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Problems interpreting Mutect2 output

laura_TLlaura_TL SpainMember
edited April 2017 in Ask the GATK team

Hello,

I am calling variants with Mutect2 (default parameters) from bulk WGS Tumor/Normal pairs following Somatic SNV Best Practices, and in the VCF outputs I am finding a lot of variants like this (the last one is the tumor):

chr1 1037759 . CTT C . PASS ECNT=1;HCNT=1;MAX_ED=.;MIN_ED=.;NLOD=4.60;RPA=12,10;RU=T;STR;TLOD=26.17 GT:AD:AF:ALT_F1R2:ALT_F2R1:QSS:REF_F1R2:REF_F2R1 0/0:31,0:NaN:0:0:0,0:0:0 0/1:13,21:1.00:8:13:0,611:0:0

The genotype suggested for the tumor is heterozygous. However, the AF is 1.00. I also see that the QSS for reference allele is 0, but with IGV I checked that the base and mapping qualities at this position are normal for both reference and alternative-supporting reads and that they are primary alignments and have their mates mapped. I got up to 14% AF=1.00, which seems very weird to me for this type of analysis.

It doesn't happen for all the deletions, though:

chr1 1128849 . CTT C . PASS ECNT=1;HCNT=2;MAX_ED=.;MIN_ED=.;NLOD=3.77;RPA=11,9;RU=T;STR;TLOD=20.27 GT:AD:AF:ALT_F1R2:ALT_F2R1:QSS:REF_F1R2:REF_F2R1 0/0:24,0:0.00:0:0:60,0:0:2 0/1:20,13:0.867:6:7:60,403:2:0

In this case QSS is not 0, and I feel that it could have some relationship ( (13403)/((2060)+(13*403))=0.814, near to the AF).

Most of the cases are indels, but not all of them:

chr2 28505882 . A G . PASS ECNT=1;HCNT=8;MAX_ED=.;MIN_ED=.;NLOD=4.00;TLOD=23.46 GT:AD:AF:ALT_F1R2:ALT_F2R1:FOXOG:QSS:REF_F1R2:REF_F2R1 0/0:16,0:0.00:0:0:.:149,0:4:1 0/1:2,15:1.00:1:0:0.00:0,30:0:0

So in summary, I don't understand the way in which AF is calculated. Am I misunderstanding the AF concept or the way it works for this type of samples? Or may I be skipping the reason of QSS=0? Should I use AD for my calculations instead?

Thank you very much!

GATK version: 3.7-0-gcfedb67
Java version: 1.8.0_31
WGS paired end samples
Bulk Tumor/Normal pairs
Sequenced with HiSeqX using TruSeq Nano DNA (350) library kit

Post edited by laura_TL on

Issue · Github
by Sheila

Issue Number
1998
State
closed
Last Updated
Assignee
Array
Milestone
Array
Closed By
chandrans

Answers

  • SheilaSheila Broad InstituteMember, Broadie, Moderator

    @laura_TL
    Hi,

    Sorry for the late response. I had to check with the team. Can you submit a bug report? Instructions are here.

    Thanks,
    Sheila

  • laura_TLlaura_TL SpainMember

    Hi Sheila,

    I uploaded the bug report with the name discussion9442.tar.gz. Please tell me if you need something else.

    I have also observed that using GATK old 3.5 version I don't have this problem, since I get this output for the first case I reported above (the first one is the tumor):

    chr1 1037759 . CTT C . PASS ECNT=1;HCNT=1;MAX_ED=.;MIN_ED=.;NLOD=4.60;RPA=12,10;RU=T;STR;TLOD=19.63 GT:AD:AF:ALT_F1R2:ALT_F2R1:FOXOG:QSS:REF_F 1R2:REF_F2R1 0/1:13,21:0.304:8:13:.:0,611:0:0 0/0:31,0:0.00:0:0:.:0,0:0:0

    Thank you very much,

    Laura

    Issue · Github
    by Sheila

    Issue Number
    2046
    State
    closed
    Last Updated
    Assignee
    Array
    Milestone
    Array
    Closed By
    vdauwera
  • SheilaSheila Broad InstituteMember, Broadie, Moderator

    @laura_TL
    Hi Laura,

    Thanks. I will have a look soon.

    -Sheila

  • SheilaSheila Broad InstituteMember, Broadie, Moderator

    @laura_TL
    Hi Laura,

    Sorry for the delay. It looks like the original issue is now fixed in GATK4 MuTect2. You can test it out here. Note the tool is in beta, but you can use it for testing purposes.

    -Sheila

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