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# Genotype and Validate, or Haplotype Caller GGA: what am I doing wrong?

Member

Hi,

My use case is quite straightforward, but has been surprisingly hard to achieve:
For each sample, I have both Omni 2.5M SNP genotype data and RNA-seq variant call data (done with GATK3).
Now I want to see how well the RNA-seq variant calling is performing, using the SNP genotypes as reference.
To do this, I need not only the variant calls in the RNA-seq data (as HC is outputting normally), but all genotypes for a given set of positions.
Ideally, I would like to keep all the normal info fields from the RNA VCF, to allow calculation of some concordance metrics based on depth of coverage and other quality parameters later.

I've tried the following:
1. GenotypeAndValidate. With SNP VCF as "truth" and BAM to evaluate. The command:

java -Xmx32g -jar ${GATK} \ -T GenotypeAndValidate \ -R${REF} \
-I ${BAM} \ -alleles${SNPVCF} \
-L ${SNPVCF} \ -o$SAMPLEID.rnasnp.vcf \
-nt 4


The results (running only chr 1, with ~185k SNPs):

(empty) ALT REF No Status
called alt 0 0 4096
called ref 0 0 12995
not called 0 0 153034

sensitivity: NaN%
specificity: 100.000000%
not confident: 3678
not covered: 149356

This runs surprisingly fast - which makes me think I'm not inputting the files as expected.

2. Haplotype Caller in GGA mode. Giving it the SNP VCF as the --alleles file. The command, adjusted for RNA-seq data:

java -Xmx32g -jar ${GATK} \ -T HaplotypeCaller \ -R${REF} \
--dbsnp ${DBSNP} \ -I${BAM} \
-L ${SNPVCF} \ -alleles${SNPVCF} \
-gt_mode GENOTYPE_GIVEN_ALLELES \
-dontUseSoftClippedBases \
-stand_call_conf 0.0 \
-stand_emit_conf 0.0 \
-o $SAMPLEID.rnasnp.vcf \ -nct 16  This almost results in what I want, in that HC starts outputting also 0/0 and ./. calls for reference and non-covered bases. But, it does so only for SNP-positions with non-reference alleles in the SNP VCF. Again, I want all positions called - including those that are homozygous reference in the SNP VCF. I am using these tools wrong? Or should I be doing this differently? Thanks in advance, Vasilios Tagged: ## Comments • Cambridge, MAMember, Administrator, Broadie Hi there, You need to run HC in -ERC GVCF mode (or BP_RESOLUTION) to get calls at all positions. • Member Thanks Geraldine, That does work if I also remove the -gt_mode, -alleles and --interval_padding (otherwise HC crashed with "code error" after a while): java -Xmx32g -jar${GATK} \
-T HaplotypeCaller \
-R ${REF} \ --dbsnp${DBSNP} \
-I ${BAM} \ -L${SNPVCF} \
-dontUseSoftClippedBases \
-stand_call_conf 0.0 \
-stand_emit_conf 0.0 \
-o \$sampleid.rnasnp.vcf \
-ERC BP_RESOLUTION \
-nct 16
`

Which also makes it run very very fast. But I assume calling only in 1bp intervals will also stop any local re-assembly (like HC normally does)? Perhaps that won't matter for my use.

Finally, any idea regarding the GenotypeAndValidate - is it not meant to deal with this type of situation?

Thanks for the help.
Vasilios

• ChinaMember

I just want to do this. Do you solve this problem ? I hope you can help me too.I have a VCF file and a mapping file (BAM),I want to konw the genotype for each site in the VCF file which also included the ref-hom . Thanks !

@vasilios, I think HC pads the interval to a minimum window size even if the original request is just one position, so you shouldn't need to worry on that front --but I will ask @vruano‌ to confirm.