(howto) Run the genotype refinement workflow
Tutorials | Created 2014-10-18 | Last updated 2015-11-26

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This tutorial describes step-by-step instruction for applying the Genotype Refinement workflow (described in this method article) to your data.

Step 1: Derive posterior probabilities of genotypes

In this first step, we are deriving the posteriors of genotype calls in our callset, recalibratedVariants.vcf, which just came out of the VQSR filtering step; it contains among other samples a trio of individuals (mother, father and child) whose family structure is described in the pedigree file trio.ped (which you need to supply). To do this, we are using the most comprehensive set of high confidence SNPs available to us, a set of sites from Phase 3 of the 1000 Genomes project (available in our resource bundle), which we pass via the --supporting argument.

 java -jar GenomeAnalysisToolkit.jar -R human_g1k_v37_decoy.fasta -T CalculateGenotypePosteriors --supporting 1000G_phase3_v4_20130502.sites.vcf -ped trio.ped -V recalibratedVariants.vcf -o recalibratedVariants.postCGP.vcf

This produces the output file recalibratedVariants.postCGP.vcf, in which the posteriors have been annotated wherever possible.

Step 2: Filter low quality genotypes

In this second, very simple step, we are tagging low quality genotypes so we know not to use them in our downstream analyses. We use Q20 as threshold for quality, which means that any passing genotype has a 99% chance of being correct.

java -jar $GATKjar -T VariantFiltration -R $bundlePath/b37/human_g1k_v37_decoy.fasta -V recalibratedVariants.postCGP.vcf -G_filter "GQ < 20.0" -G_filterName lowGQ -o recalibratedVariants.postCGP.Gfiltered.vcf

Note that in the resulting VCF, the genotypes that failed the filter are still present, but they are tagged lowGQ with the FT tag of the FORMAT field.

Step 3: Annotate possible de novo mutations

In this third and final step, we tag variants for which at least one family in the callset shows evidence of a de novo mutation based on the genotypes of the family members.

java -jar $GATKjar -T VariantAnnotator -R $bundlePath/b37/human_g1k_v37_decoy.fasta -V recalibratedVariants.postCGP.Gfiltered.vcf -A PossibleDeNovo -ped trio.ped -o recalibratedVariants.postCGP.Gfiltered.deNovos.vcf

The annotation output will include a list of the children with possible de novo mutations, classified as either high or low confidence.

See section 3 of the method article for a complete description of annotation outputs and section 4 for an example of a call and the interpretation of the annotation values.

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