Is it a bug for the Mutect2

suxxsuxx HongKongMember

Hi,

I am using the Mutect2 (gatk-4.0.2.0) paired samples to call somatic mutation.

Here is my command line:
java -jar gatk-package-4.0.2.0-local.jar Mutect2 --reference hg19.fa -I tumor.sorted.rmdup.clipper.bam -tumor test1 -I normal.sorted.rmdup.clipper.bam -normal test2 --germline-resource gnomad.exomes.2.vcf.gz --panel-of-normals pon.vcf.gz -L test.DNA.bed -O test.match.vcf.gz

Here is the result generated by Mutect2:
chr2 25457242 . C T . . DP=967;ECNT=1;NLOD=114.19;N_ART_LOD=-9.232e-01;POP_AF=2.197e-04;P_GERMLINE=-1.116e+02;TLOD=267.27 GT:AD:AF:F1R2:F2R1:MBQ:MFRL:MMQ:MPOS:SA_MAP_AF:SA_POST_PROB 0/1:406,94:0.204:199,39:207,55:38:224,229:60:15:0.182,0.172,0.188:4.684e-03,0.012,0.983 0/0:397,8:0.046:195,5:202,3:34:226,248:60:1

The AF of tumor sample is 0.204 that looks at little overestimated, bit the normal sample is 0.046 which is far overestimated compared with IGV (AF=1/(404+1), only 1 read carry this mutation. But Mutect2 result shows there are 8 reads carry this mutation).
Does mutect2 do re-alignment before calling any mutation? I cannot figure out where those extra mutant reads come from.
Thanks.

IGV for chr2:25457242

Answers

  • SheilaSheila Broad InstituteMember, Broadie, Moderator

    @suxx
    Hi,

    For the AF calculation, have a look at this thread, specifically my answer at the end.

    Indeed, Mutect2 does a reassembly step like HaplotypeCaller, so some reads may shift around. You can view the reassembled reads using the bamout.

    -Sheila

  • suxxsuxx HongKongMember

    Hi Sheila,
    Thanks for your reply.
    Would you please advice how to output reassembled reads only for normal sample when I run Mutect2 using paired samples. Because I used the bamout, Metect2 will combined tumor and normal samples in a single BAM file. I do not know which sample those reads come from when I investigate the BAM file.
    Another question is if it is possible for us to switch off reassembly function during the calling step.

  • suxxsuxx HongKongMember

    @Sheila
    Thanks for your reply.
    Would you please advice how to output reassembled reads only for normal sample when I run Mutect2 using paired samples. Because I used the bamout, Metect2 will combined tumor and normal samples in a single BAM file. I do not know which sample those reads come from when I investigate the BAM file.
    Another question is if it is possible for us to switch off reassembly function during the calling step.

  • SheilaSheila Broad InstituteMember, Broadie, Moderator

    @suxx
    Hi,

    You can view the normal and tumor reassembled reads in IGV by right clicking on the reads and choosing "color by" sample. There is no way to only output the tumor or normal reassembled reads, as they are reassembled together. There is also no way to skip reassembly step, as it is critical to the tool and making variant calls.

    -Sheila

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