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I recently received whole exome sequencing samples that were sequenced on an MGI sequencing instrument, DNBSEQ-T7. I am interested in somatic variant-calling on the paired tumor-normal samples. As this isn't the canonical Illumina sequencer, an issue that I am concerned with is that the technical artifacts arising from the sequencer are not yet well characterised and this may lead to spurious artifacts in the variants.

I asked the company that did the sequencing for a panel of normals (PON) for the instrument to run Mutect2, but they simply replied that they use the standard GATK PON for their somatic variant calls.

How should I go about assessing whether the variants are "biased" in any way, as a result of the instrument's technical properties? Is there a way to quantify the magnitude of these technical errors from the VCF files?

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  • $\begingroup$ What exactly do you have? Fastq files? Bams? You mention VCFs, how were those generated? The PONs are mostly used to try and guess if a variant is germline or somatic, as far as I know, why would they help with artifacts? $\endgroup$
    – terdon ♦
    May 10 at 15:34
  • $\begingroup$ The fastq files contain the raw seq information. They also performed some secondary analysis as "part of the package", and this includes the bwa aligned bam files and a germline haplotypecaller vcf output. The purpose of the PON is to infer possible technical artefacts - see gatk.broadinstitute.org/hc/en-us/articles/… $\endgroup$
    – kane9530
    May 11 at 4:35

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Okay it seems the company has given you the VCF (maybe), you want to re-run it via Mutect2. Mutect2 is part of the GATK suite, it's an upgrade, and they are refusing to run the appropriate PON. I think this is more personnel management than a major technical hurdle.

I would reply to the tech and CC the technical director of the company explaining Mutect2 vs. regular GATK PON. You simply use a standard process of escalation within technical justification. Maybe send them the commend to demonstrate how simple it is:

     gatk Mutect2 \
     -R reference.fa \
     -I tumor.bam \
     -I normal.bam \
     -normal normal_sample_name \
     --germline-resource af-only-gnomad.vcf.gz \
     --panel-of-normals pon.vcf.gz \
     -O somatic.vcf.gz

The company will have to justify their position regarding PONs or accommodate your requirements either now or the future. There may simply be a technical misunderstanding between you and the tech.

You've paid for the service - thats the standard you expect. It's easier for them to assist you than all the agro of a complaints procedure and I'm pretty sure they'll help.

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    $\begingroup$ Thanks! Actually fyi, the company provided me with germline VCF files as part of the secondary analysis, which isn't useful given that I am interested in the paired timor-normal comparisons. $\endgroup$
    – kane9530
    May 10 at 3:28
  • $\begingroup$ @kane9530 good luck and glad it helped. You are working in a really interesting area of research and Broad Institutes' tools/work are second-to-none (really good). I understand your concern given how meticulous the Broad Institute is and the technical issues in separating cancer cell DNA from healthy cells within the tumour (tumour) sample. $\endgroup$
    – M__ ♦
    May 10 at 4:06

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