Holiday Notice:
The Frontline Support team will be slow to respond December 17-18 due to an institute-wide retreat and offline December 22- January 1, while the institute is closed. Thank you for your patience during these next few weeks. Happy Holidays!

How does a panel of normal affect variant calling using mutect2

Hello,
I am running an analyis on tumor samples using mutect2. To investigate the effect of a PON on variant calling , I ran my analysis with and without the PON. Both analyses yield different amounts of called variants, as expected more for the analysis without the PON. But there are still variants in the analysis with PON marked as filtered on basis of the PON.

That surprises me as I thought mutect would do the calling and then filter variants marked as being site-specific artefacts. However, based on the reduced number of variants called plus variants filtered PON-wise

I conclude that mutect affects variant calling in (at least) two different ways. I would appreciate if you can clarify this.

Answers

  • shleeshlee CambridgeMember, Broadie, Moderator admin

    Hi @DanielTobias,

    Please see https://software.broadinstitute.org/gatk/documentation/article?id=11127 for some discussion on the role of the PoN in somatic short mutation calling. Remember that for certain sites, only after read reassembly can a variant allele be matched to allele representations in VCF resources. In such cases, the workflow emits such sites and filters them at the FilterMutectCalls step.

  • davidbendavidben BostonMember, Broadie, Dev ✭✭

    @DanielTobias You are correct that the PoN works in two ways:

    1) When Mutect2 genotypes a site that overlaps a PoN variant it notes this in an INFO field, which causes FilterMutectCalls to apply the panel_of_normals filter. To be clear, in this case the variant appears in the filtered vcf, it just has a filter applied.

    2) In Mutect2's initial triage step (where it decides whether to proceed with expensive assembly and alignment) sites in the PoN are skipped. In this case nothing shows up in the vcf because it happens before genotyping. Thus by default the only PoN sites that show up in a Mutect2 vcf are those that were near a possible somatic variant and came along for the ride. This is a speed optimization, and if you are investigating the role of the PoN you can turn it off with the --genotype-pon-sites argument.

Sign In or Register to comment.