Granular read analysis using --fastq_output

[kclem]

Very granular data can be produced for each read by running CRISPResso with the --fastq_output flag. If this flag is given, CRISPResso will produce a fastq file (CRISPResso_output.fastq.gz) containing the results of analysis for every input read.

This granular output is useful in investigating alignments and alignment scores for a read aligned to all reference sequences (not only the assigned reference sequence), or finding out which exact read was assigned to which reference amplicon.

The format is similar to the standard fastq file format:

• Line 1: sequence id, beginning with a '@' character
• Line 2: sequence read
• Line 3: +
• Line 4: sequence base qualities

The CRISPResso fastq_output format prints lines 1, 2, and 4 exactly as provided in the input fastq file, but adds CRISPResso analysis information after the + on the third line, including the following entries:

• ALN={the reference name the read was assigned to, or multiple reference names if ambiguous}
• ALN_SCORES={'&'-separated alignment scores for each reference - the read will be assigned to the highest-scoring reference}
• ALN_DETAILS={'&'-separated alignment details for each reference. Each detail item is a comma-separated tuple containing 1) the reference name, 2) the alignment of the read including gaps, 3) the alignment of the reference including gaps, and 4) the alignment score
• CLASS={the class name, i.e reference name and whether the read is Modified or Unmodified, or 'AMBIGUOUS'}
• MODS={a ';'-separated list of the number of deletions, insertions, and substitutions in the quantification window in the form D{#deletions};I{#insertions};S{#substitutions} e.g. D24;I0;S0 If the read is assigned to multiple references, this will be a '&'-separated list corresponding to the references in ALN}
• DEL={locations and lengths of deletions in the form location({deletion length}) e.g. 310(24) If the read is assigned to multiple references, this will be a '&'-separated list corresponding to the references in ALN}
• INS={locations. lengths, and inserted bases of insertions in the form location({insertion length}+{inserted_bases}) e.g. 310(1+T) If the read is assigned to multiple references, this will be a '&'-separated list corresponding to the references in ALN}
• SUB={locations of substitutions. If the read is assigned to multiple references, this will be a '&'-separated list corresponding to the references in ALN}
• ALN_REF={string representation of alignment of reference sequence (including gaps)}
• ALN_SEQ={string representation of alignment of read sequence (including gaps)}

For example, this is a fastq entry for a single read:

@A06433:550:HWG5EYRX2:2:2101:31937:3505 1:N:0:CAACGAGT+GCGTATCA
ACTAGTCAGCCGTGGACTGCGGCTGCCTCTTCTGTGCTGATTAGAGAGAGAGAGAGAGAGAGAGAGAGTTCCACGGTGACACCACTCCAGCCAGATTCCGCTCTCTACCGTCGTCCCGTCCTCTCCTAGATGGCCCAGCGCCGCCTCGCCGTCCTCTCTTCCCACCTCCTCCAAACCGACCCTGTACTCCCATCAGCCACGCACCGGCGGGCGGGGCCCGTCGAACCTTTGGATCTCGCCTCCACTTCATCCTGCTGCGCTGGCGGAGGCGACGCTGGACGGGATGAGGAGAAGGGATGCGTCTTTTGTAGGATCAAGGTAGAATATACTCCTTTGATCAGTTTATGCTATTTGATACCGGATTTAGTCGTACTA
+ ALN=WT ALN_SCORES=90.819&92.481 ALN_DETAILS=WT,ACTAGTCAGCCGTGGACTGCGGCTGCCTCTTCTGTGCTGATTAGAGAGAG----AGAGAGAGAGAGAGAGAGTTCCACGGTGACACCACTCCAGCCAGATTCCGCTCTCTACCGTCGTCCCGTCCTCTCCTAGATGGCCCAGCGCCGCCTCGCCGTCCTCTCTTCCCACCTCCTCCAAACCGACCCTGTACTCCCATCAGCCACGCACCGGCGGGCGGGGCCCGTCGAACCTTTGGATCTCGCCTCCACTTCATCCTGCTGCGCTGGCGGAGGCGACGCTGGACGGGATGAGGAGAAGGGATGCGTCTTTTGTAGGATCA------------------------AGGTAGAATATACTCCTTTGATCAGTTTATGCTATTTGATACCGGATTTAGTCGTACTA,------CAGCCGTGGACTGCGGCTGCCTCTCCTGTGCTGATTAGAGAGAGGGGGAGAGAGAGAGAGAGAGAGTTCCACGGTGACGCCACTCCAGCCAGATTCCGCTCTCTACCGTCGTCCCGTCCTCTCCTAGATGGCCCAGCGCCGCCTCGCCGTCCTCTCTTCCCACCTCCTCCAAACCGACCCTGTACTCCCATCAGCCACGCACCGGCGGGCGGGGTCCGTCGAACCTTTGGATCTCGCCTCCACTTCATCCTGCTGCGCTGGCGGAGGCGACGCTGGACGGGATGAGGAGAAGGGATGCGTCTTTTGTAGGATCATCCGTGATGAATCCCCCGCATTCAAGGTAGAATATACTCCTTTGATCAGTTTATGCTATTTGATACCGGATTTAGTCGTACTA,90.819&HDR,ACTAGTCAGCCGTGGACTGCGGCTGCCTCTTCTGTGCTGATTAGAGAGAGAGAGAGAGAGAGAGAGAGTTCCACGGTGACACCACTCCAGCCAGATTCCGCTCTCTACCGTCGTCCCGTCCTCTCCTAGATGGCCCAGCGCCGCCTCGCCGTCCTCTCTTCCCACCTCCTCCAAACCGACCCTGTACTCCCATCAGCCACGCACCGGCGGGCGGGGCCCGTCGAACCTTTGGATCTCGCCTCCACTTCATCCTGCTGCGCTGGCGGAGGCGACGCTGGACGGGATGAGGAGAAGGGATGCGTCTTTTGTAGGATCA------------------------AGGTAGAATATACTCCTTTGATCAGTTTATGCTATTTGATACCGGATTTAGTCGTACTA,------CAGCCGTGGACTGCGGCTGCCTCTTCTGTGCTGATTAGAGAGAGAGAGAGAGAGAGAGAGAGTTCCACGGTGACACCACTCCAGCCAGATTCCGCTCTCTACCGTCGTCCCGTCCTCTCCTAGATGGCCCAGCGCCGCCTCGCCGTCCTCTCTTCCCACCTCCTCCAAACCGACCCTGTACTCCCATCAGCCACGCACCGGCGGGCGGGGCCCGTCGAACCTTTGGATCTCGCCTCCACTTCATCCTGCTGCGCTGGCGGAGGCGACGCTGGACGGGATGAGGAGAAGGGATGCGTCTTTTGTAGGATCATCCGTGATGAATCCCCCGCATTCAAGGTAGAATATACTCCTTTGATCAGTTTATGCTATTTGATACCGGATTTAGTCGTACTA,92.481 CLASS=WT_MODIFIED MODS=D24;I0;S0 DEL=310(24) INS= SUB= ALN_REF=------CAGCCGTGGACTGCGGCTGCCTCTTCTGTGCTGATTAGAGAGAGAGAGAGAGAGAGAGAGAGTTCCACGGTGACACCACTCCAGCCAGATTCCGCTCTCTACCGTCGTCCCGTCCTCTCCTAGATGGCCCAGCGCCGCCTCGCCGTCCTCTCTTCCCACCTCCTCCAAACCGACCCTGTACTCCCATCAGCCACGCACCGGCGGGCGGGGCCCGTCGAACCTTTGGATCTCGCCTCCACTTCATCCTGCTGCGCTGGCGGAGGCGACGCTGGACGGGATGAGGAGAAGGGATGCGTCTTTTGTAGGATCATCCGTGATGAATCCCCCGCATTCAAGGTAGAATATACTCCTTTGATCAGTTTATGCTATTTGATACCGGATTTAGTCGTACTA ALN_SEQ=ACTAGTCAGCCGTGGACTGCGGCTGCCTCTTCTGTGCTGATTAGAGAGAGAGAGAGAGAGAGAGAGAGTTCCACGGTGACACCACTCCAGCCAGATTCCGCTCTCTACCGTCGTCCCGTCCTCTCCTAGATGGCCCAGCGCCGCCTCGCCGTCCTCTCTTCCCACCTCCTCCAAACCGACCCTGTACTCCCATCAGCCACGCACCGGCGGGCGGGGCCCGTCGAACCTTTGGATCTCGCCTCCACTTCATCCTGCTGCGCTGGCGGAGGCGACGCTGGACGGGATGAGGAGAAGGGATGCGTCTTTTGTAGGATCA------------------------AGGTAGAATATACTCCTTTGATCAGTTTATGCTATTTGATACCGGATTTAGTCGTACTA
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFF:FFF:FFF,FFFFFFF,:FFF:FFFFFFFFFFFFFJHJJJJJJJJHHJJJJH@JJJJJJJJJHJJJJJJJJJJJJJ;JJJJJJJHJHHJJHJJJHJHJJJJJ<JJJJHJJJJJJJHJJ@JJH<JJJH@JHJJJJHJJHHJHHJ<JJJJJJJJJJJJJJ<JFFFFF::FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF:FF:FF:FFFFFFFFFFFFF:FFFFFFFFFFF:FFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFF:FFFFF