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Create a master set of sites from your N batch VCFs that you want to genotype in all samples. At this stage you need to determine how you want to resolve disagreements among the VCFs. This is your master sites VCF.
Take the master sites VCF and genotype each sample BAM file at these sites
(Optionally) Merge the single sample VCFs into a master VCF file
The first step of batch merging is to create a master set of sites that you want to genotype in all samples. To make this problem concrete, suppose I have two VCF files:
##fileformat=VCFv4.0 #CHROM POS ID REF ALT QUAL FILTER INFO FORMAT NA12891 20 9999996 . A ATC . PASS . GT:GQ 0/1:30 20 10000000 . T G . PASS . GT:GQ 0/1:30 20 10000117 . C T . FAIL . GT:GQ 0/1:30 20 10000211 . C T . PASS . GT:GQ 0/1:30 20 10001436 . A AGG . PASS . GT:GQ 1/1:30
##fileformat=VCFv4.0 #CHROM POS ID REF ALT QUAL FILTER INFO FORMAT NA12878 20 9999996 . A ATC . PASS . GT:GQ 0/1:30 20 10000117 . C T . FAIL . GT:GQ 0/1:30 20 10000211 . C T . FAIL . GT:GQ 0/1:30 20 10000598 . T A . PASS . GT:GQ 1/1:30 20 10001436 . A AGGCT . PASS . GT:GQ 1/1:30
In order to merge these batches, I need to make a variety of bookkeeping and filtering decisions, as outlined in the merged VCF below:
20 9999996 . A ATC . PASS . GT:GQ 0/1:30 [pass in both] 20 10000000 . T G . PASS . GT:GQ 0/1:30 [only in batch 1] 20 10000117 . C T . FAIL . GT:GQ 0/1:30 [fail in both] 20 10000211 . C T . FAIL . GT:GQ 0/1:30 [pass in 1, fail in 2, choice in unclear] 20 10000598 . T A . PASS . GT:GQ 1/1:30 [only in batch 2] 20 10001436 . A AGGCT . PASS . GT:GQ 1/1:30 [A/AGG in batch 1, A/AGGCT in batch 2, including this site may be problematic]
These issues fall into the following categories:
There are two difficult situations that must be addressed by the needs of the project merging batches:
Unfortunately, we cannot determine which is actually the correct choice, especially given the goals of the project. We leave it up the project bioinformatician to handle these cases when creating the master VCF. We are hopeful that at some point in the future we'll have a consensus approach to handle such merging, but until then this will be a manual process.
The GATK tool CombineVariants can be used to merge multiple VCF files, and parameter choices will allow you to handle some of the above issues. With tools like SelectVariants one can slice-and-dice the merged VCFs to handle these complexities as appropriate for your project's needs. For example, the above master merge can be produced with the following CombineVariants:
java -jar dist/GenomeAnalysisTK.jar \ -T CombineVariants \ -R human_g1k_v37.fasta \ -V:one,VCF combine.1.vcf -V:two,VCF combine.2.vcf \ --sites_only \ -minimalVCF \ -o master.vcf
producing the following VCF:
##fileformat=VCFv4.0 #CHROM POS ID REF ALT QUAL FILTER INFO 20 9999996 . A ACT . PASS set=Intersection 20 10000000 . T G . PASS set=one 20 10000117 . C T . FAIL set=FilteredInAll 20 10000211 . C T . PASS set=filterIntwo-one 20 10000598 . T A . PASS set=two 20 10001436 . A AGG,AGGCT . PASS set=Intersection
Having created the master set of sites to genotype, along with their alleles, as in the previous section, you now use the UnifiedGenotyper to genotype each sample independently at the master set of sites. This GENOTYPE_GIVEN_ALLELES mode of the UnifiedGenotyper will jump into the sample BAM file, and calculate the genotype and genotype likelihoods of the sample at the site for each of the genotypes available for the REF and ALT alleles. For example, for site 10000211, the UnifiedGenotyper would evaluate the likelihoods of the CC, CT, and TT genotypes for the sample at this site, choose the most likely configuration, and generate a VCF record containing the genotype call and the likelihoods for the three genotype configurations.
As a concrete example command line, you can genotype the master.vcf file using in the bundle sample NA12878 with the following command:
java -Xmx2g -jar dist/GenomeAnalysisTK.jar \ -T UnifiedGenotyper \ -R bundle/b37/human_g1k_v37.fasta \ -I bundle/b37/NA12878.HiSeq.WGS.bwa.cleaned.recal.hg19.20.bam \ -alleles master.vcf \ -L master.vcf \ -gt_mode GENOTYPE_GIVEN_ALLELES \ -out_mode EMIT_ALL_SITES \ -stand_call_conf 0.0 \ -glm BOTH \ -G none \
-L master.vcf argument tells the UG to only genotype the sites in the master file. If you don't specify this, the UG will genotype the master sites in GGA mode, but it will also genotype all other sites in the genome in regular mode.
The last item,-G ` prevents the UG from computing annotations you don't need. This command produces something like the following output:
##fileformat=VCFv4.0 #CHROM POS ID REF ALT QUAL FILTER INFO FORMAT NA12878 20 9999996 . A ACT 4576.19 . . GT:DP:GQ:PL 1/1:76:99:4576,229,0 20 10000000 . T G 0 . . GT:DP:GQ:PL 0/0:79:99:0,238,3093 20 10000211 . C T 857.79 . . GT:AD:DP:GQ:PL 0/1:28,27:55:99:888,0,870 20 10000598 . T A 1800.57 . . GT:AD:DP:GQ:PL 1/1:0,48:48:99:1834,144,0 20 10001436 . A AGG,AGGCT 1921.12 . . GT:DP:GQ:PL 0/2:49:84.06:1960,2065,0,2695,222,84
Several things should be noted here:
This genotyping command can be performed independently per sample, and so can be parallelized easily on a farm with one job per sample, as in the following:
foreach sample in samples: run UnifiedGenotyper command above with -I $sample.bam -o $sample.vcf end
You can use a similar command for CombineVariants above to merge back together all of your single sample genotyping runs. Suppose all of my UnifiedGenotyper jobs have completed, and I have VCF files named sample1.vcf, sample2.vcf, to sampleN.vcf. The single command:
java -jar dist/GenomeAnalysisTK.jar -T CombineVariants -R human_g1k_v37.fasta -V:sample1 sample1.vcf -V:sample2 sample2.vcf [repeat until] -V:sampleN sampleN.vcf -o combined.vcf