Answers.Body: <p>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.</p>
  <p>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:</p>
  <pre><code>     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
  </code></pre>
  <p>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.</p>
  <p>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.</p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2023-05-10T03:28:07.190Z
Answers.Text: 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.
Answers.UserDisplayName: kane9530
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Answers.Text: @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.
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Answers.LastActivityDate: 2023-05-09T13:05:29.167Z
Answers.LastEditDate: 2023-05-09T13:05:29.167Z
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Body: <p>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.</p>
  <p>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.</p>
  <p>How should I go about assessing whether the variants are &quot;biased&quot; 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?</p>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2023-05-10T15:34:25.923Z
Comments.Text: 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?
Comments.UserDisplayName: terdon
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2023-05-11T04:35:17.307Z
Comments.Text: 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 https://gatk.broadinstitute.org/hc/en-us/articles/360035890631-Panel-of-Normals-PON-
Comments.UserDisplayName: kane9530
Tags: <phylogenetics><phylogeny><variant-calling><gatk><wes>
Title: WES variant calling with DNBSEQ-T7: technical quality assessment
ThreadText: # WES variant calling with DNBSEQ-T7: technical quality assessment
  
  (Asked by: username_0 on 2023-05-09)
  
  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?
  
  **Tags:** phylogenetics, phylogeny, variant-calling, gatk, wes
  
  > **Comments:**
  > **username_1**: 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?  
  > **username_0**: 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 https://gatk.broadinstitute.org/hc/en-us/articles/360035890631-Panel-of-Normals-PON-  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_2 on 2023-05-09. Score: 1
  
  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.
  
  > **Comments:**
  > **username_0**: 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.  
  > **username_2**: @username_0 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.  
  
  ---
  
  