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Mutect2 error

ascott3ascott3 UMHSMember

Hi,

Please below find an error with Mutect2: it terminates before it's able to even begin.

Command = [email protected]:/c/linux/gatk$ ./gatk --java-options "-Dsamjdk.sra_libraries_download=true" Mutect2 -R hg38.fa -I SRR925800.bam -O unfiltered.v.gcf

Output =
Using GATK jar /c/linux/gatk/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
-Dsamjdk.sra_libraries_download=true -jar
/c/linux/gatk/gatk-package-4.1.3.0-local.jar Mutect2 -R hg38.fa -I
SRR925800.bam -O unfiltered.v.gcf
19:50:55.488 INFO NativeLibraryLoader - Loading libgkl_compression.so
from jar:file:/c/linux/gatk/gatk-package-4.1.3.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
Aug 19, 2019 7:50:57 PM
shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials
runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.

19:50:57.707 INFO Mutect2 -

19:50:57.707 INFO Mutect2 - The Genome Analysis Toolkit (GATK) v4.1.3.0
19:50:57.707 INFO Mutect2 - For support and documentation go to
https://software.broadinstitute.org/gatk/
19:50:57.707 INFO Mutect2 - Executing as [email protected] on
Linux v4.4.0-17763-Microsoft amd64
19:50:57.707 INFO Mutect2 - Java runtime: OpenJDK 64-Bit Server VM
v11.0.4+11-post-Ubuntu-1ubuntu218.04.3
19:50:57.707 INFO Mutect2 - Start Date/Time: August 19, 2019 at 7:50:55 PM GMT

19:50:57.708 INFO Mutect2 -

19:50:57.708 INFO Mutect2 -

19:50:57.708 INFO Mutect2 - HTSJDK Version: 2.20.1
19:50:57.708 INFO Mutect2 - Picard Version: 2.20.5
19:50:57.709 INFO Mutect2 - HTSJDK Defaults.COMPRESSION_LEVEL : 2
19:50:57.709 INFO Mutect2 - HTSJDK
Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
19:50:57.709 INFO Mutect2 - HTSJDK
Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
19:50:57.709 INFO Mutect2 - HTSJDK
Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
19:50:57.709 INFO Mutect2 - Deflater: IntelDeflater
19:50:57.709 INFO Mutect2 - Inflater: IntelInflater
19:50:57.709 INFO Mutect2 - GCS max retries/reopens: 20
19:50:57.709 INFO Mutect2 - Requester pays: disabled
19:50:57.709 INFO Mutect2 - Initializing engine
19:51:03.161 INFO Mutect2 - Shutting down engine
[August 19, 2019 at 7:51:03 PM GMT]
org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2 done.
Elapsed time: 0.13 minutes.
Runtime.totalMemory()=171966464
java.lang.NullPointerException
at org.broadinstitute.hellbender.utils.SequenceDictionaryUtils.getContigNames(SequenceDictionaryUtils.java:463)
at org.broadinstitute.hellbender.utils.SequenceDictionaryUtils.getCommonContigsByName(SequenceDictionaryUtils.java:457)
at org.broadinstitute.hellbender.utils.SequenceDictionaryUtils.compareDictionaries(SequenceDictionaryUtils.java:234)
at org.broadinstitute.hellbender.utils.SequenceDictionaryUtils.validateDictionaries(SequenceDictionaryUtils.java:150)
at org.broadinstitute.hellbender.utils.SequenceDictionaryUtils.validateDictionaries(SequenceDictionaryUtils.java:98)
at org.broadinstitute.hellbender.engine.GATKTool.validateSequenceDictionaries(GATKTool.java:769)
at org.broadinstitute.hellbender.engine.GATKTool.onStartup(GATKTool.java:711)
at org.broadinstitute.hellbender.engine.AssemblyRegionWalker.onStartup(AssemblyRegionWalker.java:161)
at org.broadinstitute.hellbender.cmdline.CommandLineProgram.runTool(CommandLineProgram.java:137)
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)

Am running BAM validation as we speak

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Best Answers

Answers

  • ascott3ascott3 UMHSMember

    Also, Java version:
    openjdk version "11.0.4" 2019-07-16
    OpenJDK Runtime Environment (build 11.0.4+11-post-Ubuntu-1ubuntu218.04.3)
    OpenJDK 64-Bit Server VM (build 11.0.4+11-post-Ubuntu-1ubuntu218.04.3, mixed mode, sharing)

  • ascott3ascott3 UMHSMember

    Update:
    Here are the results of the SAM validation:

    HISTOGRAM java.lang.String

    Error Type Count
    ERROR:CIGAR_MAPS_OFF_REFERENCE 7
    ERROR:INVALID_FLAG_MATE_UNMAPPED 15419008
    ERROR:MISMATCH_FLAG_MATE_NEG_STRAND 856034
    ERROR:MISMATCH_FLAG_MATE_UNMAPPED 17112024
    ERROR:MISMATCH_MATE_ALIGNMENT_START 1693016
    ERROR:MISMATCH_MATE_REF_INDEX 1693016
    ERROR:MISSING_PLATFORM_VALUE 1
    WARNING:MISSING_TAG_NM 115312173

    [Mon Aug 19 22:22:49 GMT 2019] picard.sam.ValidateSamFile done. Elapsed time: 131.37 minutes.
    Runtime.totalMemory()=427819008

  • Tiffany_at_BroadTiffany_at_Broad Cambridge, MAMember, Administrator, Broadie, Moderator admin

    Hi @ascott3 The good news is that you've isolated that there is an issue with your input bam. This article shows how you can generate a detailed report of the specific errors so that you can fix them before using Mutect2. At the very bottom of the article you will see a list of all the errors emitted by ValidateSamFile.

    Hope this helps!

  • ascott3ascott3 UMHSMember

    Sorry, just tried a different .bam file that had no errors per "ValidateSamFile" and still got the same error as described above

  • Tiffany_at_BroadTiffany_at_Broad Cambridge, MAMember, Administrator, Broadie, Moderator admin

    Can you copy and paste the latest error stacktrace?

  • ascott3ascott3 UMHSMember
    edited August 28

    Here is the latest error:

    ./gatk --java-options "-Dsamjdk.sra_libraries_download=true" Mutect2 -R hg19.fa -I test3.bam -O unfiltered.vcf
    Using GATK jar /c/linux/gatk/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 -Dsamjdk.sra_libraries_download=true -jar /c/linux/gatk/gatk-package-4.1.3.0-local.jar Mutect2 -R hg19.fa -I test3.bam -O unfiltered.vcf
    10:33:35.145 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/c/linux/gatk/gatk-package-4.1.3.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
    Aug 28, 2019 10:33:37 AM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
    INFO: Failed to detect whether we are running on Google Compute Engine.
    10:33:37.506 INFO Mutect2 - ------------------------------------------------------------
    10:33:37.507 INFO Mutect2 - The Genome Analysis Toolkit (GATK) v4.1.3.0
    10:33:37.507 INFO Mutect2 - For support and documentation go to https://software.broadinstitute.org/gatk/
    10:33:37.508 INFO Mutect2 - Executing as [email protected] on Linux v4.4.0-17763-Microsoft amd64
    10:33:37.508 INFO Mutect2 - Java runtime: OpenJDK 64-Bit Server VM v11.0.4+11-post-Ubuntu-1ubuntu218.04.3
    10:33:37.508 INFO Mutect2 - Start Date/Time: August 28, 2019 at 10:33:35 AM GMT
    10:33:37.509 INFO Mutect2 - ------------------------------------------------------------
    10:33:37.509 INFO Mutect2 - ------------------------------------------------------------
    10:33:37.510 INFO Mutect2 - HTSJDK Version: 2.20.1
    10:33:37.510 INFO Mutect2 - Picard Version: 2.20.5
    10:33:37.510 INFO Mutect2 - HTSJDK Defaults.COMPRESSION_LEVEL : 2
    10:33:37.511 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
    10:33:37.511 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
    10:33:37.511 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
    10:33:37.513 INFO Mutect2 - Deflater: IntelDeflater
    10:33:37.513 INFO Mutect2 - Inflater: IntelInflater
    10:33:37.514 INFO Mutect2 - GCS max retries/reopens: 20
    10:33:37.514 INFO Mutect2 - Requester pays: disabled
    10:33:37.514 INFO Mutect2 - Initializing engine
    10:33:37.874 INFO Mutect2 - Shutting down engine
    [August 28, 2019 at 10:33:37 AM GMT] org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2 done. Elapsed time: 0.05 minutes.
    Runtime.totalMemory()=161480704
    java.lang.NullPointerException
    at org.broadinstitute.hellbender.utils.SequenceDictionaryUtils.getContigNames(SequenceDictionaryUtils.java:463)
    at org.broadinstitute.hellbender.utils.SequenceDictionaryUtils.getCommonContigsByName(SequenceDictionaryUtils.java:457)
    at org.broadinstitute.hellbender.utils.SequenceDictionaryUtils.compareDictionaries(SequenceDictionaryUtils.java:234)
    at org.broadinstitute.hellbender.utils.SequenceDictionaryUtils.validateDictionaries(SequenceDictionaryUtils.java:150)
    at org.broadinstitute.hellbender.utils.SequenceDictionaryUtils.validateDictionaries(SequenceDictionaryUtils.java:98)
    at org.broadinstitute.hellbender.engine.GATKTool.validateSequenceDictionaries(GATKTool.java:769)
    at org.broadinstitute.hellbender.engine.GATKTool.onStartup(GATKTool.java:711)
    at org.broadinstitute.hellbender.engine.AssemblyRegionWalker.onStartup(AssemblyRegionWalker.java:161)
    at org.broadinstitute.hellbender.cmdline.CommandLineProgram.runTool(CommandLineProgram.java:137)
    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)

    Issue · Github
    by Tiffany_at_Broad

    Issue Number
    6142
    State
    open
    Last Updated
  • ascott3ascott3 UMHSMember

    Also, the .bam files I attempted using Mutect2 were successfully read using freebayes and Strelka, so at least they were readable.

  • ascott3ascott3 UMHSMember

    Thank you so much for your help! Making a new index and dictionary of my reference sequence worked like a charm. I was then able to successfully use FilterMutectCalls on the Mutect2 output and then annotate using Funcotator.
    Thanks again!

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