Question: GATK Mutect2 Panel-Of-Normals generation
gravatar for cocchi.e89
6 months ago by
cocchi.e89120 wrote:

I am trying to generate PON for WES samples following GATK recommendations, they also have another explanation in this Mutect2 article but it's basically the same identical 3-steps procedure:

step 1. Run Mutect2 in tumor-only mode for each normal sample: gatk Mutect2 -R reference.fasta -I normal1.bam -max-mnp-distance 0 -O normal1.vcf.gz

step 2. Create a GenomicsDB from the normal Mutect2 calls: gatk GenomicsDBImport -R reference.fasta -L intervals.interval_list --genomicsdb-workspace-path pon_db -V normal1.vcf.gz -V normal2.vcf.gz -V normal3.vcf.gz -V ...

step 3. Combine the normal calls using CreateSomaticPanelOfNormals: gatk CreateSomaticPanelOfNormals -R reference.fasta --germline-resource af-only-gnomad.vcf.gz -V gendb://pon_db -O pon.vcf.gz

I am using gatk 4.1.7 (latest at the moment) but the output I got from step 2 (GenomicsDBImport) is a folder with some files in it, such as vcfheader.vcf, vidmap.json and what looks like a file for every chromosme with a $ and contig boundaries specified in the BED file (e.g. X$200786$155255277).

If I try to pass this directory in the -V option of CreateSomaticPanelOfNormals (step 3 ) I got an error that the specified input is not a regular file, and GATK documentation confirms that -V is supposed to be a VCF file.

Does anybody, that maybe has generate PONs before or worked with this, knows what is the exact file output from step 2 that I am supposed to pass in step 3 -V?

Thank you very much in advance for any help!

ADD COMMENTlink modified 6 months ago by WouterDeCoster45k • written 6 months ago by cocchi.e89120

Can you not use the PON files they make available here?

ADD REPLYlink written 6 months ago by GenoMax94k

No my samples are hg19, moreover I’d like to be sure that the PON comes from samples samples with the same kit

ADD REPLYlink written 6 months ago by cocchi.e89120
gravatar for WouterDeCoster
6 months ago by
WouterDeCoster45k wrote:

I've done this quite recently, with what I hope is the latest version.
In my case I had to do this as below:

-V gendb://pon_db

the gendb:// shouldn't be changed, and what follows is the path to the directory created by GenomicsDBImport

ADD COMMENTlink written 6 months ago by WouterDeCoster45k

Looks like it did the trick. Thank you very much @WouterDeCoster !

ADD REPLYlink written 6 months ago by cocchi.e89120
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