Hi,

I am using GATK4 (4.1.3) for "RNAseq short variant discovery (SNPs + Indels)" as given at GATK best practices workflows at: https://gatk.broadinstitute.org/hc/en-us/articles/360035531192-RNAseq-short-variant-discovery-SNPs-Indels-

As suggested, I am doing the haplotype caller per sample, and my command is:

gatk --java-options "-Xmx128g" HaplotypeCaller \
-R ${REFDIR}/Homo_sapiens_assembly38.fasta \
-I ${ID}.Aligned.sorted.split.bqsr.bam \
-O ${ID}.Aligned.sorted.split.bqsr.vcf.gz

However, when I try to validate the variants with:

gatk --java-options '-DGATK_STACKTRACE_ON_USER_EXCEPTION=true' ValidateVariants \
-R ${REFDIR}/Homo_sapiens_assembly38.fasta \
-V ${ID}.Aligned.sorted.split.bqsr.vcf.gz

I get the error:

[April 13, 2020 7:21:31 AM EDT] org.broadinstitute.hellbender.tools.walkers.variantutils.ValidateVariants done. Elapsed time: 0.70 minutes.
Runtime.totalMemory()=3643801600
Using GATK jar /mnt/beegfs/v1/js/sw/js/Pkgs/GATK/4.1.3.0/gatk-package-4.1.3.0-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 -DGATK_STACKTRACE_ON_USER_EXCEPTION=true -jar /mnt/beegfs/v1/js/sw/js/Pkgs/GATK/4.1.3.0/gatk-package-4.1.3.0-local.jar ValidateVariants -R /home/js/data/references/star_references/Homo_sapiens_assembly38.fasta -V TW27.Aligned.sorted.split.bqsr.vcf
07:21:44.635 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/mnt/beegfs/v1/js/sw/js/Pkgs/GATK/4.1.3.0/gatk-package-4.1.3.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
Apr 13, 2020 7:21:46 AM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
07:21:46.886 INFO ValidateVariants - ------------------------------------------------------------
07:21:46.888 INFO ValidateVariants - The Genome Analysis Toolkit (GATK) v4.1.3.0
07:21:46.889 INFO ValidateVariants - For support and documentation go to https://software.broadinstitute.org/gatk/
07:21:46.891 INFO ValidateVariants - Executing as js@node01.local on Linux v3.10.0-1062.18.1.el7.x86_64 amd64
07:21:46.892 INFO ValidateVariants - Java runtime: Java HotSpot(TM) 64-Bit Server VM v1.8.0_111-b14
07:21:46.894 INFO ValidateVariants - Start Date/Time: April 13, 2020 7:21:44 AM EDT
07:21:46.895 INFO ValidateVariants - ------------------------------------------------------------
07:21:46.896 INFO ValidateVariants - ------------------------------------------------------------
07:21:46.903 INFO ValidateVariants - HTSJDK Version: 2.20.1
07:21:46.904 INFO ValidateVariants - Picard Version: 2.20.5
07:21:46.905 INFO ValidateVariants - HTSJDK Defaults.COMPRESSION_LEVEL : 2
07:21:46.907 INFO ValidateVariants - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
07:21:46.908 INFO ValidateVariants - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
07:21:46.909 INFO ValidateVariants - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
07:21:46.910 INFO ValidateVariants - Deflater: IntelDeflater
07:21:46.911 INFO ValidateVariants - Inflater: IntelInflater
07:21:46.913 INFO ValidateVariants - GCS max retries/reopens: 20
07:21:46.914 INFO ValidateVariants - Requester pays: disabled
07:21:46.915 INFO ValidateVariants - Initializing engine
07:21:49.630 INFO FeatureManager - Using codec VCFCodec to read file file:///mnt/beegfs/v1/js/home/js/data/rnaseq-snp/bbarwick/SCLC/fastq.trimmed/all_vcf/TW27.Aligned.sorted.split.bqsr.vcf
07:21:49.999 INFO ValidateVariants - Done initializing engine
07:21:50.001 WARN ValidateVariants - IDS validation cannot be done because no DBSNP file was provided
07:21:50.002 WARN ValidateVariants - Other possible validations will still be performed
07:21:50.003 INFO ProgressMeter - Starting traversal
07:21:50.005 INFO ProgressMeter - Current Locus Elapsed Minutes Variants Processed Variants/Minute
07:22:00.059 INFO ProgressMeter - chr5:131343713 0.2 238000 1421885.9
07:22:09.171 INFO ValidateVariants - Shutting down engine
[April 13, 2020 7:22:09 AM EDT] org.broadinstitute.hellbender.tools.walkers.variantutils.ValidateVariants done. Elapsed time: 0.41 minutes.
Runtime.totalMemory()=3191865344
***********************************************************************

A USER ERROR has occurred: Input TW27.Aligned.sorted.split.bqsr.vcf fails strict validation: one or more of the ALT allele(s) for the record at position chr11:6479079 are not observed at all in the sample genotypes of type:

***********************************************************************
org.broadinstitute.hellbender.exceptions.UserException$FailsStrictValidation: Input TW27.Aligned.sorted.split.bqsr.vcf fails strict validation: one or more of the ALT allele(s) for the record at position chr11:6479079 are not observed at all in the sample genotypes of type:
at org.broadinstitute.hellbender.tools.walkers.variantutils.ValidateVariants.apply(ValidateVariants.java:265)
at org.broadinstitute.hellbender.engine.VariantWalker.lambda$traverse$0(VariantWalker.java:104)
at java.util.stream.ForEachOps$ForEachOp$OfRef.accept(ForEachOps.java:184)
at java.util.stream.ReferencePipeline$3$1.accept(ReferencePipeline.java:193)
at java.util.stream.ReferencePipeline$2$1.accept(ReferencePipeline.java:175)
at java.util.stream.ReferencePipeline$3$1.accept(ReferencePipeline.java:193)
at java.util.Iterator.forEachRemaining(Iterator.java:116)
at java.util.Spliterators$IteratorSpliterator.forEachRemaining(Spliterators.java:1801)
at java.util.stream.AbstractPipeline.copyInto(AbstractPipeline.java:481)
at java.util.stream.AbstractPipeline.wrapAndCopyInto(AbstractPipeline.java:471)
at java.util.stream.ForEachOps$ForEachOp.evaluateSequential(ForEachOps.java:151)
at java.util.stream.ForEachOps$ForEachOp$OfRef.evaluateSequential(ForEachOps.java:174)
at java.util.stream.AbstractPipeline.evaluate(AbstractPipeline.java:234)
at java.util.stream.ReferencePipeline.forEach(ReferencePipeline.java:418)
at org.broadinstitute.hellbender.engine.VariantWalker.traverse(VariantWalker.java:102)
at org.broadinstitute.hellbender.engine.GATKTool.doWork(GATKTool.java:1048)
at org.broadinstitute.hellbender.cmdline.CommandLineProgram.runTool(CommandLineProgram.java:139)
at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMainPostParseArgs(CommandLineProgram.java:191)
at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:210)
at org.broadinstitute.hellbender.Main.runCommandLineProgram(Main.java:162)
at org.broadinstitute.hellbender.Main.mainEntry(Main.java:205)
at org.broadinstitute.hellbender.Main.main(Main.java:291)
Using GATK jar /mnt/beegfs/v1/js/sw/js/Pkgs/GATK/4.1.3.0/gatk-package-4.1.3.0-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 -DGATK_STACKTRACE_ON_USER_EXCEPTION=true -jar /mnt/beegfs/v1/js/sw/js/Pkgs/GATK/4.1.3.0/gatk-package-4.1.3.0-local.jar ValidateVariants -R /home/js/data/references/star_references/Homo_sapiens_assembly38.fasta -V TOW0062.Aligned.sorted.split.bqsr.vcf
07:22:22.837 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/mnt/beegfs/v1/js/sw/js/Pkgs/GATK/4.1.3.0/gatk-package-4.1.3.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
Apr 13, 2020 7:22:25 AM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine

This is the corresponding line in the vcf file:

[all_vcf] % grep 6479079 TW27.Aligned.sorted.split.bqsr.vcf

chr11 6479079 . A G 38.01 . AC=0;AF=0.00;AN=2;DP=48;ExcessHet=3.0103;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;SOR=1.022 GT:AD:DP:GQ:PL 0/0:42,5:47:33:0,33,988