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split mutect2 VCF to snps, mnps and indels #170

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merged 48 commits into from
May 22, 2023

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sorelfitzgibbon
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@sorelfitzgibbon sorelfitzgibbon commented Apr 7, 2023

Description

  • Added process at end of Mutect2 workflow to split the output VCF into three VCFs with SNVs, MNVs and indels.
  • The unsplit VCF is also in the output
  • See Atomize Mutect2 MNV variants?
  • Updated and simplified Mutect2 pipeline steps in README
  • Updated Mutect2 flowchart in README

Closes #169

Testing Results

CPCG Tumor/Normal Paired Sample:

  • sample: CPCG0000000196-T001-P01 (chr1)
  • log: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/sfitz-mv-mutect-indels/mutect2.CPCG-chr1.log
  • input YAML: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/sfitz-mv-mutect-indels/CPCG.noContam.yaml
  • config: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/sfitz-mv-mutect-indels/CPCG.chr1.config
  • output: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/sfitz-mv-mutect-indels/CPCG-chr1

Variant Counts

..._pass_indels.vcf.gz 838
..._pass_mnps.vcf.gz 88
..._pass_snps.vcf.gz 1691
..._pass.vcf.gz 2617

926 non-SNVs removed from a total of 2617 chr1 variants.

A-mini:

nftest run
nftest log: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/sfitz-mv-mutect-indels/log-nftest-20230516T003258Z.log
Three log output directories:

  • /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/sfitz-mv-mutect-indels/a_mini_n2-all-tools-std-input/call-sSNV-6.0.0/TWGSAMIN000001-T001-S01-F/log-call-sSNV-6.0.0-20230516T003303Z
  • /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/sfitz-mv-mutect-indels/a_mini_n2-mutect2-tumor-only/call-sSNV-6.0.0/TWGSAMIN000001-T001-S01-F/log-call-sSNV-6.0.0-20230516T004011Z
  • /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/sfitz-mv-mutect-indels/a_mini-mutect2-multiple-samples/call-sSNV-6.0.0/TWGSAMIN000001/log-call-sSNV-6.0.0-20230516T004618Z

Checklist

  • I have read the code review guidelines and the code review best practice on GitHub check-list.

  • I have reviewed the Nextflow pipeline standards.

  • The name of the branch is meaningful and well formatted following the standards, using [AD_username (or 5 letters of AD if AD is too long)]-[brief_description_of_branch].

  • I have set up or verified the branch protection rule following the github standards before opening this pull request.

  • I have added my name to the contributors listings in the manifest block in the nextflow.config as part of this pull request; I am listed already, or do not wish to be listed. (This acknowledgement is optional.)

  • I have added the changes included in this pull request to the CHANGELOG.md under the next release version or unreleased, and updated the date.

  • I have updated the version number in the metadata.yaml and manifest block of the nextflow.config file following semver, or the version number has already been updated. (Leave it unchecked if you are unsure about new version number and discuss it with the infrastructure team in this PR.)

  • I have tested the pipeline on at least one A-mini sample.

@sorelfitzgibbon sorelfitzgibbon requested review from yashpatel6, maotian06 and tyamaguchi-ucla and removed request for maotian06 April 7, 2023 02:27
@sorelfitzgibbon sorelfitzgibbon changed the title remove mutect2 indels split mutect2 VCF to snps, mnps and indels Apr 13, 2023
@sorelfitzgibbon sorelfitzgibbon self-assigned this Apr 13, 2023
module/common.nf Outdated
"""

process index_VCF_bcftools {
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We generally use bgzip and tabix for VCF indexing, which has been modularized by Mao. https://github.com/uclahs-cds/pipeline-Nextflow-module/tree/main/modules/common/index_VCF_tabix

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I've converted it back to using the external compress_index_vcf. However, I'm wondering how important it is to use that module. The reason being that bcftools works directly on the zipped files and it seems inefficient to be uncompressing and then calling another process to compress and index it. Instead it could all be done with the one process, e.g. for filter_VCF_bcftools:

set -euo pipefail
bcftools view -f PASS --output-type z --output ${params.output_filename}_pass.vcf.gz $filtered
bcftools index --tbi ${params.output_filename}_pass.vcf.gz

For split_VCF:

set -euo pipefail
bcftools view --types $var_type --output-type z --output ${params.output_filename}_pass_${var_type}.vcf.gz ${passing_vcf}

Here it is relevant for those two steps. In the next step towards creating an intersected list of SNVs, I'm using bcftools on the output of each of the tools in order to standardize the sample names, and also again on the MuSE output to reorder the samples. That would add another 4 times the vcf would be uncompressed and recompressed.

I'm not sure but I think in this case the VCFs could be left uncompressed throughout these steps, but there are many bcftools commands that require the VCF to be zipped and indexed.

What do you think?

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I agreed with Sorel about the compressed part. If bcftools can automatically generate vcf.gz, would it be better to directly save it as the compressed form?

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I forgot to add updated testing results:
nftest run a_mini-mutect2

  • output: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output
  • log: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/log-nftest-20230420T005619Z.log

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Something to consider with BCFtools:

  • bcftools view allows for output file format specification (can choose to output compressed, uncompressed, etc) and also has a --write-index option that will automatically generate the index when the compressed output option is chosen

Given that there are several steps involving BCFtools and the commands used are capable of automatically generating the index file, we should be able to avoid having to use any external (either from the module or a specific process defined here) compression/indexing processes. How do we feel about using the built-in option from BCFtools? @tyamaguchi-ucla @sorelfitzgibbon

Also, on a side note, it's probably worth de-coupling the compression and the indexing processes in the modularized workflow to detect if an input file is already compressed and only run indexing in that case.

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I tested it with a VCF and they produced the same index (identical SHA512 checksums) so we should be ok. They're probably using the same function(s) between tabix/bcftools index

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Compression seems to produce slightly different files though, even with the same compression level. Though based on the bcftools issue, bgzip/tabix is probably the most reliant way to proceed to avoid missing records/index properly

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Even though the issue is only for lines exceeding 2GB? As noted in the issue @tyamaguchi-ucla linked, 100,000 samples with 10 alternate alleles are handled correctly, but with 120 alternate alleles has a problem. Are there any ucla-cds pipelines that are going to make sense of 120 alternate alleles? And don't you think that as data sets grow, if this becomes a notable problem bcftools will address it?

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@sorelfitzgibbon The issue that I linked was just an example.

Are there any ucla-cds pipelines that are going to make sense of 120 alternate alleles?

Regenotype gSNP and gSV pipelines. Our Regeneron dataset is going to be ~150K samples minimum. In Regenotype-gSNP pipeline, we previously had this issue https://github.com/uclahs-cds/pipeline-regenotype-gSNP/pull/48 although it was due to an issue with GATK. Also, I think it's possible for this pipeline (esp. Mutect2 multi-sample mode) to have 120 alt alleles.

In regenotype-gSNP, it looks like @stefaneng used bcftools norm and then tabix -p. Not sure if he encountered an issue with bcftools index or not. https://github.com/uclahs-cds/pipeline-regenotype-gSNP/blame/804ee2956993994a3e967bc3e9178cdacf5830da/module/joint-genotype-processes.nf @stefaneng do you happen to remember why you went with tabix -p instead of bcftools index?

And don't you think that as data sets grow, if this becomes a notable problem bcftools will address it?

Maybe or maybe not. We don't have control over this.

@yashpatel6 @sorelfitzgibbon As we're publishing call-sSNV soon, we would want to use a consistent indexing method across the algorithms if possible. If not, reviewers might ask why certain final VCFs were indexed using bcftools or tabix.

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Thanks @tyamaguchi-ucla. I guess my question wasn't so much whether we'd ever see 120 alternate alleles, but whether they would be useful. Of course every parameter choice and filtering step loses some variants and are these messy variants important to keep in this context, or are they better addressed in pipelines specific for that purpose? Is it worth possibly several extra VCF decompression and compression to disk steps in order to avoid this potential loss here, or in most of the pipelines? I may well be naive but I have to think that given the extremely wide use of bcftools, if the issue was significant it would be addressed? :-)

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@sorelfitzgibbon pipeline-Nextflow-module has been merged so we can update the submodule to point to the latest commit on the main branch from that repo

Submodule updated and merged main into this branch. Waiting for final approval to merge!

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Looks good! Anything else to add @tyamaguchi-ucla @maotian06 ?

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I don't find the log for this test case where all algorithms were tested - log_output_dir: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/a_mini_n2-all-tools-std-input/call-sSNV-6.0.0/TWGSAMIN000001-T001-S01-F/log-call-sSNV-6.0.0-20230504T212122Z Can you please point it to me? @sorelfitzgibbon

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/hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/log-nftest-20230504T212117Z.log

Ah, sorry! Because of the issue with identical SM tags causing the multiple tumor test to fail, my log cleanup script deleted it. I've now restored it from the snapshot, but in the meantime I also reran it.
[i have a simple PR waiting for approval which fixes this problem]

nftest run
log: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/log-nftest-20230509T213538Z.log
output_dir: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/a_mini_n2-all-tools-std-input/call-sSNV-6.0.0/TWGSAMIN000001-T001-S01-F
log_output_dir: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/a_mini_n2-all-tools-std-input/call-sSNV-6.0.0/TWGSAMIN000001-T001-S01-F/log-call-sSNV-6.0.0-20230509T213545Z

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I accidentally overwrote some of the output files while testing on another branch. So I reran the testing to keep it clean for reviewers.

Is it possible/desirable to add the branch name to the nftest output path?

nftest run
log: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/log-nftest-20230510T215516Z.log
output_dir: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/a_mini_n2-all-tools-std-input/call-sSNV-6.0.0/TWGSAMIN000001-T001-S01-F
log_output_dir: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/a_mini_n2-all-tools-std-input/call-sSNV-6.0.0/TWGSAMIN000001-T001-S01-F/log-call-sSNV-6.0.0-20230510T215527Z

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log: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/log-nftest-20230510T215516Z.log

Looks like the test failed?

/hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/a_mini_n2-all-tools-std-input/call-sSNV-6.0.0/TWGSAMIN000001-T001-S01-F/sample-names

Was this directory created by the test run?

Also, it looks like there are some mostly empty log directories in /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/a_mini_n2-all-tools-std-input/call-sSNV-6.0.0/TWGSAMIN000001-T001-S01-F. Do we need to keep them?

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Looks like the test failed?

No, just the multi-sample failed, which will be fixed by the other PR.

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/hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/a_mini_n2-all-tools-std-input/call-sSNV-6.0.0/TWGSAMIN000001-T001-S01-F/sample-names

No, sorry that was created by the other branch.

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Also, it looks like there are some mostly empty log directories in /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/a_mini_n2-all-tools-std-input/call-sSNV-6.0.0/TWGSAMIN000001-T001-S01-F. Do we need to keep them?

I've cleaned up those with no successful runs within them

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tyamaguchi-ucla commented May 11, 2023

I just noticed but it looks like /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output doesn't follow the standardized test directory structure as you mentioned and contained tests from two (or more) branches (e.g. sfitz-external-pipeval)? Can you please re-organize the structure (You might need some help from Yash if it's NF-test related)?

Also, it looks like we can move some test directories to corresponding released dev directories. (e.g. /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/maotian-add-muse-workflow)

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I just noticed but it looks like /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output doesn't follow the standardized test directory structure as you mentioned and contained tests from two (or more) branches (e.g. sfitz-external-pipeval)? Can you please re-organize the structure (You might need some help from Yash if it's NF-test related)?

@yashpatel6 is it possible for nftest to detect what branch it's being run from?

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Also, it looks like we can move some test directories to corresponding released dev directories. (e.g. /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/maotian-add-muse-workflow)

I do not have permission to move these files. Perhaps @maotian06 can move them?

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I don't have permission to move. Seem like @sorelfitzgibbon you are the owner of those files:

drwxr-sr-x. 3 sfitzgibbon boutrosuser 16K Mar  3 07:34 maotian-add-muse-workflow

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I don't have permission to move. Seem like @sorelfitzgibbon you are the owner of those files:

drwxr-sr-x. 3 sfitzgibbon boutrosuser 16K Mar  3 07:34 maotian-add-muse-workflow

Thanks, I got that one.

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I just noticed but it looks like /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output doesn't follow the standardized test directory structure as you mentioned and contained tests from two (or more) branches (e.g. sfitz-external-pipeval)? Can you please re-organize the structure (You might need some help from Yash if it's NF-test related)?

@yashpatel6 is it possible for nftest to detect what branch it's being run from?

It isn't, nftest isn't designed to interact with git; the output path should be set through env variables before running nftest run

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@tyamaguchi-ucla the output directories listed in the Description section, for both CPCG and Amin samples are current and ready for review.

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@tyamaguchi-ucla the output directories listed in the Description section, for both CPCG and Amin samples are current and ready for review.

  • CPCG

/hot/user/sfitzgibbon/git/pipeline-call-sSNV/sf-testing

Any reason not to use the standardized output structure in /hot/software ? If not, can you please set up the output path properly? (see https://github.com/uclahs-cds/tool-NF-test#environment-settings)

/hot/user/sfitzgibbon/git/pipeline-call-sSNV/sf-testing/mutect2.CPCG-chr1/call-sSNV-6.0.0-rc.1

Hmm, why is the version 6.0.0-rc.1 here although other tests used 6.0.0?

/hot/user/sfitzgibbon/git/pipeline-call-sSNV/sf-testing/mutect2.CPCG-chr1/call-sSNV-6.0.0-rc.1/CPCG0000000196-T001-P01/Mutect2-4.4.0.0/output/tmp2.tbi
/hot/user/sfitzgibbon/git/pipeline-call-sSNV/sf-testing/mutect2.CPCG-chr1/call-sSNV-6.0.0-rc.1/CPCG0000000196-T001-P01/Mutect2-4.4.0.0/output/tmp.tbi

Did the pipeline generate the tmp files?

  • Amini

I don't think we need this directory /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output to organize test outputs by just branch or any reason to keep the nftest-output directory?

/hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/branch_unknown

As we don't know which branch was tested here, we can probably delete this directory or any reason we shouldn't?

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@tyamaguchi-ucla, I have addressed all of your comments.

@tyamaguchi-ucla the output directories listed in the Description section, for both CPCG and Amin samples are current and ready for review.

  • CPCG

/hot/user/sfitzgibbon/git/pipeline-call-sSNV/sf-testing

Any reason not to use the standardized output structure in /hot/software ? If not, can you please set up the output path properly? (see https://github.com/uclahs-cds/tool-NF-test#environment-settings)

NF-test is set up to use Amini. I could move these results to /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/sfitz-mv-mutect-indels/?

And/or do you want me to add CPCG options to NF-test? /hot/user/sfitzgibbon/git/pipeline-call-sSNV/nftest.yml is already quite long.

If you want CPCG added to NF-test, are these suitable standard inputs for testing (and should I locate or create a contamination file to be included?):

patient_id: 'CPCG0000000196'
input:
  normal:
    - id: 'CPCG0000000196-N001-B01'
      BAM: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/input/data/CPCG0196/CPCG0196-B1.bam
  tumor:
    - id: 'CPCG0000000196-T001-P01'
      BAM: /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/input/data/CPCG0196/CPCG0000000196-T001-P01-F.bam

/hot/user/sfitzgibbon/git/pipeline-call-sSNV/sf-testing/mutect2.CPCG-chr1/call-sSNV-6.0.0-rc.1

Hmm, why is the version 6.0.0-rc.1 here although other tests used 6.0.0?

The testing with CPCG was done a while ago. It was to test the BCFtools command to split the VCF and to see the ratios of SNVs, MNPs and Indels. Amini only yields a very small number of variants. Subsequent pipeline changes have not altered that BCFtools command and so all further testing was done with amini.

/hot/user/sfitzgibbon/git/pipeline-call-sSNV/sf-testing/mutect2.CPCG-chr1/call-sSNV-6.0.0-rc.1/CPCG0000000196-T001-P01/Mutect2-4.4.0.0/output/tmp2.tbi
/hot/user/sfitzgibbon/git/pipeline-call-sSNV/sf-testing/mutect2.CPCG-chr1/call-sSNV-6.0.0-rc.1/CPCG0000000196-T001-P01/Mutect2-4.4.0.0/output/tmp.tbi

Did the pipeline generate the tmp files?

No, sorry that was me testing the identity of indices created by BCFtools or tabix.

  • Amini

I don't think we need this directory /hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output to organize test outputs by just branch or any reason to keep the nftest-output directory?

removed

/hot/software/pipeline/pipeline-call-sSNV/Nextflow/development/unreleased/nftest-output/branch_unknown

As we don't know which branch was tested here, we can probably delete this directory or any reason we shouldn't?

There are probably past PRs that refer to specific logs listed in here (with a different path but the same log-file-name). I'm OK with deleting it.

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tyamaguchi-ucla commented May 16, 2023

@sorelfitzgibbon NF-test aside, your comment makes me wonder if we're on the same page regarding the standardized test directory structure. Could I ask you why we use this particular directory structure or I'm happy to have a quick chat if that works better? https://confluence.mednet.ucla.edu/pages/viewpage.action?spaceKey=BOUTROSLAB&title=Nextflow+pipeline+standardization#Nextflowpipelinestandardization-Pipelinedevelopmenttestrundirectorystructure

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@sorelfitzgibbon NF-test aside, your comment makes me wonder if we're on the same page regarding the standardized test directory structure. Could I ask you why we use this particular directory structure or I'm happy to have a quick chat if that works better? https://confluence.mednet.ucla.edu/pages/viewpage.action?spaceKey=BOUTROSLAB&title=Nextflow+pipeline+standardization#Nextflowpipelinestandardization-Pipelinedevelopmenttestrundirectorystructure

I thought we were going to discuss this at the nextflow working group tomorrow. Whether just the final testing of a branch should be archived or all testing or something in-between.

Tangentially, I assume at least this line from that confluence page is out of date now that nftest is used?
Each ${branch-name} directory should have at least a pair of input and config files to reproduce the test run(s)

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Whether just the final testing of a branch should be archived or all testing or something in-between.

Sure but I think tests generated for reviewers should be final in general to avoid confusing reviewers (maybe except when a PR is draft and the developer specifically wants somebody to look at a test directory). Of course, developers are welcome to create tests not meant for reviewers. However, test outputs should not be generated under version controlled directories to avoid accidental push. We can discuss this topic more tomorrow.

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Looks good to me!

@sorelfitzgibbon sorelfitzgibbon merged commit 9a3ea7b into main May 22, 2023
@sorelfitzgibbon sorelfitzgibbon deleted the sfitz-mv-mutect-indels branch May 22, 2023 17:02
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mutect2 output vcf contains small indels
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