GATK HaplotypeCaller works without GVCF option, but errors with GVCF
1
0
Entering edit mode
3.0 years ago

I've extracted chromosome 4 from a whole genome bam file as follows:

samtools view -h "$BAM" chr4 > "$EXT/temp/"$PREFIX"_chr4.sam"
samtools view -bS "$EXT"/temp/$PREFIX"_chr4.sam" > "$EXT"/temp/$PREFIX"_chr4.bam"

Then added read groups, as required by GATK

picard AddOrReplaceReadGroups I="$BAM" O="$EXT"/temp/$PREFIX"_chr4_rg.bam" RGID=4 RGLB=lib1 RGPL=ILLUMINA RGPU=unit1 RGSM=20

Index the bam:

samtools index "$BAM"

Download the reference chromosome 4, index it and create the dictionary for gatk:

curl https://hgdownload.soe.ucsc.edu/goldenPath/hg19/chromosomes/chr4.fa.gz | gunzip -c > ~/refs/hg19/chr4.fa
samtools faidx $REF
bwa index $REF
bowtie2-build $REF $REF
picard CreateSequenceDictionary REFERENCE="$REF" OUTPUT=~/refs/hg19/$ACC.dict

Then I run the normal GATK variant calling with this, which works.

gatk HaplotypeCaller -R $REF -I "$BAM" -O "$DIR"/gatk/$PREFIX"_chr4_HaplotypeCaller.vcf"

But when I run it with the GVCF option, I get the error below:

gatk HaplotypeCaller -R $REF -I "$BAM" -O "$DIR"/gatk/$PREFIX"_chr4_HaplotypeCallerPGT.vcf" -ERC GVCF

The important line in the error seems to be:

A USER ERROR has occurred: Contig: chrM not found in reference dictionary.
Please check that you are using a compatible reference for your data.
Reference Contigs: [chr4]

But here is the full error:

$ gatk HaplotypeCaller -R $REF -I "$BAM" -O "$DIR"/gatk/$PREFIX"_chr4_HaplotypeCallerPGT.vcf" -ERC GVCF
Using GATK jar /Users/michaelflower/opt/anaconda3/envs/gatk4/share/gatk4-4.2.3.0-0/gatk-package-4.2.3.0-local.jar
Running:
    java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /Users/michaelflower/opt/anaconda3/envs/gatk4/share/gatk4-4.2.3.0-0/gatk-package-4.2.3.0-local.jar HaplotypeCaller -R /Users/michaelflower/refs/hg19/chr4.fa -I /Volumes/Seagate Expansion Drive/temp/125QiPSC_chr4_rg.bam -O /Users/michaelflower/Documents/ACL/Research/Projects/HTT ASO/2021.11.17 125Q vcf query/gatk/125QiPSC_chr4_HaplotypeCallerPGT.vcf -ERC GVCF
15:51:48.424 INFO  NativeLibraryLoader - Loading libgkl_compression.dylib from jar:file:/Users/michaelflower/opt/anaconda3/envs/gatk4/share/gatk4-4.2.3.0-0/gatk-package-4.2.3.0-local.jar!/com/intel/gkl/native/libgkl_compression.dylib
Nov 17, 2021 3:51:48 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
15:51:48.827 INFO  HaplotypeCaller - ------------------------------------------------------------
15:51:48.828 INFO  HaplotypeCaller - The Genome Analysis Toolkit (GATK) v4.2.3.0
15:51:48.828 INFO  HaplotypeCaller - For support and documentation go to https://software.broadinstitute.org/gatk/
15:51:48.828 INFO  HaplotypeCaller - Executing as michaelflower@Michaels-MacBook-Pro-2.local on Mac OS X v10.16 x86_64
15:51:48.828 INFO  HaplotypeCaller - Java runtime: OpenJDK 64-Bit Server VM v1.8.0_302-b08
15:51:48.828 INFO  HaplotypeCaller - Start Date/Time: 17 November 2021 15:51:48 GMT
15:51:48.829 INFO  HaplotypeCaller - ------------------------------------------------------------
15:51:48.829 INFO  HaplotypeCaller - ------------------------------------------------------------
15:51:48.829 INFO  HaplotypeCaller - HTSJDK Version: 2.24.1
15:51:48.829 INFO  HaplotypeCaller - Picard Version: 2.25.4
15:51:48.829 INFO  HaplotypeCaller - Built for Spark Version: 2.4.5
15:51:48.829 INFO  HaplotypeCaller - HTSJDK Defaults.COMPRESSION_LEVEL : 2
15:51:48.830 INFO  HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
15:51:48.830 INFO  HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
15:51:48.830 INFO  HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
15:51:48.830 INFO  HaplotypeCaller - Deflater: IntelDeflater
15:51:48.830 INFO  HaplotypeCaller - Inflater: IntelInflater
15:51:48.830 INFO  HaplotypeCaller - GCS max retries/reopens: 20
15:51:48.830 INFO  HaplotypeCaller - Requester pays: disabled
15:51:48.830 INFO  HaplotypeCaller - Initializing engine
15:51:49.333 INFO  HaplotypeCaller - Done initializing engine
15:51:49.335 INFO  HaplotypeCallerEngine - Tool is in reference confidence mode and the annotation, the following changes will be made to any specified annotations: 'StrandBiasBySample' will be enabled. 'ChromosomeCounts', 'FisherStrand', 'StrandOddsRatio' and 'QualByDepth' annotations have been disabled
15:51:49.341 INFO  HaplotypeCallerEngine - Standard Emitting and Calling confidence set to 0.0 for reference-model confidence output
15:51:49.341 INFO  HaplotypeCallerEngine - All sites annotated with PLs forced to true for reference-model confidence output
15:51:49.359 INFO  NativeLibraryLoader - Loading libgkl_utils.dylib from jar:file:/Users/michaelflower/opt/anaconda3/envs/gatk4/share/gatk4-4.2.3.0-0/gatk-package-4.2.3.0-local.jar!/com/intel/gkl/native/libgkl_utils.dylib
15:51:49.500 WARN  NativeLibraryLoader - Unable to find native library: native/libgkl_pairhmm_omp.dylib
15:51:49.500 INFO  PairHMM - OpenMP multi-threaded AVX-accelerated native PairHMM implementation is not supported
15:51:49.501 INFO  NativeLibraryLoader - Loading libgkl_pairhmm.dylib from jar:file:/Users/michaelflower/opt/anaconda3/envs/gatk4/share/gatk4-4.2.3.0-0/gatk-package-4.2.3.0-local.jar!/com/intel/gkl/native/libgkl_pairhmm.dylib
15:51:49.712 INFO  IntelPairHmm - Flush-to-zero (FTZ) is enabled when running PairHMM
15:51:49.713 WARN  IntelPairHmm - Ignoring request for 4 threads; not using OpenMP implementation
15:51:49.713 INFO  PairHMM - Using the AVX-accelerated native PairHMM implementation
15:51:49.780 INFO  ProgressMeter - Starting traversal
15:51:49.781 INFO  ProgressMeter -        Current Locus  Elapsed Minutes     Regions Processed   Regions/Minute
15:51:49.834 INFO  VectorLoglessPairHMM - Time spent in setup for JNI call : 0.0
15:51:49.834 INFO  PairHMM - Total compute time in PairHMM computeLogLikelihoods() : 0.0
15:51:49.834 INFO  SmithWatermanAligner - Total compute time in java Smith-Waterman : 0.00 sec
15:51:49.835 INFO  HaplotypeCaller - Shutting down engine
[17 November 2021 15:51:49 GMT] org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCaller done. Elapsed time: 0.02 minutes.
Runtime.totalMemory()=493879296
***********************************************************************

A USER ERROR has occurred: Contig: chrM not found in reference dictionary.
Please check that you are using a compatible reference for your data.
Reference Contigs: [chr4]

***********************************************************************
Set the system property GATK_STACKTRACE_ON_USER_EXCEPTION (--java-options '-DGATK_STACKTRACE_ON_USER_EXCEPTION=true') to print the stack trace.
(gatk4) 
picard GATK conda • 1.9k views
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Entering edit mode
3.0 years ago

The problem is unrelated to GVCF mode. you're not using the same dictionary/reference sequence that was used to map the whole BAM. It looks like your reference/fasta/dict only contains chr4

A USER ERROR has occurred: Contig: chrM not found in reference dictionary.
Please check that you are using a compatible reference for your data.
Reference Contigs: [chr4]

this is bad:

(...)  -R /Users/michaelflower/refs/hg19/chr4.fa  (...)

don't split your reference, use the original whole fasta sequence.

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0
Entering edit mode

That's true, I've added the script above, but I re downloaded just chr4 reference and used that. Shall I use the whole hg19 reference genome then?

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0
Entering edit mode

Shall I use the whole hg19 reference genome then?

yes

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0
Entering edit mode

Under way, I'll let you know how it goes

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Entering edit mode

Unfortunately it's still not working with the full hg19 reference. The vcf file produced only has one line:

#CHROM  POS ID  REF ALT QUAL    FILTER  INFO    FORMAT  20
chrM    1   .   G   <NON_REF>   .   .   END=16571   GT:DP:GQ:MIN_DP:PL  0/0:0:0:0:0,0,0

I didn't make the original bam file, but the people who did say they used hg19 from UCSC, http://hgdownload.cse.ucsc.edu/goldenPath/hg19/bigZips

The steps I used to make the vcf file above were:

Download hg19 reference, index it and create dictionary for GATK:

curl https://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/hg19.fa.gz | gunzip -c > ~/refs/hg19/hg19.fa
samtools faidx $REF
bwa index $REF
bowtie2-build $REF $REF
picard CreateSequenceDictionary REFERENCE="$REF" OUTPUT=~/refs/hg19/$ACC.dict

Then the steps above, including variant calling with:

gatk HaplotypeCaller -R $REF -I "$BAM" -O "$DIR"/gatk/$PREFIX"_chr4_HaplotypeCallerPGT.vcf" -ERC GVCF

But this gives the following output:

Using GATK jar /Users/michaelflower/opt/anaconda3/envs/gatk4/share/gatk4-4.2.3.0-0/gatk-package-4.2.3.0-local.jar
Running:
    java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /Users/michaelflower/opt/anaconda3/envs/gatk4/share/gatk4-4.2.3.0-0/gatk-package-4.2.3.0-local.jar HaplotypeCaller -R /Users/michaelflower/refs/hg19/hg19.fa -I /Volumes/Seagate Expansion Drive/temp/125QiPSC_chr4_rg.bam -O /Users/michaelflower/Documents/ACL/Research/Projects/HTT ASO/2021.11.17 125Q vcf query/gatk/125QiPSC_chr4_HaplotypeCallerPGT.vcf -ERC GVCF
18:46:41.314 INFO  NativeLibraryLoader - Loading libgkl_compression.dylib from jar:file:/Users/michaelflower/opt/anaconda3/envs/gatk4/share/gatk4-4.2.3.0-0/gatk-package-4.2.3.0-local.jar!/com/intel/gkl/native/libgkl_compression.dylib
Nov 17, 2021 6:46:42 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
18:46:42.126 INFO  HaplotypeCaller - ------------------------------------------------------------
18:46:42.127 INFO  HaplotypeCaller - The Genome Analysis Toolkit (GATK) v4.2.3.0
18:46:42.127 INFO  HaplotypeCaller - For support and documentation go to https://software.broadinstitute.org/gatk/
18:46:42.127 INFO  HaplotypeCaller - Executing as michaelflower@Michaels-MacBook-Pro-2.local on Mac OS X v10.16 x86_64
18:46:42.127 INFO  HaplotypeCaller - Java runtime: OpenJDK 64-Bit Server VM v1.8.0_302-b08
18:46:42.127 INFO  HaplotypeCaller - Start Date/Time: 17 November 2021 18:46:41 GMT
18:46:42.127 INFO  HaplotypeCaller - ------------------------------------------------------------
18:46:42.127 INFO  HaplotypeCaller - ------------------------------------------------------------
18:46:42.128 INFO  HaplotypeCaller - HTSJDK Version: 2.24.1
18:46:42.128 INFO  HaplotypeCaller - Picard Version: 2.25.4
18:46:42.128 INFO  HaplotypeCaller - Built for Spark Version: 2.4.5
18:46:42.128 INFO  HaplotypeCaller - HTSJDK Defaults.COMPRESSION_LEVEL : 2
18:46:42.128 INFO  HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
18:46:42.128 INFO  HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
18:46:42.128 INFO  HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
18:46:42.128 INFO  HaplotypeCaller - Deflater: IntelDeflater
18:46:42.128 INFO  HaplotypeCaller - Inflater: IntelInflater
18:46:42.128 INFO  HaplotypeCaller - GCS max retries/reopens: 20
18:46:42.128 INFO  HaplotypeCaller - Requester pays: disabled
18:46:42.129 INFO  HaplotypeCaller - Initializing engine
18:46:42.624 INFO  HaplotypeCaller - Done initializing engine
18:46:42.627 INFO  HaplotypeCallerEngine - Tool is in reference confidence mode and the annotation, the following changes will be made to any specified annotations: 'StrandBiasBySample' will be enabled. 'ChromosomeCounts', 'FisherStrand', 'StrandOddsRatio' and 'QualByDepth' annotations have been disabled
18:46:42.636 INFO  HaplotypeCallerEngine - Standard Emitting and Calling confidence set to 0.0 for reference-model confidence output
18:46:42.637 INFO  HaplotypeCallerEngine - All sites annotated with PLs forced to true for reference-model confidence output
18:46:42.655 INFO  NativeLibraryLoader - Loading libgkl_utils.dylib from jar:file:/Users/michaelflower/opt/anaconda3/envs/gatk4/share/gatk4-4.2.3.0-0/gatk-package-4.2.3.0-local.jar!/com/intel/gkl/native/libgkl_utils.dylib
18:46:42.849 WARN  NativeLibraryLoader - Unable to find native library: native/libgkl_pairhmm_omp.dylib
18:46:42.849 INFO  PairHMM - OpenMP multi-threaded AVX-accelerated native PairHMM implementation is not supported
18:46:42.850 INFO  NativeLibraryLoader - Loading libgkl_pairhmm.dylib from jar:file:/Users/michaelflower/opt/anaconda3/envs/gatk4/share/gatk4-4.2.3.0-0/gatk-package-4.2.3.0-local.jar!/com/intel/gkl/native/libgkl_pairhmm.dylib
18:46:43.081 INFO  IntelPairHmm - Flush-to-zero (FTZ) is enabled when running PairHMM
18:46:43.082 WARN  IntelPairHmm - Ignoring request for 4 threads; not using OpenMP implementation
18:46:43.082 INFO  PairHMM - Using the AVX-accelerated native PairHMM implementation
18:46:43.129 INFO  ProgressMeter - Starting traversal
18:46:43.130 INFO  ProgressMeter -        Current Locus  Elapsed Minutes     Regions Processed   Regions/Minute
18:46:53.131 INFO  ProgressMeter -        chr1:10191901              0.2                 34030         204180.0
18:47:03.130 INFO  ProgressMeter -        chr1:25089901              0.3                 83690         251070.0
18:47:13.130 INFO  ProgressMeter -        chr1:41172901              0.5                137300         274600.0
18:47:23.131 INFO  ProgressMeter -        chr1:50784901              0.7                169340         254003.6
18:47:33.136 INFO  ProgressMeter -        chr1:62814901              0.8                209440         251302.9
18:47:43.135 INFO  ProgressMeter -        chr1:74940901              1.0                249860         249839.2
18:47:53.136 INFO  ProgressMeter -        chr1:90363901              1.2                301270         258209.3
18:48:04.512 INFO  ProgressMeter -       chr1:105684901              1.4                352340         259767.5
18:48:14.512 INFO  ProgressMeter -       chr1:120819901              1.5                402790         264465.6
18:48:24.512 INFO  ProgressMeter -       chr1:137817901              1.7                459450         271912.2
18:48:34.657 INFO  ProgressMeter -       chr1:153876901              1.9                512980         275976.2
18:48:44.657 INFO  ProgressMeter -       chr1:168645901              2.0                562210         277572.9
18:48:54.658 INFO  ProgressMeter -       chr1:183276901              2.2                610980         278714.8
18:49:04.658 INFO  ProgressMeter -       chr1:197418901              2.4                658120         279006.3
18:49:14.659 INFO  ProgressMeter -       chr1:211458901              2.5                704920         279124.7
18:49:24.658 INFO  ProgressMeter -       chr1:223989901              2.7                746690         277360.0
18:49:34.788 INFO  ProgressMeter -       chr1:236559901              2.9                788590         275637.6
18:49:44.792 INFO  ProgressMeter -       chr1:245133901              3.0                817170         269899.4
18:50:09.670 INFO  HaplotypeCaller - Shutting down engine
[17 November 2021 18:50:09 GMT] org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCaller done. Elapsed time: 3.47 minutes.
Runtime.totalMemory()=4292870144
Exception in thread "main" java.lang.OutOfMemoryError: Java heap space
    at java.util.stream.Nodes$DoubleArrayNode.<init>(Nodes.java:1429)
    at java.util.stream.Nodes$DoubleFixedNodeBuilder.<init>(Nodes.java:1589)
    at java.util.stream.Nodes.doubleBuilder(Nodes.java:279)
    at java.util.stream.DoublePipeline.makeNodeBuilder(DoublePipeline.java:164)
    at java.util.stream.AbstractPipeline.evaluate(AbstractPipeline.java:544)
    at java.util.stream.AbstractPipeline.evaluateToArrayNode(AbstractPipeline.java:260)
    at java.util.stream.DoublePipeline.toArray(DoublePipeline.java:530)
    at org.broadinstitute.hellbender.utils.MathUtils.median(MathUtils.java:991)
    at org.broadinstitute.hellbender.utils.variant.writers.GVCFBlock.getMedianDP(GVCFBlock.java:80)
    at org.broadinstitute.hellbender.utils.variant.writers.HomRefBlock.createHomRefGenotype(HomRefBlock.java:93)
    at org.broadinstitute.hellbender.utils.variant.writers.GVCFBlock.toVariantContext(GVCFBlock.java:54)
    at org.broadinstitute.hellbender.utils.variant.writers.GVCFBlockCombiner.emitCurrentBlock(GVCFBlockCombiner.java:174)
    at org.broadinstitute.hellbender.utils.variant.writers.GVCFBlockCombiner.signalEndOfInput(GVCFBlockCombiner.java:226)
    at org.broadinstitute.hellbender.utils.variant.writers.GVCFWriter.close(GVCFWriter.java:68)
    at org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCaller.closeTool(HaplotypeCaller.java:279)
    at org.broadinstitute.hellbender.engine.GATKTool.doWork(GATKTool.java:1091)
    at org.broadinstitute.hellbender.cmdline.CommandLineProgram.runTool(CommandLineProgram.java:140)
    at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMainPostParseArgs(CommandLineProgram.java:192)
    at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMain(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)
(gatk4) 
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0
Entering edit mode

please, read the error message: the problem is unrelated to your original question.

Exception in thread "main" java.lang.OutOfMemoryError: Java heap space

set -XmX see https://stackoverflow.com/questions/37335/how-to-deal-with-java-lang-outofmemoryerror-java-heap-space-error

and "4. Adding Java arguments" in https://gatk.broadinstitute.org/hc/en-us/articles/360035531892-GATK4-command-line-syntax

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Entering edit mode

Thank you, I've had a good read of those pages. I added the argument

--java-options "-Xmx4G"

But unfortunately I still get the java.lang.OutOfMemoryError: Java heap space error

$ gatk HaplotypeCaller -R $REF -I "$BAM" -O "$DIR"/gatk/$PREFIX"_chr4_HaplotypeCallerPGT.vcf" -ERC GVCF --java-options "-Xmx4G"
Using GATK jar /Users/michaelflower/opt/anaconda3/envs/gatk4/share/gatk4-4.2.3.0-0/gatk-package-4.2.3.0-local.jar


...

Truncated for space
...



confidence mode and the annotation, the following changes will be made to any specified annotations: 'StrandBiasBySample' will be enabled. 'ChromosomeCounts', 'FisherStrand', 'StrandOddsRatio' and 'QualByDepth' annotations have been disabled
22:55:45.905 INFO  HaplotypeCallerEngine - Standard Emitting and Calling confidence set to 0.0 for reference-model confidence output
22:55:45.905 INFO  HaplotypeCallerEngine - All sites annotated with PLs forced to true for reference-model confidence output
22:55:45.940 INFO  NativeLibraryLoader - Loading libgkl_utils.dylib from jar:file:/Users/michaelflower/opt/anaconda3/envs/gatk4/share/gatk4-4.2.3.0-0/gatk-package-4.2.3.0-local.jar!/com/intel/gkl/native/libgkl_utils.dylib
22:55:46.085 WARN  NativeLibraryLoader - Unable to find native library: native/libgkl_pairhmm_omp.dylib
22:55:46.086 INFO  PairHMM - OpenMP multi-threaded AVX-accelerated native PairHMM implementation is not supported
22:55:46.086 INFO  NativeLibraryLoader - Loading libgkl_pairhmm.dylib from jar:file:/Users/michaelflower/opt/anaconda3/envs/gatk4/share/gatk4-4.2.3.0-0/gatk-package-4.2.3.0-local.jar!/com/intel/gkl/native/libgkl_pairhmm.dylib
22:55:46.268 INFO  IntelPairHmm - Flush-to-zero (FTZ) is enabled when running PairHMM
22:55:46.269 WARN  IntelPairHmm - Ignoring request for 4 threads; not using OpenMP implementation
22:55:46.269 INFO  PairHMM - Using the AVX-accelerated native PairHMM implementation
22:55:46.339 INFO  ProgressMeter - Starting traversal
22:55:46.339 INFO  ProgressMeter -        Current Locus  Elapsed Minutes     Regions Processed   Regions/Minute
22:55:56.342 INFO  ProgressMeter -         chr1:5412901              0.2                 18100         108600.0
22:56:06.341 INFO  ProgressMeter -        chr1:11346901              0.3                 37880         113634.3
22:56:16.341 INFO  ProgressMeter -        chr1:19770901              0.5                 65960         131911.2
22:56:26.342 INFO  ProgressMeter -        chr1:31377901              0.7                104650         156963.2
22:56:36.342 INFO  ProgressMeter -        chr1:43488901              0.8                145020         174013.6
22:56:46.345 INFO  ProgressMeter -        chr1:51756901              1.0                172580         172562.7
22:56:56.347 INFO  ProgressMeter -        chr1:61461901              1.2                204930         175634.2
22:57:06.585 INFO  ProgressMeter -        chr1:70170901              1.3                233960         174934.3
22:57:16.585 INFO  ProgressMeter -        chr1:80082901              1.5                267000         177516.8
22:57:26.586 INFO  ProgressMeter -        chr1:89595901              1.7                298710         178784.4
22:57:36.589 INFO  ProgressMeter -        chr1:96369901              1.8                321290         174851.7
22:57:47.026 INFO  ProgressMeter -       chr1:103842901              2.0                346200         172114.6
22:57:57.026 INFO  ProgressMeter -       chr1:110505901              2.2                368410         169141.5
22:58:07.033 INFO  ProgressMeter -       chr1:118296901              2.3                394380         168187.5
22:58:17.034 INFO  ProgressMeter -       chr1:125025901              2.5                416810         165955.1
22:58:27.038 INFO  ProgressMeter -       chr1:132084901              2.7                440340         164410.3
22:58:37.039 INFO  ProgressMeter -       chr1:138627901              2.8                462150         162442.9
22:58:47.040 INFO  ProgressMeter -       chr1:145959901              3.0                486590         161568.3
22:58:57.062 INFO  ProgressMeter -       chr1:150723901              3.2                502470         158073.2
22:59:07.397 INFO  ProgressMeter -       chr1:157704901              3.4                525740         156892.0
22:59:17.979 INFO  ProgressMeter -       chr1:163350901              3.5                544560         154382.9
22:59:27.980 INFO  ProgressMeter -       chr1:168354901              3.7                561240         151932.2
22:59:37.981 INFO  ProgressMeter -       chr1:173850901              3.9                579560         150117.9
22:59:47.982 INFO  ProgressMeter -       chr1:178047901              4.0                593550         147378.6
22:59:57.982 INFO  ProgressMeter -       chr1:183978901              4.2                613320         146235.7
23:00:07.982 INFO  ProgressMeter -       chr1:190551901              4.4                635230         145671.0
23:00:19.298 INFO  ProgressMeter -       chr1:197019901              4.5                656790         144371.1
23:00:29.298 INFO  ProgressMeter -       chr1:201786901              4.7                672680         142638.3
23:00:39.298 INFO  ProgressMeter -       chr1:208509901              4.9                695090         142359.2
23:00:49.376 INFO  ProgressMeter -       chr1:213855901              5.1                712910         141153.1
23:00:59.376 INFO  ProgressMeter -       chr1:220383901              5.2                734670         140814.7
23:01:09.501 INFO  ProgressMeter -       chr1:226479901              5.4                754990         140175.5
23:01:20.316 INFO  ProgressMeter -       chr1:231741901              5.6                772530         138787.4
23:01:30.316 INFO  ProgressMeter -       chr1:236151901              5.7                787230         137316.7
23:01:40.318 INFO  ProgressMeter -       chr1:238332901              5.9                794500         134669.4
23:01:50.319 INFO  ProgressMeter -       chr1:243987901              6.1                813350         134076.4
23:05:11.503 INFO  HaplotypeCaller - Shutting down engine
[17 November 2021 23:05:11 GMT] org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCaller done. Elapsed time: 9.45 minutes.
Runtime.totalMemory()=4292870144
Exception in thread "main" java.lang.OutOfMemoryError: Java heap space
    at java.util.stream.Nodes$DoubleArrayNode.<init>(Nodes.java:1429)
    at java.util.stream.Nodes$DoubleFixedNodeBuilder.<init>(Nodes.java:1589)
    at java.util.stream.Nodes.doubleBuilder(Nodes.java:279)
    at java.util.stream.DoublePipeline.makeNodeBuilder(DoublePipeline.java:164)
    at java.util.stream.AbstractPipeline.evaluate(AbstractPipeline.java:544)
    at java.util.stream.AbstractPipeline.evaluateToArrayNode(AbstractPipeline.java:260)
    at java.util.stream.DoublePipeline.toArray(DoublePipeline.java:530)
    at org.broadinstitute.hellbender.utils.MathUtils.median(MathUtils.java:991)
    at org.broadinstitute.hellbender.utils.variant.writers.GVCFBlock.getMedianDP(GVCFBlock.java:80)
    at org.broadinstitute.hellbender.utils.variant.writers.HomRefBlock.createHomRefGenotype(HomRefBlock.java:93)
    at org.broadinstitute.hellbender.utils.variant.writers.GVCFBlock.toVariantContext(GVCFBlock.java:54)
    at org.broadinstitute.hellbender.utils.variant.writers.GVCFBlockCombiner.emitCurrentBlock(GVCFBlockCombiner.java:174)
    at org.broadinstitute.hellbender.utils.variant.writers.GVCFBlockCombiner.signalEndOfInput(GVCFBlockCombiner.java:226)
    at org.broadinstitute.hellbender.utils.variant.writers.GVCFWriter.close(GVCFWriter.java:68)
    at org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCaller.closeTool(HaplotypeCaller.java:279)
    at org.broadinstitute.hellbender.engine.GATKTool.doWork(GATKTool.java:1091)
    at org.broadinstitute.hellbender.cmdline.CommandLineProgram.runTool(CommandLineProgram.java:140)
    at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMainPostParseArgs(CommandLineProgram.java:192)
    at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMain(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)
(gatk4) 
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