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Created 2014-09-19 14:34:45 | Updated | Tags: haplotypecaller malformedvcf genotypegvcfs
Comments (8)


I've tried my best to find the problem but without luck, so my first post...

First the background:

I have used HaplotypeCaller to make GVCFs per individual for 2 cohorts of 270 and 300 individuals each.
I then used CombineGVCFs to merged all the individuals in each cohort across 10Mb chunks across the genome.

These steps appear to have worked OK! Then I have been attempting to call genotypes from pairs GVCFs (one from each cohort) per region using GenotypeGVCFs with a command such as:

java -jar -Xmx6G /gpfs0/apps/well/gatk/3.2-2/GenomeAnalysisTK.jar \ -T GenotypeGVCFs \ -R ../hs37d5/hs37d5.fa \ --variant /well/hill/kate/exomes//haploc/cap270/cap270_1:230000000-240000000_merged_gvcf.vcf \ --variant /well/hill/kate/exomes//haploc/sepsis300/sepsis300_1:230000000-240000000_merged_gvcf.vcf \ -o /well/hill/kate/exomes//haploc/cap270-sepsis300/cap270-sepsis300_1:230000000-240000000_raw.vcf

This seems to start fine (and works perfectly to completion for some regions), but then throws a error for some of the files. For this example the error is:

ERROR MESSAGE: The provided VCF file is malformed at approximately line number 519534: ./.:29:75:25:0,63,945 is not a valid start position in the VCF format, for input source: /well/hill/kate/exomes/haploc/cap270/cap270_1:230000000-240000000_merged_gvcf.vcf

So to try and solve this I have grepped "./.:29:75:25:0,63,945 " from the VCF file (expecting to find a truncated line), but could find nothing wrong with the lines containing this expression. I looked carefully at the first line containing this after the last position that was written in the output - nothing out of the ordinary that I can see.

Any help/advice gratefully received! Kate

Comments (5)


I am having trouble calling variants using Haplotype Caller on simulated exome reads. I have been able to call reasonable-looking variants on the exome (simulated with dwgsim) with HaplotypeCaller before running it through the Best Practices Pre-Processing pipeline. The pre-processed data worked fine with UnifiedGenotyper but with HaplotypeCaller, though it runs without errors and seems to walk across the genome, only outputs a VCF header. I have tried calling variants with and without using -L to provide the exome regions (as recommended in this forum post: http://gatkforums.broadinstitute.org/discussion/1681/expected-file-size-haplotype-caller) but this hasn't made a difference - when we run the command with the pre-processed BAMs, we only get a VCF header. Everything has been tested with both 2.4-7 and 2.4-9.

Any help or guidance would be greatly appreciated!

Command Used for HaplotypeCaller:

java -Xmx4g -jar GenomeAnalysisTK.jar -T HaplotypeCaller -R ucsc.hg19.fasta -I exome.realigned.dedup.recal.bam -o exome.raw.vcf -D dbsnp_137.hg19.vcf -stand_emit_conf 10 -rf BadCigar -L Illumin_TruSeq.bed --logging_level DEBUG

Commands Used for pre-processing (run in sequence using a Perl script):

java -Xmx16g -jar GenomeAnalysisTK.jar -T RealignerTargetCreator -nt 8 -R ucsc.hg19.fasta -I exome.bam -o exome.intervals -known dbsnp_137.hg19.vcf

java -Xmx4g -jar GenomeAnalysisTK.jar -T IndelRealigner -R ucsc.hg19.fasta -I exome.bam -o exome.realigned.bam -targetIntervals intervals.bam -known dbsnp_137.hg19.vcf

java -Xmx16g -jar MarkDuplicates.jar I=exome.realigned.bam METRICS_FILE=exome.dups O=exome.realigned.dedup.bam

samtools index exome.realigned.dedup

java -Xmx4g -jar GenomeAnalysisTK.jar -T BaseRecalibrator -nct 8 -R ucsc.hg19.fasta -I exome.realigned.dedup.bam -o exome.recal_data.grp -knownSites dbsnp_137.hg19.vcf -cov ReadGroupCovariate -cov ContextCovariate -cov CycleCovariate -cov QualityScoreCovariate

java -Xmx4g -jar GenomeAnalysisTK.jar -T PrintReads -nct 8 -R ucsc.hg19.fasta -I exome.realigned.dedup.bam -BQSR exome.recal_data.grp -baq CALCULATE_AS_NECESSARY -o exome.realigned.dedup.recal.bam