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Targeted Iso Seq QC Wiki
Elizabeth Tseng edited this page Aug 23, 2022
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11 revisions
Last Updated: 01/23/2020
This wiki shows scripts used for targeted Iso-Seq analysis.
No installation is required. You just download the Cupcake repository from GitHub and add the appropriate paths.
$ git clone https://github.com/Magdoll/cDNA_Cupcake.git
$ export PYTHONPATH=$PYTHONPATH:<path_to>/cDNA_Cupcake/sequence
To see that you have successfully gotten BioPython, bx-python, and the sequence/
subdir working, try the following in Python interpreter:
$ python
Python 3.7.4 (default, Aug 13 2019, 20:35:49)
>>> import Bio # testing BioPython
>>> import bx # testing bx-python
>>> import BED # testing Cupcake sequence/
All of the above should work without error messages.
You will need:
- The input FASTA or FASTQ file
- The aligned SAM file or GTF
- A tab-delimited probe BED file with at least the first three columns and an optional 4th column of
<chrom> <start> <end> <gene_name>
. No headers allowed!
You can generate the aligned SAM file using minimap2:
minimap2 -ax splice -t 30 -uf --secondary=no \
hg38.fa input.fasta > input.fasta.sam
If your input is GTF instead of SAM, you must use the --gtf
option.
The script usage is:
python calc_probe_hit_from_sam.py [BED] [FASTA/FASTQ] [SAM/GTF]
--start_base {0 or 1}
--end_base {0 or 1}
-o [OUTPUT]
[--gtf]
Where --start_base, --end_base
indicates whether the start/end index is 0-based or 1-based.
For example:
python <path_to>/cDNA_Cupcake/sequence/calc_probe_hit_from_sam.py \
--gtf \
my-probes.bed input.fasta input.gtf \
--start_base 0 --end_base 0 \
-o input.gtf.probe_hit.txt
The output file will have the following fields:
read_id : the read ID
read_len : length of read
num_probe : number of distinct probes overlapped
num_base_overlap: total base of the read that overlaps with a probe
loci : mapped start-end of the read on the genome
genes : the 4th column of the probe BED file, if given