Genome Analysis Toolkit

Laura Sanz · May 26, 2023 02:21

Hello,

I have a multi-vcf file (10 WGS human samples, chr1) generated using HaplotypeCaller in GVCF mode, followed by GenomicsDBImport and GenotypeGVCFs. I am trying to use VariantRecalibrator but I am having an error that I have not seen before. I followed the recommendations from other posts with similar problems, but not this one with VariantRecalibrator. I tried "gzcat vcf | head -1", PrintBGZFBlockInformation and ValidateVariants on my vcf file and

hapmap_3.3.hg38.vcf.gz
1000G_omni2.5.hg38.vcf.gz
1000G_phase1.snps.high_confidence.hg38.vcf.gz
Homo_sapiens_assembly38.dbsnp138.vcf.gz

Everything looks good. The command and the program log are as follow:

REQUIRED for all errors and issues:
a) GATK version used: gatk-4.3.0.0
b) Exact command used:

#!/bin/sh

#SBATCH --time=30:00:00

#SBATCH --nodes=1

#SBATCH --ntasks=4

#SBATCH --cpus-per-task=4

#SBATCH --mem=55G

#SBATCH --job-name=testvr

#SBATCH --output=testvr.out

#SBATCH --mail-type=ALL

#SBATCH --mail-user=xxx@xxxxxx

/mnt/beegfs/users/lsanz/Software/gatk.tool/gatk-4.3.0.0/gatk --java-options "-Xmx3g -Xms3g" VariantRecalibrator \

-V/mnt/beegfs/bandit/scratch/laura/from_home/bwa/GVCF/fullpilot_chr1.vcf.gz\

--trust-all-polymorphic\

-tranche100.0-tranche99.95-tranche99.9-tranche99.8-tranche99.6-tranche99.5-tranche99.4-tranche99.3-tranche99.0-tranche98.0-tranche97.0-tranche90.0\

-anQD-anMQRankSum-anReadPosRankSum-anFS-anMQ-anSOR-anDP\

-modeSNP\

--max-gaussians6\

--resource:hapmap,known=false,training=true,truth=true,prior=15/mnt/beegfs/users/lsanz/Projects/mapping/ncbi_dataset/data/GCF_000001405.26/hapmap_3.3.hg38.vcf.gz\

--resource:omni,known=false,training=true,truth=true,prior=12/mnt/beegfs/users/lsanz/Projects/mapping/ncbi_dataset/data/GCF_000001405.26/1000G_omni2.5.hg38.vcf.gz\

--resource:1000G,known=false,training=true,truth=false,prior=10/mnt/beegfs/users/lsanz/Projects/mapping/ncbi_dataset/data/GCF_000001405.26/1000G_phase1.snps.high_confidence.hg38.vcf.gz\

--resource:dbsnp,known=true,training=false,truth=false,prior=7/mnt/beegfs/users/lsanz/Projects/mapping/ncbi_dataset/data/GCF_000001405.26/Homo_sapiens_assembly38.dbsnp138.vcf.gz\

-O/mnt/beegfs/bandit/scratch/laura/from_home/bwa/GVCF/cohort_snps.recal\

--tranches-file/mnt/beegfs/bandit/scratch/laura/from_home/bwa/GVCF/cohort_snps.tranches

c) Entire program log:

11:54:56.228 INFO VariantRecalibrator - The Genome Analysis Toolkit (GATK) v4

.3.0.0

11:54:56.229 INFO VariantRecalibrator - For support and documentation go to h

ttps://software.broadinstitute.org/gatk/

11:54:56.231 INFO VariantRecalibrator - Executing as lsanz@scn02.svi.edu.au o

n Linux v4.18.0-425.19.2.el8_7.x86_64 amd64

11:54:56.232 INFO VariantRecalibrator - Java runtime: OpenJDK 64-Bit Server V

M v1.8.0_372-b07

11:54:56.232 INFO VariantRecalibrator - Start Date/Time: 26 May 2023 11:54:55

AM

11:54:56.233 INFO VariantRecalibrator - -------------------------------------

-----------------------

11:54:56.234 INFO VariantRecalibrator - -------------------------------------

-----------------------

11:54:56.235 INFO VariantRecalibrator - HTSJDK Version: 3.0.1

11:54:56.236 INFO VariantRecalibrator - Picard Version: 2.27.5

11:54:56.236 INFO VariantRecalibrator - Built for Spark Version: 2.4.5

11:54:56.236 INFO VariantRecalibrator - HTSJDK Defaults.COMPRESSION_LEVEL : 2

11:54:56.237 INFO VariantRecalibrator - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR

_SAMTOOLS : false

11:54:56.237 INFO VariantRecalibrator - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FO

R_SAMTOOLS : true

11:54:56.238 INFO VariantRecalibrator - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FO

R_TRIBBLE : false

11:54:56.238 INFO VariantRecalibrator - Deflater: IntelDeflater

11:54:56.239 INFO VariantRecalibrator - Inflater: IntelInflater

11:54:56.239 INFO VariantRecalibrator - GCS max retries/reopens: 20

11:54:56.239 INFO VariantRecalibrator - Requester pays: disabled

11:54:56.240 INFO VariantRecalibrator - Initializing engine

11:54:56.815 INFO FeatureManager - Using codec VCFCodec to read file file:///

mnt/beegfs/users/lsanz/Projects/mapping/ncbi_dataset/data/GCF_000001405.26/hap

map_3.3.hg38.vcf.gz

11:54:57.132 INFO FeatureManager - Using codec VCFCodec to read file file:///

mnt/beegfs/users/lsanz/Projects/mapping/ncbi_dataset/data/GCF_000001405.26/100

0G_omni2.5.hg38.vcf.gz

11:54:57.301 INFO FeatureManager - Using codec VCFCodec to read file file:///

mnt/beegfs/users/lsanz/Projects/mapping/ncbi_dataset/data/GCF_000001405.26/100

0G_phase1.snps.high_confidence.hg38.vcf.gz

11:54:57.450 INFO FeatureManager - Using codec VCFCodec to read file file:///

mnt/beegfs/users/lsanz/Projects/mapping/ncbi_dataset/data/GCF_000001405.26/Hom

o_sapiens_assembly38.dbsnp138.vcf.gz

11:54:57.581 INFO FeatureManager - Using codec VCFCodec to read file file:///

mnt/beegfs/bandit/scratch/laura/from_home/bwa/GVCF/fullpilot_chr1.vcf.gz

11:54:57.718 INFO VariantRecalibrator - Done initializing engine

11:54:57.722 INFO TrainingSet - Found hapmap track: Known = false Traini

ng = true Truth = true Prior = Q15.0

11:54:57.723 INFO TrainingSet - Found omni track: Known = false Traini

ng = true Truth = true Prior = Q12.0

11:54:57.723 INFO TrainingSet - Found 1000G track: Known = false Traini

ng = true Truth = false Prior = Q10.0

11:54:57.724 INFO TrainingSet - Found dbsnp track: Known = true Traini

ng = false Truth = false Prior = Q7.0

11:54:57.743 WARN GATKVariantContextUtils - Can't determine output variant fi

le format from output file extension "recal". Defaulting to VCF.

11:54:57.768 INFO ProgressMeter - Starting traversal

11:54:57.769 INFO ProgressMeter - Current Locus Elapsed Minutes Va

riants Processed Variants/Minute

11:54:58.258 INFO VariantRecalibrator - Shutting down engine

[26 May 2023 11:54:58 AM] org.broadinstitute.hellbender.tools.walkers.vqsr.Var

iantRecalibrator done. Elapsed time: 0.04 minutes.

Runtime.totalMemory()=3087007744

org.broadinstitute.hellbender.exceptions.GATKException: Exception thrown at ch

r1:1325353 [VC /mnt/beegfs/bandit/scratch/laura/from_home/bwa/GVCF/fullpilot_c

hr1.vcf.gz @ chr1:1325353 Q1725.35 of type=SNP alleles=[A*, C] attr={AC=19, AF

=0.950, AN=20, BaseQRankSum=0.00, DP=51, ExcessHet=0.0000, FS=0.000, Inbreedin

gCoeff=-0.0850, MLEAC=18, MLEAF=0.900, MQ=60.00, MQRankSum=0.00, QD=37.92, Rea

dPosRankSum=-5.240e-01, SOR=0.823} GT=GT:AD:DP:GQ:PL 1/1:0,5:5:15:180,15,

0 0/1:2,3:5:56:92,0,56 1/1:0,7:7:21:254,21,0 1/1:0,5:5:15:180,15,0 1/

1:0,2:2:6:72,6,0 1/1:0,5:5:15:181,15,0 1/1:0,2:2:6:73,6,0 1/1:0,8:

8:24:291,24,0 1/1:0,5:5:15:180,15,0 1/1:0,5:5:15:179,15,0 filters=

at org.broadinstitute.hellbender.engine.MultiVariantWalker.lambda$trav

erse$1(MultiVariantWalker.java:145)

at java.util.stream.ForEachOps$ForEachOp$OfRef.accept(ForEachOps.java:

183)

at java.util.stream.ReferencePipeline$3$1.accept(ReferencePipeline.jav

a:193)

at java.util.stream.ReferencePipeline$2$1.accept(ReferencePipeline.jav

a:175)

at java.util.stream.ReferencePipeline$3$1.accept(ReferencePipeline.jav

a:193)

at java.util.Iterator.forEachRemaining(Iterator.java:116)

at java.util.Spliterators$IteratorSpliterator.forEachRemaining(Spliter

ators.java:1801)

at java.util.stream.AbstractPipeline.copyInto(AbstractPipeline.java:48

2)

at java.util.stream.AbstractPipeline.wrapAndCopyInto(AbstractPipeline.

java:472)

at java.util.stream.ForEachOps$ForEachOp.evaluateSequential(ForEachOps

.java:150)

at java.util.stream.ForEachOps$ForEachOp$OfRef.evaluateSequential(ForE

achOps.java:173)

at java.util.stream.AbstractPipeline.evaluate(AbstractPipeline.java:23

4)

at java.util.stream.ReferencePipeline.forEach(ReferencePipeline.java:4

85)

at org.broadinstitute.hellbender.engine.MultiVariantWalker.traverse(Mu

ltiVariantWalker.java:136)

at org.broadinstitute.hellbender.engine.GATKTool.doWork(GATKTool.java:

1095)

at org.broadinstitute.hellbender.cmdline.CommandLineProgram.runTool(Co

mmandLineProgram.java:140)

at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMa

inPostParseArgs(CommandLineProgram.java:192)

at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMa

in(CommandLineProgram.java:211)

at org.broadinstitute.hellbender.Main.runCommandLineProgram(Main.java:

160)

at org.broadinstitute.hellbender.Main.mainEntry(Main.java:203)

at org.broadinstitute.hellbender.Main.main(Main.java:289)

Caused by: htsjdk.samtools.util.RuntimeIOException: java.io.IOException: Bad a

ddress

at htsjdk.tribble.readers.TabixIteratorLineReader.readLine(TabixIterat

orLineReader.java:48)

at htsjdk.tribble.TabixFeatureReader$FeatureIterator.readNextRecord(Ta

bixFeatureReader.java:170)

at htsjdk.tribble.TabixFeatureReader$FeatureIterator.<init>(TabixFeatu

reReader.java:159)

at htsjdk.tribble.TabixFeatureReader.query(TabixFeatureReader.java:133

)

at org.broadinstitute.hellbender.engine.FeatureDataSource.refillQueryC

ache(FeatureDataSource.java:622)

at org.broadinstitute.hellbender.engine.FeatureDataSource.queryAndPref

etch(FeatureDataSource.java:591)

at org.broadinstitute.hellbender.engine.FeatureManager.getFeatures(Fea

tureManager.java:363)

at org.broadinstitute.hellbender.engine.FeatureContext.getValues(Featu

reContext.java:173)

at org.broadinstitute.hellbender.engine.FeatureContext.getValues(Featu

reContext.java:125)

at org.broadinstitute.hellbender.engine.FeatureContext.getValues(Featu

reContext.java:240)

at org.broadinstitute.hellbender.tools.walkers.vqsr.VariantDataManager

.parseTrainingSets(VariantDataManager.java:397)