In our pipeline, we use GATK on whole exome sequencing data consisting of Trios (mother, father and child). We process each sample alone (GVCF is not used and combined genotyping is not applied). All steps from BaseRecalibration to Variants recalibration to finally calling snps and indels are done. Since we are interested in calling denovo mutations, I used the GenotypeRefinement workflow and did the following for each Trio:
Step1: ReadBackedPhasing
- Command used:
java -Xmx6g -XX:ParallelGCThreads=1
-jar $CONDA_PREFIX/opt/gatk-3.8/GenomeAnalysisTK.jar
-T ReadBackedPhasing
-R hGRC37.fa
-I child.bam
--variant child.vcf
-o child_phased.vcf
--phaseQualityThresh 20.0
I assume this step worked correctly since the HP tag is added to the variants.
Same code is applied for father.vcf and mother.vcf, so that father_phased.vcf and mother_phased.vcf are produced.
Step2: Combine Variants
- Command used:
java -Xmx6g -XX:ParallelGCThreads=1
-jar $CONDA_PREFIX/opt/gatk-3.8/GenomeAnalysisTK.jar
-T CombineVariants
-R hGRC37.fa
--variant child_phased.vcf --variant mother_phased.vcf --variant father_phased.vcf
-o trio.vcf
-genotypeMergeOptions UNIQUIFY
Step3: CalculateGenotypePosteriors
- Command used:
java -Xmx6g -XX:ParallelGCThreads=1
-jar $CONDA_PREFIX/opt/gatk-3.8/GenomeAnalysisTK.jar
-T CalculateGenotypePosteriors
-R hGRC37.fa
-ped family.ped
-V trio.vcf
-o trio_GenPost.vcf
--skipPopulationPriors
Where the ped file looks like:
Family child father mother other 0
Family father 0 0 other 0
Family mother 0 0 other 0
Unfortunately, the output vcf file never gets annotated with PG tag, I tried this code on many trios and all yielded the same PG-free vcf files. What could be a possible reason?
