Mixcr to VDJtools

[pbpayal]

takara-mouse-rna-bcr-smarter

I used this module to generate results from Mixcr.

Exact MiXCR commands

mixcr analyze takara-mouse-rna-bcr-smarter 1B_fastq_R1.gz 1B_fastq_R2.gz result_1B

Import into VDJtools

I want to further investigate the results using VDJtools. Which output file to use for vdjtools? Is it the .clns file or .tsv files? Do I need to convert the file for VDJtool usage? *I tried both convertion and using the .tsv file as is in VDJtools. It gives me error.

Actual Result Files

result_1B.align.report.basicstats.txt result_1B.align.report.json result_1B.align.report.txt result_1B.assemble.report.json result_1B.assemble.report.txt result_1B.clns result_1B.clones_IGH_nondecimal.tsv result_1B.clones_IGH_nondecimal.txt result_1B.clones_IGH.tsv result_1B.clones_IGK.tsv result_1B.clones_IGL.tsv result_1B.qc.json result_1B.qc.txt result_1B.vdjca

VDJ command Convert for TSV file

java -Xmx4g -jar $VDJ_JAR Convert -S mixcr result_1B.clones_IGH.tsv result_1B.clones_IGH

Head of output TSV file (IGH)

````cloneId readCount readFraction targetSequences targetQualities allVHitsWithScore allDHitsWithScore allJHitsWithScore allCHitsWithScore allVAlignments allDAlignments allJAlignments allCAlignments nSeqFR1 minQualFR1 nSeqCDR1 minQualCDR1 nSeqFR2 minQualFR2 nSeqCDR2 minQualCDR2 nSeqFR3 minQualFR3 nSeqCDR3 minQualCDR3 nSeqFR4 minQualFR4 aaSeqFR1 aaSeqCDR1 aaSeqFR2 aaSeqCDR2 aaSeqFR3 aaSeqCDR3 aaSeqFR4 refPoints 1 207.0 0.350253807106599 GTTGTAATTAGTATCACCATCTCCAGGAAAAATCTGTCCAATCCACTCAAGACCCTTTCCAGGCCTCTGCTTCACCCAGTTCATCCAGTAATTACTGGATGAACTGGGTGAAGCAGAGGCCTGGAAAGGGTCTTGAGTGGATTGGACAGATTTTTCCTGGAGATGGTGATACTAATTACAACGGAAAATTCAAGGACAAGGCCACACTGACTGCAGACAAATCCTCCAACACAGCCTACATGCAGCTCAGCAGTCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGAGGCGGCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG [[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[ IGHV1-8000(676) IGHJ200(380) IGHG300(181.7) 77|371|391|0|293|SC77GSA78TSG79TI82AI82ASC83TSC86TST87ASG88TSG91CSG94TI95CST97CSG98ASG101ASC102AST103ASG104ASG106TSG110TST111CSG112CSG115TST118ADG119DG120DG121DG122DC123SG128ADT129SA132CSG133CSA134CST140AI142GI142CSA143TSA144CSA145TST151ASG152CSG153CST155ASA156GSC157TSG158TST162CSG168ASC169TSA231TSC253TSG265ASG273ASG306ASC331T|1199.0 30|68|68|298|336||380.0 GTTGTAATTAGTATCACCATCTCCAGGAAAAATCTGTCCAATCCACTCAAGACCCTTTCCAGGCCTCTGCTTCA 58 CCCAGTTCATCCAGTAATTACTGG 58 ATGAACTGGGTGAAGCAGAGGCCTGGAAAGGGTCTTGAGTGGATTGGACAG 58 ATTTTTCCTGGAGATGGTGATACT 58 AATTACAACGGAAAATTCAAGGACAAGGCCACACTGACTGCAGACAAATCCTCCAACACAGCCTACATGCAGCTCAGCAGTCTGACCTCTGAGGACTCTGCGGTCTATTTC 58 TGTGCAAGAGGCGGCTACTGG 58 GGCCAAGGCACCACTCTCACAGTCTCCTCAG 58 VVISITISRKNLQSTQDPFQASAS PSSSSNYW MNWVKQRPGKGLEWIGQ IFPGDGDT NYNGKFKDKATLTADKSSNTAYMQLSSLTSEDSAVYF CARGGYW GQGTTLTVSS ::::0:74:98:149:173:284:0:293:::::298:-10:305:336::````*

Error

[ERROR] java.lang.RuntimeException: Unable to parse clonotype string 207 for MiXcr input type: Some mandatory columns are absent in the input file.

[mizraelson]

Hi, VDJtools is our legacy software, so we don't support it anymore. The new MiXCR has a slightly new output format that's why vdjtools does not recognize it. Most of the postanalysis function are now included in the MiXCR so you might wanna try using it instead.

Nevertheless, there is a way to manually bypass the issue if needed:

• New MiXCR has columns named : readCount, readFraction and uniqueMoleculeCount, uniqueMoleculeFraction (in case of UMIs). For vdjtools to work correctly the following columns are required: cloneCount, cloneFraction. A simple rename will help. Keep in mind that if you have both reads and UMIs you should pick which one do you wanna use and rename the columns of interest.
• Another thing is region not covered field that sometimes is present if gene region was not covered by the alignment. We used to leave these fields blank (if the region was not covered) but that confused some of our users so we added this note. As a bypass you can run the following command for the mixcr tsv output files to remove it: sed -i 's/region_not_covered//g' sample1.tsv