GATK Mutect2 tumor vs. normal sample
Answered
a) How do I compared somatic variants called through a PoN file with the latest best-practive-guidelines between a normal-sample and a tumor-sample in order to know which one are characteristics of the latter? I don't see tumor-mode anymore in the examples etc.
Thank you very much in advance for any help!
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Hello Enrico Cocchi,
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Enrico Cocchi If I understand the question correctly, you are wondering how to compare tumor-only calls versus tumor-normal calls. The short answer is that this is pointless because calls with a matched normal are vastly superior. The reason is simple: even with the strictest germline filtering possible, namely blacklisting every germline variant in gnomAD no matter how rare, any given genome will still have 30,000 or so rare germline variants that go unfiltered without a matched normal. There is basically nothing you can do about this unless your tumor has low purity and so you can distinguish the low allele fractions of somatic variants from germline allele fractions closer to 1/2.
While we're here, let's also clear up a few things:
- The PoN is important for both tumor-only and tumor-normal calling because its job is to filter out mapping and technical artifacts.
- The germline resource (usually the AF-only gnomAD we provided in our public google bucket) is responsible for germline filtering.
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we have one normal sample and two tumor samples from the same patient, how do I set up normal-tumor sample matching? is below correct?
gatk Mutect2 \ -R reference.fa \ -I tumor1_patient1.bam \ -I tumor2_patient1.bam \
-I tumor1_patient2.bam \
-I tumor2_patient2.bam \
-I normal_patient1.bam \
-I normal_patient1.bam \
-I normal_patient2.bam \
-I normal_patient2.bam \
-normal normal_patient1 \
-normal normal_patient1 \
-normal normal_patient2 \ -normal normal_patient2 \ --germline-resource af-only-gnomad.vcf.gz \ --panel-of-normals pon.vcf.gz \ -O somatic.vcf.gz -
Jeffcgen2000 Different patients should not be combined in a single Mutect2 command. The correct command for patient1 would be:
gatk Mutect2 \ -R reference.fa \ -I tumor1_patient1.bam \ -I tumor2_patient1.bam \
-I normal_patient1.bam \
-normal normal_patient1 \ --germline-resource af-only-gnomad.vcf.gz \ --panel-of-normals pon.vcf.gz \ -O somatic.vcf.gz
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