mutect2 multi-sample

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zhao shilin

• May 07, 2020 17:27
• Edited

Hello,

I am trying to use the mutect2 multi-sample feature. As it is relatively new, I didn't find many documents about it. Would you please help to clarify a few questions?

1. If I understand it correctly, multi-sample feature is designed for multi-samples from the same patient. So inputted tumor samples were called against all inputted normal samples. There is no "matched-normal" to each tumor sample.

2. I tried to do mutect2 separately (Tumor 1 vs Normal 1, Tumor 2 vs Normal 2) or multi-sample (Tumor1 and Tumor2, vs Normal 1 and Normal 2 in one run). The result (vcf before filtering) is different. Some variants were only identified in multi-sample results, not in any of the separate results. May I know why?

3. If Point 1 is true, I am thinking that if multi-sample feature can be used in multi-samples from different patients? Seems it is possible to do so? The only issue is no "matched-normal" for tumor in each patient. But if we think any variant identified in normal samples should be excluded in somatic mutation, I think it is OK to use it for multi-samples from different patients?

4. If Point 3 is not right, what is the best way to combine mutect2 results from different patients if running separately? Seems like CombineVariants in GATK3 is a solution but it is not available in GATK4?

5. Related question. The MuTect2 Wdl in (https://github.com/broadinstitute/gatk/blob/master/scripts/mutect2_wdl/) is not very correct. (1) gatk_jar is defined as /root/gatk.jar, but in fact in gatk docker image it is /gatk/gatk.jar. (2) the mutect2_multi_sample.wdl is not multi-sample as discussed in Point 1. It just run multiple separate MuTect2 (one MuTect2 for each Tumor/Normal pair), not running one multi-sample MuTect2 for all samples.

 

Thanks for your time and answers on this.

 

 

 

 

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• 

  David Benjamin

  • August 17, 2022 14:21

  Hi Alexandre Mondaini,

  You should run Mutect2 in multi-sample mode, separately for each patient.  That is:

  gatk Mutect2 -I normal1.bam -I tumor1.bam -I tumor2.bam -I tumor3.bam -normal normal1 . . . -O patient1.vcf
  gatk Mutect2 -I normal2.bam -I tumor4.bam -I tumor5.bam -I tumor6.bam -normal normal2 . . . -O patient2.vcf

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  Alexandre Mondaini

  • August 17, 2022 14:57

  Hi David,

  Thanks for the quick answer. Is this the default mode on which samples can be run using the WDL file ?

  https://github.com/broadinstitute/gatk/blob/master/scripts/mutect2_wdl/mutect2_multi_sample.wdl

  I see details about a pair list:

  #  pair_list: a tab-separated table with no header in the following format:
  #   TUMOR_1_BAM</TAB>TUMOR_1_bai</TAB>NORMAL_1_BAM</TAB>NORMAL_1_bai
  #   TUMOR_2_BAM</TAB>TUMOR_2_bai</TAB>NORMAL_2_BAM</TAB>NORMAL_2_bai

  With a subsequent scatter over each row of this tsv file:

  scatter( row in pairs )

  So should I replicate the normal file over the rows for all combinations of tumors as such ?

  TUMOR_1_BAM</TAB>TUMOR_1_bai</TAB>NORMAL_1_BAM</TAB>NORMAL_1_bai
  
  TUMOR_2_BAM</TAB>TUMOR_2_bai</TAB>NORMAL_1_BAM</TAB>NORMAL_1_bai
  
  TUMOR_3_BAM</TAB>TUMOR_3_bai</TAB>NORMAL_1_BAM</TAB>NORMAL_1_bai

  Thank you for the support.

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  David Benjamin

  • August 17, 2022 17:36

  Alexandre Mondaini That WDL file is misleading.  It invokes multiple independent runs of Mutect2 on tumor-normal pairs, and each pair has a single tumor and normal.  Given that Mutect2 should be run using the featured workflow on Terra, where scattering over multiple pairs is built into the UI and is not part of the WDL itself, this workflow (which predated Terra) is obsolete (see https://github.com/broadinstitute/gatk/pull/7992).

  We do not yet have a WDL for genuine multi-sample mode with multiple tumor samples from a single patient.

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  Anthony Dias-Ciarla

  • October 28, 2022 19:51

  Hi Alexandre Mondaini,

  Thank you for writing to the GATK forum! I hope that we can help you sort this out.

  I brought your inquiry to our developers and received some feedback to share with you.

  Firstly, you should run Mutect2 separately for each person. When you have a matched normal, you should use it, but you shouldn't use one patient’s normal with another patient’s tumor. Likewise, you shouldn't combine two different patients’ tumors in the same run of Mutect2.

  I hope this helps clarify your questions! Please let me know if any other questions arise in the future. Thank you for being a valued contributor to the GATK community.

  Best,
  Anthony

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  Anthony Dias-Ciarla

  • November 04, 2022 16:14

  Hi Alexandre Mondaini,

  We haven't heard from you in a while so we're going to close out this ticket. If you still require assistance, simply respond to this email and we'll be happy to pick up where we left off!

  Kind regards,

  Anthony​

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