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Mutect2 not detecting variant visible in tumor and matched cell line bam

cobrinik_labcobrinik_lab Los AngelesMember

I am unable to detect a particular variant when running Mutect2 on a tumor and tumor-derived cell line exome data. I ran each with the corresponding matched normal. The image below displays, from top to bottom: 1) input bam of cell line 2) bamout of cell line 3) input bam of tumor 4) bamout of tumor.

I have had some success in cases where mutect2 didn't detect a visible variant by running again with a focal interval file. No such luck here. Do you have guidance on troubleshooting?

Answers

  • cobrinik_labcobrinik_lab Los AngelesMember

    I discovered this post which suggested enabling -forceActive upon variant calling. This recovered the previously undetected variant.

    Why did this work?

    I also enabled -debug to examine the actual haplotype assembly, but couldn't identify any particular error. Do you have guidance on interpreting debug output?

    Thanks for your help,

  • SheilaSheila Broad InstituteMember, Broadie admin
    edited September 2017

    @cobrinik_lab
    Hi,

    The -forceActive flag tags all bases in your intervals as "active". You can read more about active regions here. I am guessing there was either bad quality data in the region, causing those bases in the region to not be tagged as active originally. Or, the normal samples could also have evidence for the variant in them.

    Do your matched normals show evidence for the variant? Also, can you test this site with GATK4 and see if it is called by default?

    Thanks,
    Sheila

  • cobrinik_labcobrinik_lab Los AngelesMember

    Thanks for this response, Sheila,

    I'll try running that region with GATK. Matched normal does not show evidence of the variant. I haven't investigated base quality scores in the region.

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