`tests/data/test_guide_info.csv` above) included per-gRNA chromosome & position and you have bigWig file with accessibility signal, - ``` - bean-run sorting variant \ - tests/data/bean_count_${screen_id}_masked.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl \ - --scale-by-acc \ - --accessibility-bw accessibility.bw - ``` - -2. From **reporter** - ``` - bean-run sorting variant \ - tests/data/bean_count_${screen_id}_masked.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl - ``` -3. No reporter information, assume the same editing efficiency of all gRNAs. - Use this option if your data don't have editing rate information. - ``` - bean-run sorting variant \ - tests/data/bean_count_${screen_id}_masked.h5ad \ - -o tests/test_res/var/ \ - --fit-negctrl \ - --uniform-edit - ``` -See [full argument list](../../README#optional-parameters) to accommodate different input sample & guide metadata columns/values and run options. \ No newline at end of file From 7bb40e15fa9414029e4a3a6eb958d49cbe6b9208 Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 15:19:18 -0400 Subject: [PATCH 104/107] resolve merge conflict --- README.md | 500 ++++-------------------------------------------------- 1 file changed, 37 insertions(+), 463 deletions(-) mode change 100644 => 100755 README.md diff --git a/README.md b/README.md old mode 100644 new mode 100755 index 80255c0..54337f3 --- a/README.md +++ b/README.md @@ -1,502 +1,76 @@ -# crispr-bean +# crispr-bean [![PyPI pyversions](https://img.shields.io/pypi/pyversions/crispr-bean)](https://pypi.org/project/crispr-bean/) [![PyPI version](https://img.shields.io/pypi/v/crispr-bean)](https://pypi.org/project/crispr-bean/) [![Code style](https://img.shields.io/badge/code%20style-black-black)](https://github.com/psf/black) -**CRISPR** **B**ayesian **E**stimation of variant effect (from **B**ase **E**diting reporter screens) with guide **A**ctivity **N**ormalization -This is an analysis toolkit for the pooled CRISPR reporter or sensor data. The reporter technique transfects cells with plasmid with not only sgRNA but with the **target sequence surrogate** which we call **reporter** or **sensor**. +`bean` unconfounds variant effect from variable editing outcome of CRISPR screens by considering genotypic outcome from *reporter* sequence. -Reporter construct +Reporter construct ## Overview -`crispr-bean` supports end-to-end analysis of pooled sorting screens, with or without reporter. - -dag_bean_v2.svg - -1. [`bean-count-sample`](#bean-count-samples-count-reporter-screen-data): Base-editing-aware **mapping** of guide, optionally with reporter from `.fastq` files. - * [`bean-count-create`](#bean-create-screen-create-reporterscreen-object-from-flat-files) creates minimal ReporterScreen object from flat gRNA count file. Note that this way, allele counts are not included and many functionalities involving allele and edit counts are not supported. -2. [`bean-profile`](#bean-profile-profile-editing-patterns): Profile editing preferences of your editor. -3. [`bean-qc`](#bean-qc-qc-of-reporter-screen-data): Quality control report and filtering out / masking of aberrant sample and guides -4. [`bean-filter`](#bean-filter-filtering-and-optionally-translating-alleles): Filter reporter alleles; essential for `tiling` mode that allows for all alleles generated from gRNA. -5. [`bean-run`](#bean-run-quantify-variant-effects): Quantify targeted variants' effect sizes from screen data. - -### Screen data is saved as *ReporterScreen* object in the pipeline. -BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). See [Data Structure](#data-structure) section for more information. - -

      - -## Examples -| Tutorial link | [Library design](#pipeline-run-options-by-library-design) | Selection | Reporter | -|---------------| -------------- | --------- | -------- | -| [LDL-C GWAS](docs/tutorials/ldl_var.md) | GWAS variant library | FACS sorting | Yes/No | -| [LDL-C LDLR CDS](docs/tutorials/ldl_cds.md) | Coding sequence tiling libarary | FACS sorting | Yes/No | -| TKO simulated (Coming soon!) | GWAS variant library | Survival / Proliferation | Yes/No | -| TKO simulated, tiling (Coming soon!) | Coding sequence tiling libarary | Survival / Proliferation | Yes/No | - -We also provide example scripts in `tests/`. Running `pytest --sparse-ordering` will generate example input/output files. - -### Pipeline run options by library design -The `bean-filter` and `bean-run` steps depend on the type of gRNA library design, where BEAN supports two modes of running. -1. `variant` library: Several gRNAs tile each of the targeted variants - Ex) - variant library design - -2. `tiling` library: gRNA densely tiles a long region (e.g. gene(s), exon(s), coding sequence(s)) - Ex) - tiling library design - -

      - +`bean` supports end-to-end analysis of pooled sorting screens, with or without reporter. + +dag_bean_v2.svg + +`bean` subcommands include the following: Click on the [`links`]() to see the full documentation. +1. [`count`](https://pinellolab.github.io/crispr-bean/count.html), [`count-samples`](https://pinellolab.github.io/crispr-bean/count_samples.html): Base-editing-aware **mapping** of guide, optionally with reporter from `.fastq` files. + * [`create-screen`](https://pinellolab.github.io/crispr-bean/create_screen.html) creates minimal ReporterScreen object from flat gRNA count file. Note that this way, allele counts are not included and many functionalities involving allele and edit counts are not supported. +2. [`profile`](https://pinellolab.github.io/crispr-bean/profile.html): Profile editing preferences of your editor. +3. [`qc`](https://pinellolab.github.io/crispr-bean/qc.html): Quality control report and filtering out / masking of aberrant sample and guides +4. [`filter`](https://pinellolab.github.io/crispr-bean/filter.html): Filter reporter alleles; essential for `tiling` mode that allows for all alleles generated from gRNA. +5. [`run`](https://pinellolab.github.io/crispr-bean/run.html): Quantify targeted variants' effect sizes from screen data. +* Screen data is saved as [`ReporterScreen` object](https://pinellolab.github.io/crispr-bean/reporterscreen.html) in the pipeline. +BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). ## Installation -### Full installation -First install [pytorch](https://pytorch.org/) >=12.1,<2. +First install [PyTorch](https://pytorch.org/get-started/). Then download from PyPI: ``` pip install crispr-bean[model] ``` -### Mapping and data wrangling, without variant effect quantification +Following installation without PyTorch dependency wouldn't have variant effect size quantification (`bean run`) functionality. ``` pip install crispr-bean ``` -This wouldn't have variant effect size quantification (`bean-run`) functionality. - -

      - -## `bean-count-samples`: Count (reporter) screen data -`bean-count-samples` (or `bean-count` for a single sample) maps guide into guide counts, **allowing for base transition in spacer sequence**. When the matched reporter information is provided, it can count the **target site edits** and **alleles produced by each guide**. Mapping is efficiently done based on [CRISPResso2](https://github.com/pinellolab/CRISPResso2) modified for base-edit-aware mapping. - - - -```python -bean-count-samples \ - --input sample_list.csv `# sample with lines 'R1_filepath,R2_filepath,sample_name\n'` \ - -b A `# base that is being edited (A/G)` \ - -f sgRNA_info_table.csv `# sgRNA information` \ - -o . `# output directory` \ - -r `# read edit/allele information from reporter` \ - -t 12 `# number of threads` \ - --name my_sorting_screen `# name of this sample run` \ -``` -```python -bean-count \ - --R1 read1.fq[.gz] - --R2 read2.fq[.gz] - -b A `# base that is being edited (A/G)` \ - -f sgRNA_info_table.csv `# sgRNA information` \ - -o . `# output directory` \ - -r `# read edit/allele information from reporter` \ - --name my_sample `# name of this sample run` -``` - -By default, `bean-count[-samples]` assume R1 and R2 are trimmed off of the adapter sequence. You may need to adjust the command arguments according to your read structure. - Read structuren -See full detail in [Parameters](#parameters). - -### Input file format -#### 1. gRNA_library.csv -File should contain following columns. -* `name`: gRNA ID column -* `sequence`: gRNA sequence -* `barcode`: R2 barcode to help match reporter to gRNA, written in the sense direction (as in R1) -* In order to use accessibility in the [variant effect quantification](#bean-run-quantify-variant-effects), provide accessibility information in one of two options. (For non-targeting guides, provide NA values (empty cell).) - * Option 1: `chrom` & `genomic_pos`: Chromosome (ex. `chr19`) and genomic position of guide sequence. You will have to provide the path to the bigwig file with matching reference version in `bean-run`. - * Option 2: `accessibility_signal`: ATAC-seq signal value of the target loci of each guide. -* For variant library (gRNAs are designed to target specific variants and ignores bystander edits) - * `target`: This column denotes which target variant/element of each gRNA. This is not used in `bean-count[-samples]` but required to run `bean-run` in later steps. - * `target_group`: If negative/positive control gRNA will be considered in `bean-qc` and/or `bean-run`, specify as "NegCtrl"/"PosCtrl" in this column. - * `target_pos`: If `--match_target_pos` flag is used, input file needs `target_pos` which specifies 0-based relative position of targeted base within Reporter sequence. -* For tiling library (gRNAs tile coding / noncoding sequences) - * `strand`: Specifies gRNA strand information relative to the reference genome. - * `chrom`: Chromosome of gRNA targeted locus. - * `start_pos`: gRNA starting position in the genome. Required when you provide `strand` column. Should specify the smaller coordinate value among start and end position regardless of gRNA strandedness. - -Also see examples for [variant library](tests/data/test_guide_info.csv) and [tiling library](tests/data/test_guide_info_tiling.csv). - -#### 2. sample_list.csv -File should contain following columns with header. -* `R1_filepath`: Path to read 1 `.fastq[.gz]` file -* `R2_filepath`: Path to read 1 `.fastq[.gz]` file -* `sample_id`: ID of sequencing sample -* `replicate`: Replicate # of this sample (Should NOT contain `.`) -* `condition`: Name of the sorting bin (ex. `top`, `bot`), or label of timepoint (ex. `D5`, `D18`) - -For FACS sorting screens: -* `upper_quantile`: FACS sorting upper quantile -* `lower_quantile`: FACS sorting lower quantile - -For proliferation / survival screens: -* `time`: Numeric time following the base editing of each sample. - - -Also see examples for [FACS sorting screen](tests/data/sample_list.csv). - -### Output file format -`count` or `count-samples` produces `.h5ad` and `.xlsx` file with guide and per-guide allele counts. -* `.h5ad`: This output file follows annotated matrix format compatible with `AnnData` and is based on `Screen` object in [purturb_tools](https://github.com/pinellolab/perturb-tools). See [Data Structure](#data-structure) section for more information. -* `.xlsx`: This output file contains `.guides`, `.samples`, `.X[_bcmatch,_edits]`. (`allele_tables` are often too large to write into an Excel!) - -### Parameters -* `-b`, `--edited-base` (`str`, default: `None`): For base editors, the base that should be ignored when matching the gRNA sequence -* `-i`, `--input`: List of fastq and sample ids. See above for the format. -* `-f`, `--sgRNA-filename` (`str`, default: `None`): sgRNA description file. (See above) - -Output formats -* `-n`, `--name`: Name of the output file will be `bean_count_{name}.h5ad`. -* `-o`, `--output-folder`: Output folder -* `--tiling`: Specify that the guide library is tiling library -* `-r`, `--count-reporter` (default: `False`): Count edited alleles in reporters. -* `--string-allele` (default: `False`): Store allele as quality filtered string instead of Allele object -* `-g`, `--count-guide-edits` (default: `False`): count the self editing of guides (default: False) -* `-m`, `--count-guide-reporter-alleles`: count the matched allele of guide and reporter edit -* `--match-target-pos` (default: `False`): Count the edit in the exact target position. -* `--target-pos-col` (default: `target_pos`): Column name specifying the relative target position within *reporter* sequence. -* `--offset` (default: `False`): Guide file has `offest` column that will be added to the relative position of reporters. -* `--align-fasta` (default: ''): gRNA is aligned to this sequence to infer the offset. Can be used when the exact offset is not provided. - - -Read structure -* `--guide-start-seq` (default: ''): Guide starts after this sequence in R1 -* `--guide-end-seq` (default: ''): Guide ends after this sequence in R1 -* `--barcode-start-seq` (default: ''): Barcode + reporter starts after this sequence in R2, denoted as the sense direction (the same sequence direction as R1). -* `--guide-start-seqs-file` (default: `None`): CSV file path with per-sample `guide_start_seq` to be used, if provided. Formatted as `sample_id, guide_start_seq` -* `--guide-end-seqs-file` (default: `None`): CSV file path with per-sample `guide_end_seq` to be used, if provided. Formatted as `sample_id,guide_end_seq` -* `--guide-start-seqs-file` (default: `None`): CSV file path with per-sample `barcode_start_seq` to be used, if provided. Formatted as `sample_id, guide_start_seq` -* `-l`, `--reporter-length` (default: `32`): Length of the reporter sequence. -* `--gstart-reporter` (default: `6`): Start position of the guide sequence in the reporter -* `--guide-bc` (default: `True`): Construct has guide barcode -* `--guide-bc-len` (default: `4`): Guide barcode sequence length at the beginning of the R2 - -Mapping quality filters -* `-q`, `--min-average-read-quality` (default: `30`): Minimum average quality score -(phred33) to keep a read -* `--keep-intermediate` (default: `False`): Keep all intermediate files generated from filtering. -* `-s`, `--min-single-bp-quality` (default: `0`): Minimum single bp score (phred33) to keep a read (default: 0) -* `--qstart-R1` (default: `0`): Start position of the read when filtering for quality score of the read 1 -* `--qend-R1` (default: `47`): End position of the read when filtering for quality score of the read 1 -* `--qstart-R2` (default: `0`): Start position of the read when filtering for quality score of the read 2 -* `--qend-R2` (default: `36`): End position of the read when filtering for quality score of the read 2 - -Run options -* `-t`, `--threads` (default: `10`): Number of threads to use -* `--rerun` (default: `False`): Recount each sample. If `False`, existing count for each sample is taken. - - -## `bean-create-screen`: Create ReporterScreen object from flat files -```bash -bean-create-screen gRNA_library.csv sample_list.csv gRNA_counts_table.csv -``` -### Input - * [gRNA_library.csv](#1-gRNA_librarycsv) - * [sample_list.csv](#2-sample_listcsv) - * gRNA_counts_table.csv: Table with gRNA ID in the first column and sample IDs as the column names (first row) - -### Full Parameters - * `-e`, `--edits` (default: `None`): Path to edit counts .csv table, with index at first column and column names at the first row. - * `-o`, `--output-prefix` (default: `None`): Output file prefix (output will be saved as `output_prefix.h5ad`). If not provided, `gRNA_counts_table_csv` file prefix is used. - -

      - -## `bean-profile`: Profile editing patterns -```bash -bean-profile my_sorting_screen.h5ad -o output_prefix `# Prefix for editing profile report` -``` -### Output -Above command produces `prefix_editing_preference.[html,ipynb]` as editing preferences ([see example](notebooks/profile_editing_preference.ipynb)). -Allele translation -### Parameters - * `-o`, `--output-prefix` (default: `None`): Output prefix of editing pattern report (prefix.html, prefix.ipynb). If not provided, base name of `bdata_path` is used. - * `--replicate-col` (default: `"replicate"`): Column name in `bdata.samples` that describes replicate ID. - * `--condition-col` (default: `"condition"`): Column name in `bdata.samples` that describes experimental condition. (sorting bin, time, etc.) - * `--pam-col` (default: `None`): Column name describing PAM of each gRNA in `bdata.guides`. - * `--control-condition` (default: `"bulk"`): Control condition where editing preference would be profiled at. Pre-filters data where `bdata.samples[condition_col] == control_condition`. DO NOT use plasmid library as control here where we do not expect editing. - * `-w`, `--window-length` (default: `6`): Window length of editing window of maximal editing efficiency to be identified. This window is used to quantify context specificity within the window. +## Documentaton +See the [documentation](https://pinellolab.github.io/crispr-bean/) for tutorials and API references. +## Tutorials +| [Library design](#pipeline-run-options-by-library-design) | Selection | Reporter | Tutorial link | +|---------------| -------------- | --------- | -------- | +| GWAS variant library | FACS sorting | Yes/No | [GWAS variant screen](https://pinellolab.github.io/crispr-bean/gwas) +| Coding sequence tiling libarary | FACS sorting | Yes/No | [Coding sequence tiling screen](https://pinellolab.github.io/crispr-bean/cds) +| GWAS variant library | Survival / Proliferation | Yes/No | [GWAS variant screen](https://pinellolab.github.io/crispr-bean/prolif_gwas) +| Coding sequence tiling libarary | Survival / Proliferation | Yes/No | Coming soon! -

      - -## `bean-qc`: QC of reporter screen data -```bash -bean-qc \ - my_sorting_screen.h5ad `# Input ReporterScreen .h5ad file path` \ - -o my_sorting_screen_masked.h5ad `# Output ReporterScreen .h5ad file path` \ - -r qc_report_my_sorting_screen `# Prefix for QC report` \ - --ctrl-cond presort `# "condition" column in the control sample before selection. Mean gRNA editing rates in these samples are reported. ` \ -# Inspect the output qc_report_my_sorting_screen.html to tweak QC threshold - -bean-qc \ - my_sorting_screen.h5ad \ - -o my_sorting_screen_masked.h5ad \ - -r qc_report_my_sorting_screen \ - #[--count-correlation-thres 0.7 ...]\ - -b -``` - -`bean-qc` supports following quality control and masks samples with low quality. Specifically: - -Allele translation - -* Plots guide coverage and the uniformity of coverage -* Guide count correlation between samples -* Log fold change correlation when positive controls are provided -* Plots editing rate distribution -* Identify samples with low guide coverage/guide count correlation/editing rate and mask the sample in `bdata.samples.mask` -* Identify outlier guides to filter out - -#### Output -Above command produces -* `my_sorting_screen_masked.h5ad` without problematic replicate and guides and with sample masks, and -* `qc_report_my_sorting_screen.[html,ipynb]` as QC report. - - -#### Additional Parameters -##### Optional arguments: -* `-o OUT_SCREEN_PATH`, `--out-screen-path OUT_SCREEN_PATH` - Path where quality-filtered ReporterScreen object to be written to -* `-r OUT_REPORT_PREFIX`, `--out-report-prefix OUT_REPORT_PREFIX` - Output prefix of qc report (prefix.html, prefix.ipynb) - -##### QC thresholds: -* `--count-correlation-thres COUNT_CORRELATION_THRES` - Correlation threshold to mask out. -* `--edit-rate-thres EDIT_RATE_THRES` - Mean editing rate threshold per sample to mask out. -* `--lfc-thres LFC_THRES` - Positive guides' correlation threshold to filter out. - -##### Run options: -* `-b`, `--remove-bad-replicates` - Remove replicates with at least two of its samples meet the QC threshold (bean-run does not support having only one sorting bin sample for a replicate). -* `-i`, `--ignore-missing-samples` - If the flag is not provided, if the ReporterScreen object does not contain all condiitons for - each replicate, make fake empty samples. If the flag is provided, don't add dummy samples. -* `--no-editing` Ignore QC about editing. Can be used for QC of other editing modalities. -* `--dont-recalculate-edits` - When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from - ReporterScreen.uns['allele_count']. - -##### Input `.h5ad` formatting: -Note that these arguements will change the way the QC metrics are calculated for guides, samples, or replicates. -* `--tiling TILING` Specify that the guide library is tiling library without 'n guides per target' design -* `--replicate-label REPLICATE_LABEL` - Label of column in `bdata.samples` that describes replicate ID. -* `--sample-covariates SAMPLE_COVARIATES` - Comma-separated list of column names in `bdata.samples` that describes non-selective - experimental condition. (drug treatment, etc.) -* `--condition-label CONDITION_LABEL` - Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time, - etc.) -###### Editing rate calculation - * `--control-condition CTRL_COND` - Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate - to be calculated. Default is `None`, which considers all samples. - * `--rel-pos-is-reporter` - Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If - `False`, those are relative to spacer position. - Editing rate is calculated with following parameters in - * Variant screens: - * `--target-pos-col TARGET_POS_COL` - Target position column in `bdata.guides` specifying target edit position in reporter - * tiling screens: - * `--edit-start-pos EDIT_START_POS` - Edit start position to quantify editing rate on, 0-based inclusive. - * `--edit-end-pos EDIT_END_POS` - Edit end position to quantify editing rate on, 0-based exclusive. -###### LFC of positive controls - * `--posctrl-col POSCTRL_COL` - Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all - gRNAs, feed empty string ''. - * `--posctrl-val POSCTRL_VAL` - Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the - positive control in calculating log fold change. - * `--lfc-conds LFC_CONDS` - Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated - between, delimited by comma - -

      - - -## `bean-filter`: Filtering (and optionally translating) alleles -As `tiling` mode of `bean-run` accounts for any robustly observed alleles, `bean-filter` filters for such alleles. -```bash -bean-filter my_sorting_screen_masked.h5ad \ --o my_sorting_screen_filtered.h5ad `# Output file path` \ -``` - -### Output -Above command produces -* `my_sorting_screen_filtered.h5ad` with filtered alleles stored in `.uns`, -* `my_sorting_screen_filtered.filtered_allele_stats.pdf`, and `my_sorting_screen_filtered.filter_log.txt` that report allele count stats in each filtering step. - -You may want to adjust the flitering parameters to obtain optimal balance between # guides per variant & # variants that are scored. See example outputs of filtering step [here](docs/example_filtering_output/). - - -### Translating alleles -If you want to obtain **amino acid level variant** for coding sequence tiling screens, provide coding sequence positions which variants occuring within the coding sequence will be translated. *This is optional, but **highly recommended** to increase per-(coding)variant support.* - -Allele translation - - -```bash -bean-filter my_sorting_screen.h5ad \ --o my_sorting_screen_masked.h5ad \ ---translate `# Translate coding variants` \ -[ --translate-gene-name GENE_SYMBOL OR - --translate-genes-list path_to_gene_names_file.txt OR - --translate-fasta gene_exon.fa, OR - --translate-fastas-csv gene_exon_fas.csv] -``` -* When library covers a single gene, do either of the following: - 1. Feed `--translate-gene-name GENE_SYMBOL` if your `genomic_pos` column of `sgRNA_info_tbl` is compatible with [MANE transcript](https://useast.ensembl.org/info/genome/genebuild/mane.html)'s reference genome. (Per 10/23/2023, GRCh38). This will automatically load the exon positions based on MANE transcript annotation. - 2. To use your custom coding sequence and exon positions, feed `--translate-fasta gene_exon.fa` argument where `gene_exon.fa` is the FASTA file with entries of exons. [See full details here](docs/exon_fa_format.md). -* When library covers multiple genes, do either of the following: - 1. Feed `--translate-genes-list path_to_gene_names_file.txt` where `path_to_gene_names_file.txt` is file with one gene symbol per line. - 2. Feed `--translate-fastas-csv gene_exon_fas.csv` where `gene_exon_fas.csv` is the csv file with lines `gene_id,gene_exon_fasta_path` without header. Each FASTA file in `gene_exon_fasta_path` is formatted [as the single-gene FASTA file](docs/exon_fa_format.md). -* Translation will keep the variants outside the coding sequence as nucleotide-level variants, while aggregating variants leading to the same coding sequence variants. - -### Full list of parameters -* `-o`, `--output-prefix` (default: `None`): Output prefix for log and ReporterScreen file with allele assignment. -* `-p`, `--plasmid-path` (default: `None`): Plasmid `ReporterScreen` object path. - * If provided, alleles are filtered based on if a nucleotide edit is more significantly enriched in sample compared to the plasmid data. - * Negative control data where no edit is expected can be fed in instead of plasmid library. -* `-w`, `--filter-window` (default: `False`): "Only consider edit within window provided by (`edit_start_pos`, `edit_end_pos`). If this flag is not provided, `--edit-start-pos` and `--edit-end-pos` flags are ignored. -* `-s`, `--edit-start-pos` (default: `2`): 0-based start posiiton (inclusive) of edit relative to the start of guide spacer. -* `-e`, `--edit-end-pos` (default: `7`): 0-based end position (exclusive) of edit relative to the start of guide spacer. -* `-b`, `--filter-target-basechange` (default: `False`): Filter for target (intended) base change of edits (stored in `bdata.uns['target_base_change']`) -* `-j`, `--jaccard-threshold` (default: `0.3`): Jaccard Index threshold when the alleles are mapped to the most similar alleles. In each filtering step, allele counts of filtered out alleles will be mapped to the most similar allele only if they have Jaccard Index of shared edit higher than this threshold. -* `--translate` (default: `False`): Translate nucleotide-level variants prior to allele proportion filtering. -* `-f`, `--translate-fasta` (defulat: `None`): fasta file path with exon positions. If not provided and `--translate` flag is provided, LDLR hg19 coordinates will be used. -* `-fs`, `--translate-fastas-csv` (defulat: `None`): .csv with two columns with gene IDs and FASTA file path corresponding to each gene. -* `-g`, `--translate-gene-name` (default: `None`): Gene symbol for translation -* `-gs`, `--translate-genes-list` (default: `None`): Path to the text file with gene symbols in each line -* `-ap`, `--filter-allele-proportion` (default: `0.05`): If provided, only the alleles that exceed `filter_allele_proportion` in `filter-sample-proportion` will be retained. -* `-ac`, `--filter-allele-count` (default: `5`): If provided, alleles that exceed `filter_allele_proportion` AND `filter_allele_count` in `filter-sample-proportion` will be retained. -* `sp`, `--filter-sample-proportion` (default: `0.2`): "If `filter_allele_proportion` is provided, alleles that exceed `filter_allele_proportion` in `filter-sample-proportion` will be retained. - - -

      - -## `bean-run`: Quantify variant effects -BEAN uses Bayesian network to incorporate gRNA editing outcome to provide posterior estimate of variant phenotype. The Bayesian network reflects data generation process. Briefly, -1. Cellular phenotype (either for cells are sorted upon for sorting screen, or log(proliferation rate)) is modeled as the Gaussian mixture distribution of wild-type phenotype and variant phenotype. -2. The weight of the mixture components are inferred from the reporter editing outcome and the chromatin accessibility of the loci. -3. Cells with each gRNA, formulated as the mixture distribution, is sorted by the phenotypic quantile to produce the gRNA counts. - -For the full detail, see the method section of the [BEAN manuscript](https://www.medrxiv.org/content/10.1101/2023.09.08.23295253v1). - -model - -

      -```bash -bean-run sorting[survival] variant[tiling] my_sorting_screen_filtered.h5ad \ -[--uniform-edit, --scale-by-acc [--acc-bw-path accessibility_signal.bw, --acc-col accessibility]] \ --o output_prefix/ \ ---fit-negctrl -``` - -### Input -`my_sorting_screen_filtered.h5ad` can be produced by one of the following: -1. [`bean-count-samples`]((#bean-count-samples-count-reporter-screen-data)) when you have raw `.fastq` file -2. (Limited to `bean-run variant` mode) `bean-create-screen` when you have flat `.csv` tables of gRNA metadata table, sample metadata table, gRNA counts table (# guides x # samples), and optionally # edits table. - ```bash - bean-create-screen gRNA_info_table.csv sample_info_table.csv gRNA_counts_table.csv \ - [--edits edit_counts_table.csv -o output.h5ad] - ``` - * `gRNA_info_table.csv` should have following columns. - * `name`: gRNA ID column - * `target`: This column denotes which target variant/element of each gRNA. - * `target_group [Optional]`: If negative control gRNA will be used, specify as "NegCtrl" in this column. - * `sample_info_table.csv` should have following columns. - * `sample_id`: ID of sequencing sample - * `replicate`: Replicate # of this sample - * `bin`: Name of the sorting bin - * `upper_quantile`: FACS sorting upper quantile - * `lower_quantile`: FACS sorting lower quantile - * `gRNA_counts_table.csv` should be formatted as follows. - * Columns include one of `sample_id` columns in `sample_info_table.csv` file. - * 1st row (row index) follows `name` (gRNA ID) in `gRNA_info_table.csv` file. -3. You can manually create the `AnnData` object with more annotations including allele counts: see [API tutorial](#using-bean-as-python-module) for full detail. - - -### Output -model - -Above command produces -* `output_prefix/bean_element_result.[model_type].csv` with following columns: - * Estimated variant effect sizes - * `mu` (Effect size): Mean of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. - * `mu_sd`: Mean of variant phenotype `mu` is modeled as normal distribution. The column shows fitted standard deviation of `mu` that quantify the uncertainty of the variant effect. - * `mu_z`: z-score of `mu` - * `sd`: Standard deviation of variant phenotype, given the wild type has standard normal phenotype distribution of `mu = 0, sd = 1`. - * `CI[0.025`, `0.975]`: Credible interval of `mu` - * When negative control is provided, above columns with `_adj` suffix are provided, which are the corresponding values adjusted for negative control. - * Metrics on per-variant evidence provided in input (provided in `tiling` mode) - * `effective_edit_rate`: Sum of per-variant editing rates over all alleles observed in the input. Allele-level editing rate is divided by the number of variants observed in the allele prior to summing up. - * `n_guides`: # of guides covering the variant. - * `n_coocc`: # of cooccurring variants with a given variant in any alleles observed in the input. -* `output_prefix/bean_sgRNA_result.[model_type].csv`: - * `edit_rate`: Estimated editing rate at the target loci. - -### Optional parameters -* Run options - * `-o`, `--outdir`: Directory to save the run result. - * `--result-suffix` (default: ""): Suffix of the output files. - * `--uniform-edit` (default: `False`): Ignores variable editing - * `--scale-by-acc` (default: `False`): Scale guide editing efficiency by the target loci accessibility. Needs one of `acc_bw_path` or `acc_col` that provides per-guide accessibility information. - * `--acc-bw-path` : Accessibility .bigWig file to be used to assign accessibility of guides. To read accessibility information from the .bigWig file, input `.h5ad` file needs `chr` and `genomic_pos` column in `.guides` DataFrame. - * `--fit-negctrl` (default: `False`): Fit the shared negative control distribution to normalize the fitted parameters. - * `--negctrl-col` (default: `target_group`): Column in bdata.obs specifying if a guide is negative control. If the `bdata.guides[negctrl_col].tolower() == negctrl_col_value`, it is treated as negative control guide. - * `--negctrl-col-value` (default: `negctrl`): Column value in bdata.guides specifying if a guide is negative control. If the `bdata.guides[negctrl_col].tolower() == negctrl_col_value`, it is treated as negative control guide. - * `--repguide-mask` (default: `repguide_mask`): n_replicate x n_guide mask to mask the outlier guides. bdata.uns[repguide_mask] will be used. This is calculated with `bean-qc`. - * `--cuda` (default: `False`): Run on GPU - * `--device`: Optionally use GPU if provided valid GPU device name (ex. cuda:0) - * `--ignore-bcmatch` (default: `False`): If provided, even if the screen object has .X_bcmatch, ignore the count when fitting. - * `--allele-df-key` (default: `allele_counts`): bdata.uns[allele_df_key] will be used as the allele count. - * `--control-guide-tag` (default: `None`): (Relevant in bean-run *tiling* mode) If this string is in guide name, treat each guide separately not to mix the position. Used for non-targeting negative controls. -* Guide annotations (`bdata.guides` column keys) - * `--acc-col`: Column name in bdata.guides that specify raw ATAC-seq signal. - * `--target-column` (default: `target`): Column key in `bdata.guides` that describes the target element of each guide. - * `--guide-activity-col`: Column in `bdata.guides` DataFrame showing the editing rate estimated via external tools. -* Sample annotations (`bdata.samples` column keys) - * `--condition-column` (default: `condition`): Column key in `bdata.samples` that describes experimental condition. - * `--control-condition` (default: `bulk`): Value in `bdata.samples[condition_col]` that indicates control experimental condition. - * `-uq`, `--sorting-bin-upper-quantile-column` (default: `upper_quantile`): Column name with upper quantile values of each sorting bin in bdata.samples - * `-lq`, `--sorting-bin-lower-quantile-column` (default: `lower_quantile`): Column name with lower quantile values of each sorting bin in bdata.samples - -

      -## Data Structure -### ReporterScreen object -BEAN stores mapped gRNA and allele counts in `ReporterScreen` object which is compatible with [AnnData](https://anndata.readthedocs.io/en/latest/index.html). +### Library design: variant or tiling? +The `bean filter` and `bean run` steps depend on the type of gRNA library design, where BEAN supports two modes of running. +variant library design -ReporterScreen object structure +1. `variant` library: Several gRNAs tile each of the targeted variants. Only the editing rate of the target variant is considered and the bystander effects are ignored. - * `.guides`: guide information provided in input (`gRNA_library.csv` in above example) - * `.samples`: sample information provided in input (`sample_list.csv` in above example) - * `.X`: Main guide count matrix, where row corresponds to each guide in `.guides` and columns correspond to samples in `.samples`. -Following attributes are included if matched reporter is provided and you chose to read edit/allele information from the reporter using `-r` option. - * `.X_bcmatch [Optional]`: Contains information about number of barcode-matched reads. Information about R2 barcode should be specified as `barcode` column in your `gRNA_library.csv` file. - * `.X_edits [Optional]`: If target position of each guide is specified as `target_pos` in input `gRNA_library.csv` file and `--match-target-position` option is provided, the result has the matrix with the number of target edit at the specified positions. - * `.allele_tables [Optional]`: Dictionary with a single allele count table that counts per guide and allele combination, what is the count per sample. +2. `tiling` library: gRNA densely tiles a long region (e.g. gene(s), exon(s), coding sequence(s)). Bystander edits are considered to obtain alleles with significant fractions. Edited alleles can be "translated" to output coding variants. -### Using BEAN as Python module +## Using BEAN as Python module ``` import bean as be cdata = be.read_h5ad("bean_counts_sample.h5ad") ``` Python package `bean` supports multiple data wrangling functionalities for `ReporterScreen` objects. See the [**ReporterScreen API tutorial**](docs/ReporterScreen_api.ipynb) for more detail. -

      - ## Run time * Installation takes 14.4 mins after pytorch installation with pytorch in Dell XPS 13 Ubuntu WSL. -* `bean-run` takes 4.6 mins with `--scale-by-acc` tag in Dell XPS 13 Ubuntu WSL for variant screen dataset with 3455 guides and 6 replicates with 4 sorting bins. +* `bean run` takes 4.6 mins with `--scale-by-acc` tag in Dell XPS 13 Ubuntu WSL for variant screen dataset with 3455 guides and 6 replicates with 4 sorting bins. * Full pipeline takes 90.1s in GitHub Action for toy dataset of 2 replicates and 30 guides. +## Contributing +If you have questions or feature request, please open an issue. Please feel free to send a pull request. + ## Citation If you have used BEAN for your analysis, please cite: Ryu, J. et al. Joint genotypic and phenotypic outcome modeling improves base editing variant effect quantification. medRxiv (2023) doi:10.1101/2023.09.08.23295253 From 2ae0192df765bf5309e94f36e4e62d50d6a4a5db Mon Sep 17 00:00:00 2001 From: jykr Date: Sat, 30 Mar 2024 15:21:33 -0400 Subject: [PATCH 105/107] add docs --- .github/workflows/CI.yml | 4 +- .gitignore | 1 + LICENSE | 0 MANIFEST.in | 0 bean/__init__.py | 0 bean/annotate/__init__.py | 0 bean/annotate/_supporting_fn.py | 0 bean/annotate/filter_alleles.py | 0 bean/annotate/ldlr_exons.fa | 0 bean/annotate/translate_allele.py | 0 bean/annotate/utils.py | 24 +- bean/framework/AminoAcidEdit.py | 0 bean/framework/Edit.py | 0 bean/framework/ReporterScreen.py | 0 bean/framework/__init__.py | 0 bean/framework/__pycache__/ReporterScreen_.py | 0 bean/framework/allele_counts.py | 0 bean/framework/read_from_csvs.py | 45 - bean/mapping/CRISPResso2Align.pyx | 0 bean/mapping/GuideEditCounter.py | 0 bean/mapping/__init__.py | 0 bean/mapping/_supporting_fn.py | 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actions/setup-python@v3 with: - python-version: '3.8' + python-version: '3.x' - name: Install dependencies run: | python -m pip install --upgrade pip - pip install torch==1.12.1+cpu torchvision==0.13.1+cpu torchaudio==0.12.1 --extra-index-url https://download.pytorch.org/whl/cpu + pip install torch torchvision torchaudio pip install -r requirements.txt pip install -e . - name: Test with pytest diff --git a/.gitignore b/.gitignore index 0d20b64..00cb0e6 100644 --- a/.gitignore +++ b/.gitignore @@ -1 +1,2 @@ *.pyc +*.fastq diff --git a/LICENSE b/LICENSE old mode 100644 new mode 100755 diff --git a/MANIFEST.in b/MANIFEST.in old mode 100644 new mode 100755 diff --git a/bean/__init__.py b/bean/__init__.py old mode 100644 new mode 100755 diff --git a/bean/annotate/__init__.py b/bean/annotate/__init__.py old mode 100644 new mode 100755 diff --git a/bean/annotate/_supporting_fn.py b/bean/annotate/_supporting_fn.py old mode 100644 new mode 100755 diff --git a/bean/annotate/filter_alleles.py b/bean/annotate/filter_alleles.py old mode 100644 new mode 100755 diff --git a/bean/annotate/ldlr_exons.fa b/bean/annotate/ldlr_exons.fa old mode 100644 new mode 100755 diff --git a/bean/annotate/translate_allele.py b/bean/annotate/translate_allele.py old mode 100644 new mode 100755 diff --git a/bean/annotate/utils.py b/bean/annotate/utils.py old mode 100644 new mode 100755 index 2252187..dd1358f --- a/bean/annotate/utils.py +++ b/bean/annotate/utils.py @@ -218,22 +218,12 @@ def get_cds_seq_pos_from_gene_name(gene_name: str, ref_version: str = "GRCh38"): return cds_chrom, cds_seq, cds_pos, strand -def parse_args(): - """Get the input arguments""" - print( - r""" - _ _ - / \ '\ __ _ _ _ - | \ \ / _(_) | |_ ___ _ _ - \ \ | | _| | | _/ -_) '_| - `.__|/ |_| |_|_|\__\___|_| - """ - ) - print("bean-filter: filter alleles") - parser = argparse.ArgumentParser( - prog="allele_filter", - description="Filter alleles based on edit position in spacer and frequency across samples.", - ) +def parse_args(parser=None): + if parser is None: + parser = argparse.ArgumentParser( + prog="allele_filter", + description="Filter alleles based on edit position in spacer and frequency across samples.", + ) parser.add_argument( "bdata_path", type=str, @@ -349,7 +339,7 @@ def parse_args(): action="store_true", help="Load temporary file and work from there.", ) - return parser.parse_args() + return parser def check_args(args): diff --git a/bean/framework/AminoAcidEdit.py b/bean/framework/AminoAcidEdit.py old mode 100644 new mode 100755 diff --git a/bean/framework/Edit.py b/bean/framework/Edit.py old mode 100644 new mode 100755 diff --git a/bean/framework/ReporterScreen.py b/bean/framework/ReporterScreen.py old mode 100644 new mode 100755 diff --git a/bean/framework/__init__.py b/bean/framework/__init__.py old mode 100644 new mode 100755 diff --git a/bean/framework/__pycache__/ReporterScreen_.py b/bean/framework/__pycache__/ReporterScreen_.py old mode 100644 new mode 100755 diff --git a/bean/framework/allele_counts.py b/bean/framework/allele_counts.py old mode 100644 new mode 100755 diff --git a/bean/framework/read_from_csvs.py b/bean/framework/read_from_csvs.py old mode 100644 new mode 100755 index 8331a3f..2a3bd9a --- a/bean/framework/read_from_csvs.py +++ b/bean/framework/read_from_csvs.py @@ -4,51 +4,6 @@ from perturb_tools import Screen -def get_input_parser() -> argparse.Namespace: - """Add multi-sample specific arguments to the base parser.""" - print( - r""" - _ _ - / \ '\ _ - | \ \ __ _ _ ___ __ _| |_ ___ - \ \ | / _| '_/ -_) _` | _/ -_) - `.__|/ \__|_| \___\__,_|\__\___| - """ - ) - parser = argparse.ArgumentParser( - description="bean-create-screen parameters", - formatter_class=argparse.ArgumentDefaultsHelpFormatter, - ) - parser.add_argument( - "gRNA_info_table_csv", - type=str, - help="Path to gRNA info .csv table, with index at first column and column names at the first row.", - ) - parser.add_argument( - "samples_info_table_csv", - type=str, - help="Path to sample info .csv table, with index at first column and column names at the first row.", - ) - parser.add_argument( - "gRNA_counts_table_csv", - type=str, - help="Path to gRNA counts .csv table, with index at first column and column names at the first row.", - ) - parser.add_argument( - "-e", - "--edits", - type=str, - help="Path to edit counts .csv table, with index at first column and column names at the first row.", - default=None, - ) - parser.add_argument( - "-o", - "--output-prefix", - type=str, - help="Output file prefix (output will be saved as `output_prefix.h5ad`). If not provided, `gRNA_counts_table_csv` file prefix is used.", - default=None, - ) - return parser def create_screen(args: argparse.Namespace) -> Screen: diff --git a/bean/mapping/CRISPResso2Align.pyx b/bean/mapping/CRISPResso2Align.pyx old mode 100644 new mode 100755 diff --git a/bean/mapping/GuideEditCounter.py b/bean/mapping/GuideEditCounter.py old mode 100644 new mode 100755 diff --git a/bean/mapping/__init__.py b/bean/mapping/__init__.py old mode 100644 new mode 100755 diff --git a/bean/mapping/_supporting_fn.py b/bean/mapping/_supporting_fn.py old mode 100644 new mode 100755 diff --git a/bean/mapping/utils.py b/bean/mapping/utils.py old mode 100644 new mode 100755 index 34054ce..4bd87c1 --- a/bean/mapping/utils.py +++ b/bean/mapping/utils.py @@ -30,23 +30,77 @@ def _check_library(library_name): ) from exc -def _get_input_parser(): - """Get the input data""" - print( - r""" - _ _ - / \ '\ _ - | \ \ __ ___ _ _ _ _| |_ - \ \ | / _/ _ \ || | ' \ _| - `.__|/ \__\___/\_,_|_||_\__| - """ - ) - - parser = argparse.ArgumentParser( - description="bean-count parameters", - formatter_class=argparse.ArgumentDefaultsHelpFormatter, +def get_input_parser(parser=None): + """Add multi-sample specific arguments to the base parser.""" + if parser is None: + parser = argparse.ArgumentParser() + parser.add_argument( + "-i", + "--sample-list", + type=str, + help="List of fastq and sample ids. Formatted as `R1_filepath,R2_filepath,sample_id`", + required=True, + ) + parser = _get_input_parser(parser) + parser.add_argument( + "-t", "--threads", type=int, help="Number of threads", default=10 + ) + parser.add_argument( + "--guide-start-seqs-file", + type=str, + help="CSV file path with per-sample `guide_start_seq` to be used." + + "Formatted as `sample_id, guide_start_seq`", + default=None, + ) + parser.add_argument( + "--guide-end-seqs-file", + type=str, + help="CSV file path with per-sample `guide_end_seq` to be used." + + "Formatted as `sample_id,guide_end_seq`", + default=None, + ) + parser.add_argument( + "--barcode-start-seqs-file", + type=str, + help="CSV file path with per-sample `barcode_start_seq` to be used." + + "Formatted as `sample_id,guide_end_seq`", + default=None, + ) + + parser.add_argument( + "--rerun", help="Recount each sample", action="store_true", default=False ) + return parser + + +def get_input_parser_count(parser=None): + """Get single-sample specific argument parser.""" + if parser is None: + parser = argparse.ArgumentParser() + parser.add_argument( + "--R1", + type=str, + help="FASTQ file path for read 1", + required=True, + ) + parser.add_argument( + "--R2", + type=str, + help="FASTQ file path for read 2.", + required=True, + ) + parser = _get_input_parser(parser) + return parser + + +def _get_input_parser(parser=None): + if parser is None: + parser = argparse.ArgumentParser( + description="bean-count parameters", + formatter_class=argparse.ArgumentDefaultsHelpFormatter, + ) + parser.add_argument( "-b", "--edited-base", diff --git a/bean/model/__init__.py b/bean/model/__init__.py old mode 100644 new mode 100755 diff --git a/bean/model/model.py b/bean/model/model.py old mode 100644 new mode 100755 diff --git a/bean/model/readwrite.py b/bean/model/readwrite.py old mode 100644 new mode 100755 index a36493b..c7cf181 --- a/bean/model/readwrite.py +++ b/bean/model/readwrite.py @@ -52,6 +52,7 @@ def write_result_table( model_label: str, prefix: str = "", suffix: str = "", + negctrl_params=None, write_fitted_eff: bool = True, adjust_confidence_by_negative_control: bool = True, adjust_confidence_negatives: np.ndarray = None, @@ -62,19 +63,19 @@ def write_result_table( return_result: bool = False, ) -> Union[pd.DataFrame, None]: """Combine target information and scores to write result table to a csv file or return it.""" - if param_hist_dict["params"]["mu_loc"].dim() == 2: - mu = param_hist_dict["params"]["mu_loc"].detach()[:, 0].cpu().numpy() - mu_sd = param_hist_dict["params"]["mu_scale"].detach()[:, 0].cpu().numpy() + if param_hist_dict["mu_loc"].dim() == 2: + mu = param_hist_dict["mu_loc"].detach()[:, 0].cpu().numpy() + mu_sd = param_hist_dict["mu_scale"].detach()[:, 0].cpu().numpy() if sd_is_fitted: - sd = param_hist_dict["params"]["sd_loc"].detach().exp()[:, 0].cpu().numpy() - elif param_hist_dict["params"]["mu_loc"].dim() == 1: - mu = param_hist_dict["params"]["mu_loc"].detach().cpu().numpy() - mu_sd = param_hist_dict["params"]["mu_scale"].detach().cpu().numpy() + sd = param_hist_dict["sd_loc"].detach().exp()[:, 0].cpu().numpy() + elif param_hist_dict["mu_loc"].dim() == 1: + mu = param_hist_dict["mu_loc"].detach().cpu().numpy() + mu_sd = param_hist_dict["mu_scale"].detach().cpu().numpy() if sd_is_fitted: - sd = param_hist_dict["params"]["sd_loc"].detach().exp().cpu().numpy() + sd = param_hist_dict["sd_loc"].detach().exp().cpu().numpy() else: raise ValueError( - f'`mu_loc` has invalid shape of {param_hist_dict["params"]["mu_loc"].shape}' + f'`mu_loc` has invalid shape of {param_hist_dict["mu_loc"].shape}' ) param_dict = { "mu": mu, @@ -85,34 +86,26 @@ def write_result_table( param_dict["sd"] = sd if sample_covariates is not None: assert ( - "mu_cov_loc" in param_hist_dict["params"] - and "mu_cov_scale" in param_hist_dict["params"] - ), param_hist_dict["params"].keys() + "mu_cov_loc" in param_hist_dict and "mu_cov_scale" in param_hist_dict + ), param_hist_dict.keys() for i, sample_cov in enumerate(sample_covariates): param_dict[f"mu_{sample_cov}"] = ( - mu + param_hist_dict["params"]["mu_cov_loc"].detach().cpu().numpy()[i] + mu + param_hist_dict["mu_cov_loc"].detach().cpu().numpy()[i] ) param_dict[f"mu_sd_{sample_cov}"] = np.sqrt( mu_sd**2 - + param_hist_dict["params"]["mu_cov_scale"].detach().cpu().numpy()[i] - ** 2 + + param_hist_dict["mu_cov_scale"].detach().cpu().numpy()[i] ** 2 ) param_dict[f"mu_z_{sample_cov}"] = ( param_dict[f"mu_{sample_cov}"] / param_dict[f"mu_sd_{sample_cov}"] ) fit_df = pd.DataFrame(param_dict) - if "negctrl" in param_hist_dict.keys(): + if negctrl_params is not None: print("Normalizing with common negative control distribution") - mu0 = param_hist_dict["negctrl"]["params"]["mu_loc"].detach().cpu().numpy() + mu0 = negctrl_params["mu_loc"].detach().cpu().numpy() if sd_is_fitted: - sd0 = ( - param_hist_dict["negctrl"]["params"]["sd_loc"] - .detach() - .exp() - .cpu() - .numpy() - ) + sd0 = negctrl_params["sd_loc"].detach().exp().cpu().numpy() else: sd0 = 1.0 print(f"Fitted mu0={mu0}" + (f", sd0={sd0}." if sd_is_fitted else "")) @@ -185,10 +178,10 @@ def write_result_table( ) if write_fitted_eff or guide_acc is not None: - if "alpha_pi" not in param_hist_dict["params"].keys(): + if "alpha_pi" not in param_hist_dict.keys(): pi = 1.0 else: - a_fitted = param_hist_dict["params"]["alpha_pi"].detach().cpu().numpy() + a_fitted = param_hist_dict["alpha_pi"].detach().cpu().numpy() pi = a_fitted[..., 1:].sum(axis=1) / a_fitted.sum(axis=1) sgRNA_df = pd.DataFrame({"edit_eff": pi}, index=guide_index) if guide_acc is not None: @@ -196,7 +189,7 @@ def write_result_table( sgRNA_df["scaled_edit_eff"] = _scale_pi( pi, guide_acc, - fitted_noise_logit=param_hist_dict["params"]["noise_scale"] + fitted_noise_logit=param_hist_dict["noise_scale"] .detach() .cpu() .numpy(), diff --git a/bean/model/run.py b/bean/model/run.py old mode 100644 new mode 100755 index f8bde98..e2347e4 --- a/bean/model/run.py +++ b/bean/model/run.py @@ -1,6 +1,6 @@ import os import sys -import argparse + from tqdm import tqdm import pickle as pkl import pandas as pd @@ -26,242 +26,6 @@ pyro.set_rng_seed(101) -def none_or_str(value): - if value == "None": - return None - return value - - -def parse_args(): - print( - r""" - _ _ - / \ '\ - | \ \ _ _ _ _ _ _ - \ \ | | '_| || | ' \ - `.__|/ |_| \_,_|_||_| - """ - ) - print("bean-run: Run model to identify targeted variants and their impact.") - parser = argparse.ArgumentParser(description="Run model on data.") - parser.add_argument( - "selection", - type=str, - choices=["sorting", "survival"], - help="Screen selection type whether cells are sorted based on continuous phenotype ('sorting') or proliferated based on their viability ('survival').", - ) - parser.add_argument( - "library_design", - type=str, - choices=["variant", "tiling"], - help="Library design type whether to run variant or tiling screen model.\nVariant library design assumes gRNA has specific target variant and bystander edits are ignored. Tiling library design considers all alleles generated by gRNA in reporter.", - ) - parser.add_argument("bdata_path", type=str, help="Path of an ReporterScreen object") - parser.add_argument( - "--rep-pi", - "-r", - action="store_true", - default=False, - help="Fit replicate specific scaling factor. Recommended to set as True if you expect variable editing activity across biological replicates.", - ) - parser.add_argument( - "--uniform-edit", - "-p", - action="store_true", - default=False, - help="Assume uniform editing rate for all guides.", - ) - parser.add_argument( - "--scale-by-acc", - action="store_true", - default=False, - help="Scale guide editing efficiency by the target loci accessibility", - ) - parser.add_argument( - "--acc-bw-path", - type=str, - default=None, - help="Accessibility .bigWig file to be used to assign accessibility of guides.", - ) - parser.add_argument( - "--acc-col", - type=str, - default=None, - help="Column name in bdata.guides that specify raw ATAC-seq signal.", - ) - parser.add_argument( - "--const-pi", - default=False, - action="store_true", - help="Use constant pi provided in --guide-activity-col (instead of fitting from reporter data)", - ) - parser.add_argument( - "--shrink-alpha", - default=False, - action="store_true", - help="Instead of using the trend-fitted alpha values, use estimated alpha values for each gRNA that are shrunk towards the fitted trend.", - ) - parser.add_argument( - "--condition-col", - default="condition", - type=str, - help="Column key in `bdata.samples` that describes experimental condition.", - ) - parser.add_argument( - "--time-col", - default="time", - type=str, - help="Column key in `bdata.samples` that describes time elapsed.", - ) - parser.add_argument( - "--control-condition", - default="bulk", - type=str, - help="Value in `bdata.samples[condition_col]` that indicates control experimental condition.", - ) - parser.add_argument( - "--include-control-condition-for-inference", - "-ic", - default=False, - action="store_true", - help="Include control conditions for inference. Currently only supported for survival screens.", - ) - parser.add_argument( - "--replicate-col", - default="replicate", - type=str, - help="Column key in `bdata.samples` that describes experimental replicates.", - ) - parser.add_argument( - "--target-col", - default="target", - type=str, - help="Column key in `bdata.guides` that describes the target element of each guide.", - ) - parser.add_argument( - "--guide-activity-col", - "-a", - type=str, - default=None, - help="Column in ReporterScreen.guides DataFrame showing the editing rate estimated via external tools", - ) - parser.add_argument( - "--outdir", - "-o", - default=".", - type=str, - help="Directory to save the run result.", - ) - parser.add_argument( - "--result-suffix", - default="", - type=str, - help="Suffix of the output files", - ) - parser.add_argument( - "--sorting-bin-upper-quantile-col", - "-uq", - help="Column name with upper quantile values of each sorting bin in [Reporter]Screen.samples (or AnnData.var)", - default="upper_quantile", - ) - parser.add_argument( - "--sorting-bin-lower-quantile-col", - "-lq", - help="Column name with lower quantile values of each sorting bin in [Reporter]Screen.samples (or AnnData var)", - default="lower_quantile", - ) - parser.add_argument( - "--alpha-if-overdispersion-fitting-fails", - "-af", - default=None, - type=str, - help="Comma-separated regression coefficient (b0, b1) of log(a0) ~ log(q) that will be used if fitting dispersion on the data fails.", - ) - parser.add_argument("--cuda", action="store_true", default=False, help="run on GPU") - parser.add_argument( - "--sample-mask-col", - type=str, - default=None, - help="Name of the column indicating the sample mask in [Reporter]Screen.samples (or AnnData.var). Sample is ignored if the value in this column is 0. This can be used to mask out low-quality samples.", - ) - parser.add_argument( - "--fit-negctrl", - action="store_true", - default=False, - help="Fit the shared negative control distribution to normalize the fitted parameters", - ) - parser.add_argument( - "--negctrl-col", - type=str, - default="target_group", - help="Column in bdata.obs specifying if a guide is negative control. If the `bdata.guides[negctrl_col].lower() == negctrl_col_value`, it is treated as negative control guide.", - ) - parser.add_argument( - "--negctrl-col-value", - type=str, - default="negctrl", - help="Column value in bdata.guides specifying if a guide is negative control. If the `bdata.guides[negctrl_col].lower() == negctrl_col_value`, it is treated as negative control guide.", - ) - parser.add_argument( - "--repguide-mask", - type=none_or_str, - default="repguide_mask", - help="n_replicate x n_guide mask to mask the outlier guides. screen.uns[repguide_mask] will be used.", - ) - parser.add_argument( - "--device", - type=str, - default=None, - help="Optionally use GPU if provided valid GPU device name (ex. cuda:0)", - ) - parser.add_argument( - "--ignore-bcmatch", - action="store_true", - default=False, - help="If provided, even if the screen object has .X_bcmatch, ignore the count when fitting.", - ) - parser.add_argument( - "--allele-df-key", - type=str, - default=None, - help="screen.uns[allele_df_key] will be used as the allele count.", - ) - parser.add_argument( - "--splice-site-path", - type=str, - default=None, - help="Path to splicing site", - ) - parser.add_argument( - "--control-guide-tag", - type=none_or_str, - default=None, - help="If this string is in guide name, treat each guide separately not to mix the position. Used for negative controls.", - ) - parser.add_argument( - "--dont-fit-noise", # TODO: add check args - action="store_true", - ) - parser.add_argument( - "--dont-adjust-confidence-by-negative-control", - action="store_true", - help="Adjust confidence by negative controls. For variant library_design, this uses negative control variants. For tiling library_design, adjusts confidence by synonymous edits.", - ) - parser.add_argument( - "--n-iter", # TODO: add check args - type=int, - default=2000, - help="# of SVI steps taken for inference.", - ) - parser.add_argument( - "--load-existing", # TODO: add check args - action="store_true", - help="Load existing .pkl file if present.", - ) - - return parser.parse_args() - - def check_args(args, bdata): args.adjust_confidence_by_negative_control = ( not args.dont_adjust_confidence_by_negative_control @@ -423,9 +187,9 @@ def run_inference( raise ValueError( f"Fitting halted for command: {' '.join(sys.argv)} with following error: \n {exc}" ) - return { + return pyro.get_param_store(), { "loss": losses, - "params": pyro.get_param_store(), + "params": pyro.get_param_store().get_state(), } diff --git a/bean/model/survival_model.py b/bean/model/survival_model.py old mode 100644 new mode 100755 index ddfb362..88d7d10 --- a/bean/model/survival_model.py +++ b/bean/model/survival_model.py @@ -151,15 +151,17 @@ def ControlNormalModel(data, mask_thres=10, use_bcmatch=True): with replicate_plate2: with pyro.plate("guide_plate3", data.n_guides, dim=-1): a = get_alpha(expected_guide_p, data.size_factor, data.sample_mask, data.a0) - - assert ( - data.X.shape - == data.X_bcmatch.shape - == ( + assert data.X.shape == ( + data.n_reps, + data.n_condits, + data.n_guides, + ), ( + data.X.shape, + ( data.n_reps, data.n_condits, data.n_guides, - ) + ), ) with poutine.mask( mask=torch.logical_and( @@ -307,7 +309,7 @@ def MixtureNormalModel( # assert a.shape == a_bcmatch.shape == (data.n_reps, data.n_guides, data.n_condits) assert ( data.X.shape - == data.X_bcmatch.shape + == data.X_bcmatch_masked.shape == ( data.n_reps, data.n_condits, @@ -563,7 +565,7 @@ def MultiMixtureNormalModel( # assert a.shape == a_bcmatch.shape == (data.n_reps, data.n_guides, data.n_condits) assert ( data.X.shape - == data.X_bcmatch.shape + == data.X_bcmatch_masked.shape == ( data.n_reps, data.n_condits, diff --git a/bean/model/utils.py b/bean/model/utils.py old mode 100644 new mode 100755 diff --git a/bean/plotting/__init__.py b/bean/plotting/__init__.py old mode 100644 new mode 100755 diff --git a/bean/plotting/allele_stats.py b/bean/plotting/allele_stats.py old mode 100644 new mode 100755 diff --git a/bean/plotting/editing_patterns.py b/bean/plotting/editing_patterns.py old mode 100644 new mode 100755 diff --git a/bean/plotting/utils.py b/bean/plotting/utils.py old mode 100644 new mode 100755 index 8080d11..ef84816 --- a/bean/plotting/utils.py +++ b/bean/plotting/utils.py @@ -1,19 +1,9 @@ import argparse -def parse_args(): - print(" \n~~~BEAN Profile~~~") - print("-Profile editing patterns of your editor-") - print( - r""" - _ _ __ _ _ - / \ '\ _ __ _ _ ___ / _(_) |___ - | \ \ | '_ \ '_/ _ \ _| | / -_) - \ \ | | .__/_| \___/_| |_|_\___| - `.__|/ |_| - """ - ) - parser = argparse.ArgumentParser() +def parse_args(parser=None): + if parser is None: + parser = argparse.ArgumentParser() parser.add_argument( "bdata_path", help="Path to the ReporterScreen object to run QC on", type=str ) @@ -55,10 +45,7 @@ def parse_args(): default=6, ) - args = parser.parse_args() - if args.output_prefix is None: - args.output_prefix = f"{args.bdata_path.rsplit('.h5ad', 1)[0]}" - return args + return parser def check_args(args): diff --git a/bean/preprocessing/__init__.py b/bean/preprocessing/__init__.py old mode 100644 new mode 100755 diff --git a/bean/preprocessing/data_class.py b/bean/preprocessing/data_class.py old mode 100644 new mode 100755 index 1418b39..0c37fb2 --- a/bean/preprocessing/data_class.py +++ b/bean/preprocessing/data_class.py @@ -1296,6 +1296,20 @@ def __init__( screen, ) + def __getitem__(self, guide_idx): + ndata = super().__getitem__(guide_idx) + if hasattr(ndata, "X_bcmatch"): + ndata.X_bcmatch = ndata.X_bcmatch[:, :, guide_idx] + if hasattr(ndata, "X_bcmatch_masked"): + ndata.X_bcmatch_masked = ndata.X_bcmatch_masked[:, :, guide_idx] + if hasattr(ndata, "X_bcmatch_control"): + ndata.X_bcmatch_control = ndata.X_bcmatch_control[:, :, guide_idx] + if hasattr(ndata, "X_bcmatch_control_masked"): + ndata.X_bcmatch_control_masked = ndata.X_bcmatch_control_masked[ + :, :, guide_idx + ] + return ndata + def set_bcmatch(self, screen): screen.samples["size_factor_bcmatch"] = self.get_size_factor( screen.layers["X_bcmatch"] diff --git a/bean/preprocessing/get_alpha0.py b/bean/preprocessing/get_alpha0.py old mode 100644 new mode 100755 diff --git a/bean/preprocessing/get_pi_alpha0.py b/bean/preprocessing/get_pi_alpha0.py old mode 100644 new mode 100755 diff --git a/bean/preprocessing/utils.py b/bean/preprocessing/utils.py old mode 100644 new mode 100755 diff --git a/bean/qc/__init__.py b/bean/qc/__init__.py old mode 100644 new mode 100755 diff --git a/bean/qc/guide_qc.py b/bean/qc/guide_qc.py old mode 100644 new mode 100755 diff --git a/bean/qc/sample_qc.py b/bean/qc/sample_qc.py old mode 100644 new mode 100755 diff --git a/bean/qc/utils.py b/bean/qc/utils.py old mode 100644 new mode 100755 index 82c8bf8..b56738a --- a/bean/qc/utils.py +++ b/bean/qc/utils.py @@ -1,166 +1,8 @@ -import distutils from typing import Union, List import numpy as np import pandas as pd from copy import deepcopy -import argparse -from ..framework.ReporterScreen import ReporterScreen, concat - - -def parse_args(): - print(" \n~~~BEANQC~~~") - print("-Check guide/sample level quality and mask / discard-") - print( - r""" - _ _ - / \ '\ ___ ___ - | \ \ / _ \ / __| - \ \ | | (_) | (__ - `.__|/ \__\_\\___| - """ - ) - parser = argparse.ArgumentParser() - parser.add_argument( - "bdata_path", help="Path to the ReporterScreen object to run QC on", type=str - ) - thres_parser = parser.add_argument_group("QC thresholds") - run_parser = parser.add_argument_group("Run options") - input_parser = parser.add_argument_group("Input .h5ad formatting") - - thres_parser.add_argument( - "--count-correlation-thres", - help="Correlation threshold to mask out.", - type=float, - default=0.7, - ) - thres_parser.add_argument( - "--edit-rate-thres", - help="Mean editing rate threshold per sample to mask out.", - type=float, - default=0.1, - ) - thres_parser.add_argument( - "--lfc-thres", - help="Positive guides' correlation threshold to filter out.", - type=float, - default=-0.1, - ) - - parser.add_argument( - "-o", - "--out-screen-path", - help="Path where quality-filtered ReporterScreen object to be written to", - type=str, - ) - parser.add_argument( - "-r", - "--out-report-prefix", - help="Output prefix of qc report (prefix.html, prefix.ipynb)", - type=str, - ) - - run_parser.add_argument( - "-b", - "--remove-bad-replicates", - help="Remove replicates with at least two of its samples meet the QC threshold.", - action="store_true", - ) - run_parser.add_argument( - "-i", - "--ignore-missing-samples", - help="If the flag is not provided, if the ReporterScreen object does not contain all condiitons for each replicate, make fake empty samples. If the flag is provided, don't add dummy samples.", - action="store_true", - ) - run_parser.add_argument( - "--no-editing", - help="Ignore QC about editing. Can be used for QC of other editing modalities.", - action="store_true", - ) - run_parser.add_argument( - "--dont-recalculate-edits", - help="When ReporterScreen.layers['edit_count'] exists, do not recalculate the edit counts from ReporterScreen.uns['allele_count'].", - action="store_true", - ) - - input_parser.add_argument( - "--tiling", - dest="tiling", - type=lambda x: bool(distutils.util.strtobool(x)), - help="Specify that the guide library is tiling library without 'n guides per target' design", - ) - input_parser.add_argument( - "--replicate-label", - help="Label of column in `bdata.samples` that describes replicate ID.", - type=str, - default="replicate", - ) - input_parser.add_argument( - "--sample-covariates", - help="Comma-separated list of column names in `bdata.samples` that describes non-selective experimental condition. (drug treatment, etc.)", - type=str, - default=None, - ) - input_parser.add_argument( - "--condition-label", - help="Label of column in `bdata.samples` that describes experimental condition. (sorting bin, time, etc.)", - type=str, - default="condition", - ) - input_parser.add_argument( - "--target-pos-col", - help="Target position column in `bdata.guides` specifying target edit position in reporter", - type=str, - default="target_pos", - ) - input_parser.add_argument( - "--rel-pos-is-reporter", - help="Specifies whether `edit_start_pos` and `edit_end_pos` are relative to reporter position. If `False`, those are relative to spacer position.", - action="store_true", - default=False, - ) - input_parser.add_argument( - "--edit-start-pos", - help="Edit start position to quantify editing rate on, 0-based inclusive.", - default=2, - ) - input_parser.add_argument( - "--edit-end-pos", - help="Edit end position to quantify editing rate on, 0-based exclusive.", - default=7, - ) - - input_parser.add_argument( - "--posctrl-col", - help="Column name in ReporterScreen.guides DataFrame that specifies guide category. To use all gRNAs, feed empty string ''.", - type=str, - default="target_group", - ) - input_parser.add_argument( - "--posctrl-val", - help="Value in ReporterScreen.guides[`posctrl_col`] that specifies guide will be used as the positive control in calculating log fold change.", - type=str, - default="PosCtrl", - ) - - input_parser.add_argument( - "--lfc-conds", - help="Values in of column in `ReporterScreen.samples[condition_label]` for LFC will be calculated between, delimited by comma", - type=str, - default="top,bot", - ) - input_parser.add_argument( - "--control-condition", - help="Values in of column in `ReporterScreen.samples[condition_label]` for guide-level editing rate to be calculated", - type=str, - default="bulk", - ) - - args = parser.parse_args() - if args.out_screen_path is None: - args.out_screen_path = f"{args.bdata_path.rsplit('.h5ad', 1)[0]}.filtered.h5ad" - if args.out_report_prefix is None: - args.out_report_prefix = f"{args.bdata_path.rsplit('.h5ad', 1)[0]}.qc_report" - return args +from bean.framework.ReporterScreen import ReporterScreen, concat def check_args(args): diff --git a/bean/utils/__init__.py b/bean/utils/__init__.py old mode 100644 new mode 100755 diff --git a/bean/utils/arithmetric.py b/bean/utils/arithmetric.py old mode 100644 new mode 100755 diff --git a/bean/utils/plot.py b/bean/utils/plot.py old mode 100644 new mode 100755 diff --git a/bin/bean-count b/bin/bean-count deleted file mode 100644 index 49a7153..0000000 --- a/bin/bean-count +++ /dev/null @@ -1,105 +0,0 @@ -#!/usr/bin/env python -# -*- coding: utf-8 -*- -"""Count guides, optionally with reporter and alleles of a single sequencing sample.""" - -import logging -import os -import sys - -import bean -from bean.mapping.utils import ( - _check_arguments, - _get_input_parser, - _check_file, - _get_first_read_length, - _check_read_length, -) - -logging.basicConfig( - level=logging.INFO, - format="%(levelname)-5s @ %(asctime)s:\n\t %(message)s \n", - datefmt="%a, %d %b %Y %H:%M:%S", - stream=sys.stderr, - filemode="w", -) -error = logging.critical -warn = logging.warning -debug = logging.debug -info = logging.info - - -_ROOT = os.path.abspath(os.path.dirname(__file__)) - - -def get_input_parser(): - """Get single-sample specific argument parser.""" - parser = _get_input_parser() - parser.add_argument( - "--R1", - type=str, - help="fastq file for read 1", - required=True, - default="Fastq filename", - ) - parser.add_argument( - "--R2", - type=str, - help="fastq file for read 2, sorted as the same name order as in --R1 file.", - required=True, - default="Fastq filename", - ) - return parser - - -def check_arguments(args, info_logger, warn_logger, error_logger): - args = _check_arguments( - args, info_logger=info, warn_logger=warn, error_logger=error - ) - _check_file(args.R1) - _check_file(args.R2) - read_length = _get_first_read_length(args.R1) - _check_read_length(args, read_length, warn) - return args - - -def main(): - parser = get_input_parser() - args = parser.parse_args() - - args = check_arguments(args, info_logger=info, warn_logger=warn, error_logger=error) - args_dict = vars(args) - - edited_from = args_dict["edited_base"] - match_target_pos = args_dict["match_target_pos"] - - counter = bean.mp.GuideEditCounter(**args_dict) - counter.check_filter_fastq() - - counter.get_counts() - if counter.count_reporter_edits: - counter.screen.uns["allele_counts"] = counter.screen.uns["allele_counts"].loc[ - counter.screen.uns["allele_counts"].allele.map(str) != "", : - ] - if match_target_pos: - base_editing_map = {"A": "G", "C": "T"} - edited_to = base_editing_map[edited_from] - counter.screen.get_edit_mat_from_uns( - edited_from, edited_to, match_target_pos - ) - counter.screen.write(f"{counter.output_dir}.h5ad") - counter.screen.to_Excel(f"{counter.output_dir}.xlsx") - info(f"Output written at:\n {counter.output_dir}.h5ad,\n {counter.output_dir}.xlsx") - info("All Done!") - print( - r""" - _ _ - / \ '\ _ - | \ \ __ ___ _ _ _ _| |_ - \ \ | / _/ _ \ || | ' \ _| - `.__|/ \__\___/\_,_|_||_\__| - """ - ) - - -if __name__ == "__main__": - main() diff --git a/bin/bean-count-samples b/bin/bean-count-samples deleted file mode 100644 index 06c3cdb..0000000 --- a/bin/bean-count-samples +++ /dev/null @@ -1,236 +0,0 @@ -#!/usr/bin/env python -"""Count guides, optionally with reporter and alleles for multiple samples that share the same guide library.""" - - -import argparse -import logging -from copy import deepcopy -import os -import sys -from multiprocessing import Pool - -import bean -import pandas as pd -from bean.mapping.utils import ( - InputFileError, - _check_arguments, - _get_input_parser, - _get_first_read_length, - _check_read_length, -) - -logging.basicConfig( - level=logging.INFO, - format="%(levelname)-5s @ %(asctime)s:\n\t %(message)s \n", - datefmt="%a, %d %b %Y %H:%M:%S", - stream=sys.stderr, - filemode="w", -) -error = logging.critical -warn = logging.warning -debug = logging.debug -info = logging.info - - -def get_input_parser() -> argparse.Namespace: - """Add multi-sample specific arguments to the base parser.""" - parser = _get_input_parser() - parser.add_argument( - "-i", - "--input", - type=str, - help="List of fastq and sample ids. Formatted as `R1_filepath,R2_filepath,sample_id`", - required=True, - ) - parser.add_argument( - "-t", "--threads", type=int, help="Number of threads", default=10 - ) - parser.add_argument( - "--guide-start-seqs-file", - type=str, - help="CSV file path with per-sample `guide_start_seq` to be used." - + "Formatted as `sample_id, guide_start_seq`", - default=None, - ) - parser.add_argument( - "--guide-end-seqs-file", - type=str, - help="CSV file path with per-sample `guide_end_seq` to be used." - + "Formatted as `sample_id,guide_end_seq`", - default=None, - ) - parser.add_argument( - "--barcode-start-seqs-file", - type=str, - help="CSV file path with per-sample `barcode_start_seq` to be used." - + "Formatted as `sample_id,guide_end_seq`", - default=None, - ) - - parser.add_argument( - "--rerun", help="Recount each sample", action="store_true", default=False - ) - - return parser - - -def count_sample(R1: str, R2: str, sample_id: str, args: argparse.Namespace): - """Count single sample given R1 and R2 paths. - Arguments are modified accordingly to the provided sample_id before being passed to GuideEditCounter. - """ - args_dict = deepcopy(vars(args)) - args_dict["R1"] = R1 - args_dict["R2"] = R2 - args_dict["name"] = sample_id - args_dict["output_folder"] = os.path.join(args.output_folder, sample_id) - - base_editing_map = {"A": "G", "C": "T"} - edited_from = args_dict["edited_base"] - edited_to = base_editing_map[edited_from] - match_target_pos = args_dict["match_target_pos"] - if ( - "guide_start_seqs_tbl" in args_dict - and args_dict["guide_start_seqs_tbl"] is not None - ): - args_dict["guide_start_seq"] = str(args_dict["guide_start_seqs_tbl"][sample_id]) - if ( - "guide_end_seqs_tbl" in args_dict - and args_dict["guide_end_seqs_tbl"] is not None - ): - args_dict["guide_end_seq"] = args_dict["guide_end_seqs_tbl"][sample_id] - if ( - "barcode_start_seqs_tbl" in args_dict - and args_dict["barcode_start_seqs_tbl"] is not None - ): - args_dict["barcode_start_seq"] = str(args_dict["barcode_start_seqs_tbl"][sample_id]) - counter = bean.mp.GuideEditCounter(**args_dict) - if os.path.exists(f"{counter.output_dir}.h5ad") and not args_dict["rerun"]: - screen = bean.read_h5ad(f"{counter.output_dir}.h5ad") - if counter.count_reporter_edits and match_target_pos: - screen.uns["allele_counts"] = screen.uns["allele_counts"].loc[ - screen.uns["allele_counts"].allele.map(str) != "", : - ] - try: - screen.get_edit_from_allele("allele_counts", "allele") - except ValueError as exc: - raise ValueError( - f"File {counter.output_dir}.h5ad doesn't have alllele information stored." - ) from exc - screen.get_edit_mat_from_uns(edited_from, edited_to, match_target_pos) - info( - f"Reading already existing data for {sample_id} from \n\ - {counter.output_dir}.h5ad" - ) - - else: - info(f"Counting {sample_id}") - counter.check_filter_fastq() - counter.get_counts() - counter.screen.write(f"{counter.output_dir}.h5ad") - screen = counter.screen - if screen.X.max() == 0: - warn(f"Nothing counted for {sample_id}. Check your input.") - if counter.count_reporter_edits and match_target_pos: - screen.uns["allele_counts"] = screen.uns["allele_counts"].loc[ - screen.uns["allele_counts"].allele.map(str) != "", : - ] - screen.get_edit_from_allele("allele_counts", "allele") - screen.get_edit_mat_from_uns(edited_from, edited_to, match_target_pos) - info( - f"Done for {sample_id}. \n\ - Output written at {counter.output_dir}.h5ad" - ) - del counter - return screen - - -def check_arguments(args: argparse.Namespace) -> argparse.Namespace: - """Checks the validity of the argument.""" - args = _check_arguments(args, info, warn, error) - sample_tbl = pd.read_csv(args.input) - if len(sample_tbl.iloc[:, 2].unique()) != len(sample_tbl.iloc[:, 2]): - raise InputFileError( - f"Sample ID not unique. Please check your input file {args.input}." - ) - first_read_lengths = [ - _get_first_read_length(fastq_R1) for fastq_R1 in sample_tbl.iloc[:, 0] - ] - for read_length in first_read_lengths: - _check_read_length(args, read_length, warn) - - def _check_return_guide_seqs_tbl(guide_seqs_file, sample_tbl, label): - """Check if the provided `guide_[start,end]_seqs_file` contains information about all samples in `sample_tbl`.""" - guide_seqs_tbl = pd.read_csv(guide_seqs_file, header=None, dtype=str).fillna("") - if len(guide_seqs_tbl.columns) == 1: - info("No guide start seq provided. Ignoring the file.") - return None - sample_has_seq = sample_tbl.iloc[:, 2].isin(guide_seqs_tbl[0]) - if not sample_has_seq.all(): - raise InputFileError( - f"Sample ID(s) {sample_tbl.iloc[:,2][~sample_has_seq]} not in {label}_seqs_file {guide_seqs_tbl}" - ) - guide_seqs_tbl.columns = ["sample", "seq"] - return guide_seqs_tbl.set_index("sample")["seq"] - - if args.guide_start_seqs_file is not None: - args.guide_start_seqs_tbl = _check_return_guide_seqs_tbl( - args.guide_start_seqs_file, sample_tbl, "guide_start" - ) - if args.guide_end_seqs_file is not None: - args.guide_end_seqs_tbl = _check_return_guide_seqs_tbl( - args.guide_end_seqs_file, sample_tbl, "guide_end" - ) - if args.barcode_start_seqs_file is not None: - args.barcode_start_seqs_tbl = _check_return_guide_seqs_tbl( - args.barcode_start_seqs_file, sample_tbl, "barcode_start" - ) - return args - - -def main(): - parser = get_input_parser() - args = parser.parse_args() - args = check_arguments(args) - sample_tbl = pd.read_csv(args.input) # R1_filepath, R2_filepath, sample_name - sample_tbl_input = sample_tbl.iloc[:, :3] - sample_info_tbl = sample_tbl.iloc[:, 2:].set_index(sample_tbl.columns[2]) - with Pool(processes=args.threads, maxtasksperchild=1) as p: - result = p.starmap( - count_sample, - [ - list(tup) + [args] - for tup in list(sample_tbl_input.to_records(index=False)) - ], - ) - # result = p.starmap(count_sample, sample_tbl[0], sample_tbl[1], sample_tbl[2]) - - screen = bean.concat(result, axis=1) - database_id = args.name or os.path.basename(args.input).split(".")[0] - output_path = os.path.join( - os.path.abspath(args.output_folder), f"bean_count_{database_id}" - ) - if not os.path.exists(args.output_folder): - os.makedirs(args.output_folder) - try: - screen.samples = screen.samples.join(sample_info_tbl, how="left") - except TypeError: - print(screen.samples) - print(sample_info_tbl) - # screen.guides = result[0].guides.loc[screen.guides.index, :] - screen.write(f"{output_path}.h5ad") - screen.to_Excel(f"{output_path}.xlsx") - - info("All Done!") - print( - r""" - _ _ - / \ '\ _ - | \ \ __ ___ _ _ _ _| |_ - \ \ | / _/ _ \ || | ' \ _| - `.__|/ \__\___/\_,_|_||_\__| - """ - ) - - -if __name__ == "__main__": - main() diff --git a/bin/bean-create-screen b/bin/bean-create-screen deleted file mode 100644 index 596b5c9..0000000 --- a/bin/bean-create-screen +++ /dev/null @@ -1,33 +0,0 @@ -#!/usr/bin/env python -"""Create Screen object (AnnData) from gRNA metadata, sample metadata, and count tables.""" - -import os -import sys -import logging -from bean.framework.read_from_csvs import get_input_parser, create_screen - -logging.basicConfig( - level=logging.INFO, - format="%(levelname)-5s @ %(asctime)s:\n\t %(message)s \n", - datefmt="%a, %d %b %Y %H:%M:%S", - stream=sys.stderr, - filemode="w", -) -error = logging.critical -warn = logging.warning -debug = logging.debug -info = logging.info - - -def main(): - parser = get_input_parser() - args = parser.parse_args() - screen = create_screen(args) - info(f"Done obtaining screen:\n{screen}\nWriting result...") - output_path = f"{args.output_prefix if args.output_prefix else os.path.splitext(args.gRNA_counts_table_csv)[0]}.h5ad" - screen.write(output_path) - info(f"Done writing screen object to {output_path}.") - - -if __name__ == "__main__": - main() diff --git a/bin/bean-filter b/bin/bean-filter deleted file mode 100644 index 128ef42..0000000 --- a/bin/bean-filter +++ /dev/null @@ -1,191 +0,0 @@ -#!/usr/bin/env python -# -*- coding: utf-8 -*- -import sys - -import logging -import pandas as pd -import bean as be -from bean.plotting.allele_stats import ( - plot_n_alleles_per_guide, - plot_n_guides_per_edit, - plot_allele_stats, -) -from bean.annotate.translate_allele import get_mismatch_df -from bean.annotate.utils import parse_args, check_args -import matplotlib.pyplot as plt - -plt.style.use("default") -logging.basicConfig( - level=logging.INFO, - format="%(levelname)-5s @ %(asctime)s:\n\t %(message)s \n", - datefmt="%a, %d %b %Y %H:%M:%S", - stream=sys.stderr, - filemode="w", -) -error = logging.critical -warn = logging.warning -debug = logging.debug -info = logging.info - - -if __name__ == "__main__": - args = parse_args() - args = check_args(args) - if not args.load_tmp: - bdata = be.read_h5ad(args.bdata_path) - allele_df_keys = ["allele_counts"] - info( - f"Starting from .uns['allele_counts'] with {len(bdata.uns['allele_counts'])} alleles." - ) - if args.plasmid_path is not None: - info( - "Filtering significantly more edited nucleotide per guide compared to plasmid library..." - ) - plasmid_adata = be.read_h5ad(args.plasmid_path) - plasmid_adata.uns[allele_df_keys[-1]] = plasmid_adata.uns[ - allele_df_keys[-1] - ].loc[plasmid_adata.uns[allele_df_keys[-1]].allele.map(str) != "", :] - - ( - q_val_each, - sig_allele_df, - ) = be.an.filter_alleles.filter_alleles( - bdata, plasmid_adata, filter_each_sample=True, run_parallel=True - ) - bdata.uns["sig_allele_counts"] = sig_allele_df.reset_index(drop=True) - allele_df_keys.append("sig_allele_counts") - info(f"Filtered down to {len(bdata.uns['sig_allele_counts'])} alleles.") - - print(len(bdata.uns[allele_df_keys[-1]])) - if len(bdata.uns[allele_df_keys[-1]]) >= 1: - info("Filtering out edits outside spacer position...") - bdata.uns[ - f"{allele_df_keys[-1]}_spacer" - ] = bdata.filter_allele_counts_by_pos( - rel_pos_start=0, - rel_pos_end=20, - rel_pos_is_reporter=False, - map_to_filtered=True, - allele_uns_key=allele_df_keys[-1], - jaccard_threshold=0.2, - ).reset_index( - drop=True - ) - info( - f"Filtered down to {len(bdata.uns[f'{allele_df_keys[-1]}_spacer'])} alleles." - ) - allele_df_keys.append(f"{allele_df_keys[-1]}_spacer") - bdata.write(f"{args.output_prefix}.tmp.h5ad") - else: - bdata = be.read_h5ad(f"{args.output_prefix}.tmp.h5ad") - allele_df_keys = ["allele_counts"] - if "sig_allele_counts" in bdata.uns.keys(): - allele_df_keys += ["sig_allele_counts"] - if f"{allele_df_keys[-1]}_spacer" in bdata.uns.keys(): - allele_df_keys += [f"{allele_df_keys[-1]}_spacer"] - - if len(bdata.uns[allele_df_keys[-1]]) > 0 and args.filter_window: - info( - f"Filtering out edits based on relatvie position in spacer: 0-based [{args.edit_start_pos},{args.edit_end_pos})..." - ) - filtered_key = f"{allele_df_keys[-1]}_{args.edit_start_pos}_{args.edit_end_pos}" - bdata.uns[filtered_key] = bdata.filter_allele_counts_by_pos( - rel_pos_start=args.edit_start_pos, - rel_pos_end=args.edit_end_pos, - rel_pos_is_reporter=False, - map_to_filtered=True, - allele_uns_key=allele_df_keys[-1], - jaccard_threshold=args.jaccard_threshold, - ).reset_index(drop=True) - allele_df_keys.append(filtered_key) - info(f"Filtered down to {len(bdata.uns[filtered_key])} alleles.") - - if len(bdata.uns[allele_df_keys[-1]]) > 0 and not args.keep_indels: - filtered_key = f"{allele_df_keys[-1]}_noindels" - info(f"Filtering out indels...") - bdata.uns[filtered_key] = bdata.filter_allele_counts_by_base( - ["A", "T", "G", "C"], - ["A", "T", "G", "C"], - map_to_filtered=False, - allele_uns_key=allele_df_keys[-1], - ).reset_index(drop=True) - info(f"Filtered down to {len(bdata.uns[filtered_key])} alleles.") - allele_df_keys.append(filtered_key) - - if len(bdata.uns[allele_df_keys[-1]]) > 0 and args.filter_target_basechange: - filtered_key = ( - f"{allele_df_keys[-1]}_{bdata.base_edited_from}.{bdata.base_edited_to}" - ) - info(f"Filtering out non-{bdata.uns['target_base_change']} edits...") - bdata.uns[filtered_key] = bdata.filter_allele_counts_by_base( - bdata.base_edited_from, - bdata.base_edited_to, - map_to_filtered=False, - allele_uns_key=allele_df_keys[-1], - ).reset_index(drop=True) - info(f"Filtered down to {len(bdata.uns[filtered_key])} alleles.") - allele_df_keys.append(filtered_key) - - if len(bdata.uns[allele_df_keys[-1]]) > 0 and args.translate: - if args.translate_fastas_csv: - fasta_df = pd.read_csv( - args.translate_fastas_csv, - header=None, - ) - fasta_dict = {row[0]: row[1] for i, row in fasta_df.iterrows()} - else: - fasta_dict = None - info( - "Translating alleles..." - ) # TODO: Check & document custom fasta file for translation - filtered_key = f"{allele_df_keys[-1]}_translated" - bdata.uns[filtered_key] = be.translate_allele_df( - bdata.uns[allele_df_keys[-1]], - gene_name=args.translate_gene, - gene_names=args.translate_genes_list, - fasta_file=args.translate_fasta, - fasta_file_dict=fasta_dict, - ).rename(columns={"allele": "aa_allele"}) - get_mismatch_df().to_csv(f"{args.output_prefix}.translation_ref_mismatches.csv") - - allele_df_keys.append(filtered_key) - if not any(bdata.uns[filtered_key].aa_allele.map(lambda a: a.has_coding)): - warn("WARNING: No alleles have been translated. Check your gene input(s).") - info(f"Filtered down to {len(bdata.uns[filtered_key])} alleles.") - - if ( - len(bdata.uns[allele_df_keys[-1]]) > 0 - and args.filter_allele_proportion is not None - ): - info( - f"Filtering alleles for those have allele fraction {args.filter_allele_proportion} in at least {args.filter_sample_proportion*100}% of samples..." - ) - filtered_key = f"{allele_df_keys[-1]}_prop{args.filter_allele_proportion}_{args.filter_sample_proportion}" - bdata.uns[filtered_key] = be.an.filter_alleles.filter_allele_prop( - bdata, - allele_df_keys[-1], - allele_prop_thres=args.filter_allele_proportion, - allele_count_thres=args.filter_allele_count, - sample_prop_thres=args.filter_sample_proportion, - map_to_filtered=True, - retain_max=True, - allele_col=bdata.uns[allele_df_keys[-1]].columns[1], - distribute=True, - jaccard_threshold=args.jaccard_threshold, - ) - allele_df_keys.append(filtered_key) - info(f"Filtered down to {len(bdata.uns[filtered_key])} alleles.") - info("Done filtering!") - info(f"Saving ReporterScreen with filtered alleles at {args.output_prefix}.h5ad...") - bdata.write(f"{args.output_prefix}.h5ad") - - info("Plotting allele stats for each filtering step...") - plot_allele_stats( - bdata, allele_df_keys, f"{args.output_prefix}.filtered_allele_stats.pdf" - ) - info( - f"Saving plotting result and log at {args.output_prefix}.[filtered_allele_stats.pdf, filter_log.txt]." - ) - with open(f"{args.output_prefix}.filter_log.txt", "w") as out_log: - for key in allele_df_keys: - out_log.write(f"{key}\t{len(bdata.uns[key])}\n") diff --git a/bin/bean-profile b/bin/bean-profile deleted file mode 100644 index 8b48daa..0000000 --- a/bin/bean-profile +++ /dev/null @@ -1,34 +0,0 @@ -#!/usr/bin/env python -import os -import papermill as pm -import bean as be -from bean.plotting.utils import parse_args, check_args - - -def main(): - args = parse_args() - args = check_args(args) - os.system( - "python -m ipykernel install --user --name bean_python3 --display-name bean_python3" - ) - pm.execute_notebook( - f"{os.path.dirname(be.__file__)}/../notebooks/profile_editing_preference.ipynb", - f"{args.output_prefix}.ipynb", - parameters=dict( - bdata_path=args.bdata_path, - output_prefix=args.output_prefix, - replicate_col=args.replicate_col, - condition_col=args.condition_col, - control_condition=args.control_condition, - max_editing_window_length=args.window_length, - pam_col=args.pam_col, - ), - kernel_name="bean_python3", - ) - os.system( - f"jupyter nbconvert --to html {args.output_prefix}_editing_preference.ipynb" - ) - - -if __name__ == "__main__": - main() diff --git a/bin/bean-qc b/bin/bean-qc deleted file mode 100644 index a6880bf..0000000 --- a/bin/bean-qc +++ /dev/null @@ -1,46 +0,0 @@ -#!/usr/bin/env python -import os -import papermill as pm -import bean as be -from bean.qc.utils import parse_args, check_args - - -def main(): - args = parse_args() - args = check_args(args) - os.system( - "python -m ipykernel install --user --name bean_python3 --display-name bean_python3" - ) - pm.execute_notebook( - f"{os.path.dirname(be.__file__)}/../notebooks/sample_quality_report.ipynb", - f"{args.out_report_prefix}.ipynb", - parameters=dict( - bdata_path=args.bdata_path, - out_bdata_path=args.out_screen_path, - tiling=args.tiling, - edit_quantification_start_pos=args.edit_start_pos, - edit_quantification_end_pos=args.edit_end_pos, - target_pos_col=args.target_pos_col, - rel_pos_is_reporter=args.rel_pos_is_reporter, - corr_X_thres=args.count_correlation_thres, - edit_rate_thres=args.edit_rate_thres, - posctrl_col=args.posctrl_col, - posctrl_val=args.posctrl_val, - lfc_thres=args.lfc_thres, - replicate_label=args.replicate_label, - condition_label=args.condition_label, - comp_cond1=args.lfc_cond1, - comp_cond2=args.lfc_cond2, - ctrl_cond=args.control_condition, - exp_id=args.out_report_prefix, - recalculate_edits=~args.dont_recalculate_edits, - base_edit_data=args.base_edit_data, - remove_bad_replicates=args.remove_bad_replicates, - ), - kernel_name="bean_python3", - ) - os.system(f"jupyter nbconvert --to html {args.out_report_prefix}.ipynb") - - -if __name__ == "__main__": - main() diff --git a/bin/bean-run b/bin/bean-run deleted file mode 100644 index 540bd37..0000000 --- a/bin/bean-run +++ /dev/null @@ -1,202 +0,0 @@ -#!/usr/bin/env python -import os -import sys -import logging -import warnings -from functools import partial -from copy import deepcopy -import numpy as np -import pandas as pd - -import torch -import pyro -import pyro.infer -import pyro.optim -import pickle as pkl - -import bean.model.model as m -from bean.model.readwrite import write_result_table -from bean.preprocessing.data_class import DATACLASS_DICT -from bean.preprocessing.utils import ( - prepare_bdata, - _obtain_effective_edit_rate, - _obtain_n_guides_alleles_per_variant, - _obtain_n_cooccurring_variants, -) - -import bean as be -from bean.model.run import ( - run_inference, - _get_guide_target_info, - parse_args, - check_args, - identify_model_guide, - identify_negctrl_model_guide, -) - -logging.basicConfig( - level=logging.INFO, - format="%(levelname)-5s @ %(asctime)s:\n\t %(message)s \n", - datefmt="%a, %d %b %Y %H:%M:%S", - stream=sys.stderr, - filemode="w", -) -error = logging.critical -warn = logging.warning -debug = logging.debug -info = logging.info -pyro.set_rng_seed(101) - -warnings.filterwarnings( - "ignore", - category=FutureWarning, - message=r".*is_categorical_dtype is deprecated and will be removed in a future version.*", -) - -warnings.filterwarnings( - "ignore", - category=FutureWarning, - message=r".*FutureWarning: The default of observed=False is deprecated and will be changed to True in a future version of pandas.*", -) - - -def main(args, bdata): - if args.cuda: - os.environ["CUDA_VISIBLE_DEVICES"] = "1" - torch.set_default_tensor_type(torch.cuda.FloatTensor) - else: - torch.set_default_tensor_type(torch.FloatTensor) - prefix = ( - args.outdir - + "/bean_run_result." - + os.path.basename(args.bdata_path).rsplit(".", 1)[0] - ) - os.makedirs(prefix, exist_ok=True) - model_label, model, guide = identify_model_guide(args) - info("Done loading data. Preprocessing...") - bdata = prepare_bdata(bdata, args, warn, prefix) - guide_index = bdata.guides.index.copy() - ndata = DATACLASS_DICT[args.selection][model_label]( - screen=bdata, - device=args.device, - repguide_mask=args.repguide_mask, - sample_mask_column=args.sample_mask_col, - accessibility_col=args.acc_col, - accessibility_bw_path=args.acc_bw_path, - use_const_pi=args.const_pi, - condition_column=args.condition_col, - time_column=args.time_col, - control_condition=args.control_condition, - control_can_be_selected=args.include_control_condition_for_inference, - allele_df_key=args.allele_df_key, - control_guide_tag=args.control_guide_tag, - target_col=args.target_col, - shrink_alpha=args.shrink_alpha, - popt=args.popt, - replicate_col=args.replicate_col, - use_bcmatch=(not args.ignore_bcmatch), - ) - adj_negctrl_idx = None - if args.library_design == "variant": - if not args.uniform_edit: - if "edit_rate" not in ndata.screen.guides.columns: - ndata.screen.get_edit_from_allele() - ndata.screen.get_edit_mat_from_uns(rel_pos_is_reporter=True) - ndata.screen.get_guide_edit_rate( - unsorted_condition_label=args.control_condition - ) - target_info_df = _get_guide_target_info( - ndata.screen, args, cols_include=[args.negctrl_col] - ) - if args.adjust_confidence_by_negative_control: - adj_negctrl_idx = np.where( - target_info_df[args.negctrl_col].map(lambda s: s.lower()) - == args.negctrl_col_value.lower() - )[0] - else: - if args.splice_site_path is not None: - splice_site = pd.read_csv(args.splice_site_path).pos - target_info_df = be.an.translate_allele.annotate_edit( - pd.DataFrame(pd.Series(ndata.edit_index)) - .reset_index() - .rename(columns={"index": "edit"}), - control_tag=args.control_guide_tag, - splice_sites=None if args.splice_site_path is None else splice_site, - ) - target_info_df["effective_edit_rate"] = _obtain_effective_edit_rate(ndata).cpu() - target_info_df["n_guides"] = _obtain_n_guides_alleles_per_variant(ndata).cpu() - target_info_df["n_coocc"] = _obtain_n_cooccurring_variants(ndata) - if args.adjust_confidence_by_negative_control: - adj_negctrl_idx = np.where( - (target_info_df.ref == target_info_df.alt) - & (target_info_df.coding == "coding") - )[0] - print("syn idx: ", adj_negctrl_idx) - - guide_info_df = ndata.screen.guides - - info(f"Running inference for {model_label}...") - - if args.load_existing: - with open(f"{prefix}/{model_label}.result.pkl", "rb") as handle: - param_history_dict = pkl.load(handle) - else: - param_history_dict = deepcopy( - run_inference(model, guide, ndata, num_steps=args.n_iter) - ) - if args.fit_negctrl: - negctrl_model, negctrl_guide = identify_negctrl_model_guide( - args, "X_bcmatch" in bdata.layers - ) - negctrl_idx = np.where( - guide_info_df[args.negctrl_col].map(lambda s: s.lower()) - == args.negctrl_col_value.lower() - )[0] - info( - f"Using {len(negctrl_idx)} negative control elements to adjust phenotypic effect sizes..." - ) - ndata_negctrl = ndata[negctrl_idx] - param_history_dict["negctrl"] = run_inference( - negctrl_model, negctrl_guide, ndata_negctrl, num_steps=args.n_iter - ) - - outfile_path = ( - f"{prefix}/bean_element[sgRNA]_result.{model_label}{args.result_suffix}.csv" - ) - info(f"Done running inference. Writing result at {outfile_path}...") - if not os.path.exists(prefix): - os.makedirs(prefix) - with open(f"{prefix}/{model_label}.result{args.result_suffix}.pkl", "wb") as handle: - try: - pkl.dump(param_history_dict, handle) - except TypeError as exc: - print(exc.message) - # print(param_history_dict) - write_result_table( - target_info_df, - param_history_dict, - model_label=model_label, - prefix=f"{prefix}/", - suffix=args.result_suffix, - guide_index=guide_index, - guide_acc=( - ndata.guide_accessibility.cpu().numpy() - if hasattr(ndata, "guide_accessibility") - and ndata.guide_accessibility is not None - else None - ), - adjust_confidence_by_negative_control=args.adjust_confidence_by_negative_control, - adjust_confidence_negatives=adj_negctrl_idx, - sd_is_fitted=(args.selection == "sorting"), - sample_covariates=( - ndata.sample_covariates if hasattr(ndata, "sample_covariates") else None - ), - ) - info("Done!") - - -if __name__ == "__main__": - args = parse_args() - bdata = be.read_h5ad(args.bdata_path) - args, bdata = check_args(args, bdata) - main(args, bdata) diff --git a/docs/ReporterScreen_api.ipynb b/docs/ReporterScreen_api.ipynb old mode 100644 new mode 100755 diff --git a/docs/ReporterScreen_api.rst b/docs/ReporterScreen_api.rst old mode 100644 new mode 100755 index d333908..d489ffc --- a/docs/ReporterScreen_api.rst +++ b/docs/ReporterScreen_api.rst @@ -1,4 +1,4 @@ -Tutorial +ReporterScreen API tutorial ================== Load the required packages. (Anndata import isn't required to use the package). diff --git a/docs/editing_pattern_analysis.ipynb b/docs/editing_pattern_analysis.ipynb old mode 100644 new mode 100755 diff --git a/docs/exon_fa_format.md b/docs/exon_fa_format.md old mode 100644 new mode 100755 diff --git a/docs/index.md b/docs/index.md deleted file mode 100644 index 45e2414..0000000 --- a/docs/index.md +++ /dev/null @@ -1,4 +0,0 @@ ---- -layout: default -title: CRISPR-BEAN ---- diff --git a/docs/qc_report_LDLvar.html b/docs/qc_report_LDLvar.html old mode 100644 new mode 100755 diff --git a/docs/qc_report_LDLvar.ipynb b/docs/qc_report_LDLvar.ipynb old mode 100644 new mode 100755 diff --git a/docs/sample_quality_report.ipynb b/docs/sample_quality_report.ipynb old mode 100644 new mode 100755 diff --git a/environment_bean.yml b/environment_bean.yml old mode 100644 new mode 100755 diff --git a/imgs/anbe.svg b/imgs/anbe.svg deleted file mode 100644 index b39b191..0000000 --- a/imgs/anbe.svg +++ /dev/null @@ -1 +0,0 @@ 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