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Empty VCF file from Mutect2 v. GATK 4.1.6.0 in tumor only mode

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    Genevieve Brandt

    Hello P M, is there any chance that your input BAM or reference files are empty?

    Could you send the error log of just running Mutect2?

    We do not provide support for minimap2 so I am not able to determine if there are issues with that part of your pipeline. Here is our support policy.

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    P M

    Thanks Genevieve Brandt for your reply.

    Both my BAM & reference files are not empty. 

    From the log, the problem line appears to be

    265 read(s) filtered by: WellformedReadFilter

    265 total reads filtered

    However, I couldn't understand, why my reads are failing this filter. Would you have further insights?

    Here's my log from Mutect2. 

        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 /sysapps/cluster/software/GATK/4.1.8.1-Java-1.8.0_92/gatk-package-4.1.8.1-local.jar Mutect2 -R reference.fa -I sample1_rg.bam -O sample1_rg.vcf.gz

    17:14:03.165 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/sysapps/cluster/software/GATK/4.1.8.1-Java-1.8.0_92/gatk-package-4.1.8.1-local.jar!/com/intel/gkl/native/libgkl_compression.so

    Sep 29, 2020 5:14:03 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine

    INFO: Failed to detect whether we are running on Google Compute Engine.

    17:14:03.572 INFO  Mutect2 - ------------------------------------------------------------

    17:14:03.572 INFO  Mutect2 - The Genome Analysis Toolkit (GATK) v4.1.8.1

    17:14:03.573 INFO  Mutect2 - For support and documentation go to https://software.broadinstitute.org/gatk/

    17:14:03.588 INFO  Mutect2 - Executing as mudvarip2@ai-hpcn106.cm.cluster on Linux v3.10.0-327.36.1.el7.x86_64 amd64

    17:14:03.588 INFO  Mutect2 - Java runtime: Java HotSpot(TM) 64-Bit Server VM v1.8.0_92-b14

    17:14:03.588 INFO  Mutect2 - Start Date/Time: September 29, 2020 5:14:03 PM EDT

    17:14:03.588 INFO  Mutect2 - ------------------------------------------------------------

    17:14:03.588 INFO  Mutect2 - ------------------------------------------------------------

    17:14:03.589 INFO  Mutect2 - HTSJDK Version: 2.23.0

    17:14:03.589 INFO  Mutect2 - Picard Version: 2.22.8

    17:14:03.589 INFO  Mutect2 - HTSJDK Defaults.COMPRESSION_LEVEL : 2

    17:14:03.589 INFO  Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false

    17:14:03.589 INFO  Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true

    17:14:03.589 INFO  Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false

    17:14:03.589 INFO  Mutect2 - Deflater: IntelDeflater

    17:14:03.589 INFO  Mutect2 - Inflater: IntelInflater

    17:14:03.590 INFO  Mutect2 - GCS max retries/reopens: 20

    17:14:03.590 INFO  Mutect2 - Requester pays: disabled

    17:14:03.590 INFO  Mutect2 - Initializing engine

    17:14:04.265 INFO  Mutect2 - Done initializing engine

    17:14:04.311 INFO  NativeLibraryLoader - Loading libgkl_utils.so from jar:file:/sysapps/cluster/software/GATK/4.1.8.1-Java-1.8.0_92/gatk-package-4.1.8.1-local.jar!/com/intel/gkl/native/libgkl_utils.so

    17:14:04.316 INFO  NativeLibraryLoader - Loading libgkl_pairhmm_omp.so from jar:file:/sysapps/cluster/software/GATK/4.1.8.1-Java-1.8.0_92/gatk-package-4.1.8.1-local.jar!/com/intel/gkl/native/libgkl_pairhmm_omp.so

    17:14:04.356 WARN  NativeLibraryLoader - Unable to load libgkl_pairhmm_omp.so from native/libgkl_pairhmm_omp.so (/tmp/libgkl_pairhmm_omp90150183072861089.so: /sysapps/cluster/software/GCC/4.8.4/lib64/libgomp.so.1: version `GOMP_4.0' not found (required by /tmp/libgkl_pairhmm_omp90150183072861089.so))

    17:14:04.356 INFO  PairHMM - OpenMP multi-threaded AVX-accelerated native PairHMM implementation is not supported

    17:14:04.356 INFO  NativeLibraryLoader - Loading libgkl_pairhmm.so from jar:file:/sysapps/cluster/software/GATK/4.1.8.1-Java-1.8.0_92/gatk-package-4.1.8.1-local.jar!/com/intel/gkl/native/libgkl_pairhmm.so

    17:14:04.472 INFO  IntelPairHmm - Flush-to-zero (FTZ) is enabled when running PairHMM

    17:14:04.473 WARN  IntelPairHmm - Ignoring request for 4 threads; not using OpenMP implementation

    17:14:04.473 INFO  PairHMM - Using the AVX-accelerated native PairHMM implementation

    17:14:04.546 INFO  ProgressMeter - Starting traversal

    17:14:04.546 INFO  ProgressMeter -        Current Locus  Elapsed Minutes     Regions Processed   Regions/Minute

    17:14:04.812 INFO  Mutect2 - 0 read(s) filtered by: MappingQualityReadFilter

    0 read(s) filtered by: MappingQualityAvailableReadFilter

    0 read(s) filtered by: MappingQualityNotZeroReadFilter

    0 read(s) filtered by: MappedReadFilter

    0 read(s) filtered by: NotSecondaryAlignmentReadFilter

    0 read(s) filtered by: NotDuplicateReadFilter

    0 read(s) filtered by: PassesVendorQualityCheckReadFilter

    0 read(s) filtered by: NonChimericOriginalAlignmentReadFilter

    0 read(s) filtered by: NonZeroReferenceLengthAlignmentReadFilter

    0 read(s) filtered by: ReadLengthReadFilter

    0 read(s) filtered by: GoodCigarReadFilter

    265 read(s) filtered by: WellformedReadFilter

    265 total reads filtered

    17:14:04.813 INFO  ProgressMeter -       Reference:2701              0.0                    19           4285.7

    17:14:04.813 INFO  ProgressMeter - Traversal complete. Processed 19 total regions in 0.0 minutes.

    17:14:04.841 INFO  VectorLoglessPairHMM - Time spent in setup for JNI call : 0.0

    17:14:04.841 INFO  PairHMM - Total compute time in PairHMM computeLogLikelihoods() : 0.0

    17:14:04.841 INFO  SmithWatermanAligner - Total compute time in java Smith-Waterman : 0.00 sec

    17:14:04.841 INFO  Mutect2 - Shutting down engine

    [September 29, 2020 5:14:04 PM EDT] org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2 done. Elapsed time: 0.03 minutes.

    Runtime.totalMemory()=2491940864

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    Genevieve Brandt

    P M here is documentation on the WelformedReadFilter. You can check if your BAM file has any issues with ValidateSamFile

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    P M

    Genevieve Brandt Yes I have looked at the description of the WellformedReadFilter. I don't know which criteria my reads are not fulfilling. I have also used ValidateSamFile to check my BAM. There were no issues there. 

    I ran Mutect with the WellformedReadFilter disabled. I got a java.lang.ArrayIndexOutOfBoundsException: 251 error. Here's the log when I run with the filter disabled. 

    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 /sysapps/cluster/software/GATK/4.1.8.1-Java-1.8.0_92/gatk-package-4.1.8.1-local.jar Mutect2 -DF WellformedReadFilter -R reference.fa -I sample1_rg.bam -O sample1_rg.vcf.gz

    22:12:32.393 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/sysapps/cluster/software/GATK/4.1.8.1-Java-1.8.0_92/gatk-package-4.1.8.1-local.jar!/com/intel/gkl/native/libgkl_compression.so

    Sep 29, 2020 10:12:32 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine

    INFO: Failed to detect whether we are running on Google Compute Engine.

    22:12:32.660 INFO  Mutect2 - ------------------------------------------------------------

    22:12:32.660 INFO  Mutect2 - The Genome Analysis Toolkit (GATK) v4.1.8.1

    22:12:32.660 INFO  Mutect2 - For support and documentation go to https://software.broadinstitute.org/gatk/

    22:12:32.663 INFO  Mutect2 - Executing as mudvarip2@ai-hpcn157.cm.cluster on Linux v3.10.0-327.36.1.el7.x86_64 amd64

    22:12:32.663 INFO  Mutect2 - Java runtime: Java HotSpot(TM) 64-Bit Server VM v1.8.0_92-b14

    22:12:32.663 INFO  Mutect2 - Start Date/Time: September 29, 2020 10:12:32 PM EDT

    22:12:32.663 INFO  Mutect2 - ------------------------------------------------------------

    22:12:32.663 INFO  Mutect2 - ------------------------------------------------------------

    22:12:32.663 INFO  Mutect2 - HTSJDK Version: 2.23.0

    22:12:32.664 INFO  Mutect2 - Picard Version: 2.22.8

    22:12:32.664 INFO  Mutect2 - HTSJDK Defaults.COMPRESSION_LEVEL : 2

    22:12:32.664 INFO  Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false

    22:12:32.664 INFO  Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true

    22:12:32.664 INFO  Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false

    22:12:32.664 INFO  Mutect2 - Deflater: IntelDeflater

    22:12:32.664 INFO  Mutect2 - Inflater: IntelInflater

    22:12:32.664 INFO  Mutect2 - GCS max retries/reopens: 20

    22:12:32.664 INFO  Mutect2 - Requester pays: disabled

    22:12:32.664 INFO  Mutect2 - Initializing engine

    22:12:33.171 INFO  Mutect2 - Done initializing engine

    22:12:33.188 INFO  NativeLibraryLoader - Loading libgkl_utils.so from jar:file:/sysapps/cluster/software/GATK/4.1.8.1-Java-1.8.0_92/gatk-package-4.1.8.1-local.jar!/com/intel/gkl/native/libgkl_utils.so

    22:12:33.194 INFO  NativeLibraryLoader - Loading libgkl_pairhmm_omp.so from jar:file:/sysapps/cluster/software/GATK/4.1.8.1-Java-1.8.0_92/gatk-package-4.1.8.1-local.jar!/com/intel/gkl/native/libgkl_pairhmm_omp.so

    22:12:33.226 WARN  NativeLibraryLoader - Unable to load libgkl_pairhmm_omp.so from native/libgkl_pairhmm_omp.so (/tmp/libgkl_pairhmm_omp1727963993330396650.so: /sysapps/cluster/software/GCC/4.8.4/lib64/libgomp.so.1: version `GOMP_4.0' not found (required by /tmp/libgkl_pairhmm_omp1727963993330396650.so))

    22:12:33.226 INFO  PairHMM - OpenMP multi-threaded AVX-accelerated native PairHMM implementation is not supported

    22:12:33.227 INFO  NativeLibraryLoader - Loading libgkl_pairhmm.so from jar:file:/sysapps/cluster/software/GATK/4.1.8.1-Java-1.8.0_92/gatk-package-4.1.8.1-local.jar!/com/intel/gkl/native/libgkl_pairhmm.so

    22:12:33.305 INFO  IntelPairHmm - Flush-to-zero (FTZ) is enabled when running PairHMM

    22:12:33.306 WARN  IntelPairHmm - Ignoring request for 4 threads; not using OpenMP implementation

    22:12:33.306 INFO  PairHMM - Using the AVX-accelerated native PairHMM implementation

    22:12:33.387 INFO  ProgressMeter - Starting traversal

    22:12:33.389 INFO  ProgressMeter -        Current Locus  Elapsed Minutes     Regions Processed   Regions/Minute

    22:12:33.498 INFO  VectorLoglessPairHMM - Time spent in setup for JNI call : 0.0

    22:12:33.499 INFO  PairHMM - Total compute time in PairHMM computeLogLikelihoods() : 0.0

    22:12:33.499 INFO  SmithWatermanAligner - Total compute time in java Smith-Waterman : 0.00 sec

    22:12:33.500 INFO  Mutect2 - Shutting down engine

    [September 29, 2020 10:12:33 PM EDT] org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2 done. Elapsed time: 0.02 minutes.

    Runtime.totalMemory()=2138046464

    java.lang.ArrayIndexOutOfBoundsException: 251

    at org.broadinstitute.hellbender.utils.read.SAMRecordToGATKReadAdapter.getBaseQuality(SAMRecordToGATKReadAdapter.java:304)

    at org.broadinstitute.hellbender.utils.pileup.PileupElement.getQual(PileupElement.java:161)

    at org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2Engine.isNextToUsefulSoftClip(Mutect2Engine.java:537)

    at org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2Engine.altQuals(Mutect2Engine.java:465)

    at org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2Engine.isActive(Mutect2Engine.java:375)

    at org.broadinstitute.hellbender.engine.AssemblyRegionIterator.loadNextAssemblyRegion(AssemblyRegionIterator.java:136)

    at org.broadinstitute.hellbender.engine.AssemblyRegionIterator.<init>(AssemblyRegionIterator.java:96)

    at org.broadinstitute.hellbender.engine.AssemblyRegionWalker.processReadShard(AssemblyRegionWalker.java:188)

    at org.broadinstitute.hellbender.engine.AssemblyRegionWalker.traverse(AssemblyRegionWalker.java:173)

    at org.broadinstitute.hellbender.engine.GATKTool.doWork(GATKTool.java:1049)

    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)

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    Genevieve Brandt

    P M I don't think the best solution is to disable that filter, from our documentation, if a read fails that filter it has "major internal inconsistencies and issues that could lead to errors downstream". 

    How many reads are in your input file for Mutect2?

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    P M

    Genevieve Brandt I have 265 reads for this sample. It is amplicon sequencing. 

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    Genevieve Brandt

    P M do you have reads that pass the criteria listed in the Welformed Read Filter documentation but are being filtered?

    • Alignment coordinates: start larger than 0 and end after the start position.
    • Alignment agrees with header: contig exists and start is within its range.
    • Read Group and Sequence are present
    • Consistent read length: bases match in length with the qualities and the CIGAR string.
    • Do not contain skipped regions: represented by the 'N' operator in the CIGAR string.
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