Mutect2 cannot generate stats file and f1r2.tar.gz file
Answered
REQUIRED for all errors and issues:
a) GATK version used:4.2.6.1
b) Exact command used:
gatk Mutect2 -R $ref -L b37_Broad.human.exome.b37.interval_list -I $tumor1 -I $tumor2 -I $normal -normal W00139_b --germline-resource somatic-b37_af-only-gnomad.raw.sites.vcf --panel-of-normals $home/gatk/pon/pon.vcf.gz --f1r2-tar-gz f1r2.tar.gz -O somatic.vcf.gz
c) Entire program log:I can get the somatic.vcf.gz and somatic.vcf.gz.tbi, but it didn't product the stats file and f1r2.tar.gz file.
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Hi Yi Huang,
Thank you for writing to the GATK forum! I hope that we can help you sort this out.
I appreciate that you have included the GATK version and the exact command you used.
To accurately identify the issue you are encountering, I'll need to see the entire program log/stack trace. Please include all error messages/exceptions so we may best diagnose the issue and determine the next steps.
I look forward to hearing from you! If any other questions arise in the meantime, please do not hesitate to include them in your response.
Best,
Anthony -
My entire program file:
15:46:33.133 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/home/huangyi/anaconda/share/gatk4-4.2.6.1-1/gatk-package-4.2.6.1-local.jar!/com/intel/gkl/native/libgkl_compression.so
15:46:38.580 INFO Mutect2 - ------------------------------------------------------------
15:46:38.580 INFO Mutect2 - The Genome Analysis Toolkit (GATK) v4.2.6.1
15:46:38.580 INFO Mutect2 - For support and documentation go to https://software.broadinstitute.org/gatk/
15:46:38.580 INFO Mutect2 - Executing as huangyi@localhost.localdomain on Linux v3.10.0-1160.el7.x86_64 amd64
15:46:38.580 INFO Mutect2 - Java runtime: OpenJDK 64-Bit Server VM v1.8.0_121-b15
15:46:38.581 INFO Mutect2 - Start Date/Time: 2022年8月26日 下午03时46分33秒
15:46:38.581 INFO Mutect2 - ------------------------------------------------------------
15:46:38.581 INFO Mutect2 - ------------------------------------------------------------
15:46:38.581 INFO Mutect2 - HTSJDK Version: 2.24.1
15:46:38.581 INFO Mutect2 - Picard Version: 2.27.1
15:46:38.581 INFO Mutect2 - Built for Spark Version: 2.4.5
15:46:38.581 INFO Mutect2 - HTSJDK Defaults.COMPRESSION_LEVEL : 2
15:46:38.581 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
15:46:38.581 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
15:46:38.581 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
15:46:38.581 INFO Mutect2 - Deflater: IntelDeflater
15:46:38.581 INFO Mutect2 - Inflater: IntelInflater
15:46:38.581 INFO Mutect2 - GCS max retries/reopens: 20
15:46:38.582 INFO Mutect2 - Requester pays: disabled
15:46:38.582 INFO Mutect2 - Initializing engine
WARNING: BAM index file /home/huangyi/data/recal/W00139_2_recal.bai is older than BAM /home/huangyi/data/recal/W00139_2_recal.bam
15:46:38.953 INFO FeatureManager - Using codec VCFCodec to read file file:///home/huangyi/data/somatic-b37_Mutect2-exome-panel.vcf
15:46:39.026 INFO FeatureManager - Using codec VCFCodec to read file file:///home/huangyi/data/somatic-b37_af-only-gnomad.raw.sites.vcf
15:46:40.232 WARN IndexUtils - Feature file "file:///home/huangyi/data/somatic-b37_af-only-gnomad.raw.sites.vcf" appears to contain no sequence dictionary. Attempting to retrieve a sequence dictionary from the associated index file
15:46:41.231 WARN IndexUtils - Index file /home/huangyi/data/somatic-b37_af-only-gnomad.raw.sites.vcf.idx is out of date (index older than input file). Use IndexFeatureFile to make a new index.
15:46:41.255 INFO Mutect2 - Done initializing engine
15:46:41.278 INFO NativeLibraryLoader - Loading libgkl_utils.so from jar:file:/home/huangyi/anaconda/share/gatk4-4.2.6.1-1/gatk-package-4.2.6.1-local.jar!/com/intel/gkl/native/libgkl_utils.so
15:46:41.279 INFO NativeLibraryLoader - Loading libgkl_pairhmm_omp.so from jar:file:/home/huangyi/anaconda/share/gatk4-4.2.6.1-1/gatk-package-4.2.6.1-local.jar!/com/intel/gkl/native/libgkl_pairhmm_omp.so
15:46:41.309 INFO IntelPairHmm - Using CPU-supported AVX-512 instructions
15:46:41.309 INFO IntelPairHmm - Flush-to-zero (FTZ) is enabled when running PairHMM
15:46:41.310 INFO IntelPairHmm - Available threads: 72
15:46:41.310 INFO IntelPairHmm - Requested threads: 4
15:46:41.310 INFO PairHMM - Using the OpenMP multi-threaded AVX-accelerated native PairHMM implementation
15:46:41.373 INFO ProgressMeter - Starting traversal
15:46:41.374 INFO ProgressMeter - Current Locus Elapsed Minutes Regions Processed Regions/Minute
15:46:51.421 INFO ProgressMeter - 1:812957 0.2 3140 18753.7
15:47:02.204 INFO ProgressMeter - 1:897406 0.3 3620 10427.3
15:47:12.467 INFO ProgressMeter - 1:951245 0.5 3910 7545.1
15:47:23.035 INFO ProgressMeter - 1:987798 0.7 4100 5904.8
15:47:33.099 INFO ProgressMeter - 1:1119170 0.9 4750 5509.9
15:47:43.279 INFO ProgressMeter - 1:1201347 1.0 5230 5069.1
15:47:55.072 INFO ProgressMeter - 1:1250234 1.2 5480 4461.5
15:48:05.846 INFO ProgressMeter - 1:1278995 1.4 5610 3984.8
15:48:16.177 INFO ProgressMeter - 1:1374120 1.6 6100 3860.6
15:48:26.881 INFO ProgressMeter - 1:1445416 1.8 6470 3679.4
15:48:38.241 INFO ProgressMeter - 1:1563451 1.9 7090 3640.006:54:13.481 INFO VectorLoglessPairHMM - Time spent in setup for JNI call : 32.203864026000005
06:54:13.481 INFO PairHMM - Total compute time in PairHMM computeLogLikelihoods() : 3106.289570751
06:54:13.482 INFO SmithWatermanAligner - Total compute time in java Smith-Waterman : 9328.66 sec
06:54:13.483 INFO Mutect2 - Shutting down engine
[2022年8月27日 上午06时54分13秒] org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2 done. Elapsed time: 907.67 minutes.
Runtime.totalMemory()=14369685504
java.nio.BufferUnderflowException
at java.nio.Buffer.nextGetIndex(Buffer.java:506)
at java.nio.DirectByteBuffer.getInt(DirectByteBuffer.java:681)
at htsjdk.samtools.MemoryMappedFileBuffer.readInteger(MemoryMappedFileBuffer.java:39)
at htsjdk.samtools.AbstractBAMFileIndex.readInteger(AbstractBAMFileIndex.java:443)
at htsjdk.samtools.AbstractBAMFileIndex.query(AbstractBAMFileIndex.java:272)
at htsjdk.samtools.DiskBasedBAMFileIndex.getSpanOverlapping(DiskBasedBAMFileIndex.java:61)
at htsjdk.samtools.BAMFileReader.getFileSpan(BAMFileReader.java:914)
at htsjdk.samtools.BAMFileReader.createIndexIterator(BAMFileReader.java:931)
at htsjdk.samtools.BAMFileReader.query(BAMFileReader.java:612)
at htsjdk.samtools.SamReader$PrimitiveSamReaderToSamReaderAdapter.query(SamReader.java:550)
at htsjdk.samtools.SamReader$PrimitiveSamReaderToSamReaderAdapter.queryOverlapping(SamReader.java:417)
at org.broadinstitute.hellbender.utils.iterators.SamReaderQueryingIterator.loadNextIterator(SamReaderQueryingIterator.java:130)
at org.broadinstitute.hellbender.utils.iterators.SamReaderQueryingIterator.<init>(SamReaderQueryingIterator.java:69)
at org.broadinstitute.hellbender.engine.ReadsPathDataSource.prepareIteratorsForTraversal(ReadsPathDataSource.java:412)
at org.broadinstitute.hellbender.engine.ReadsPathDataSource.iterator(ReadsPathDataSource.java:336)
at org.broadinstitute.hellbender.engine.MultiIntervalLocalReadShard.iterator(MultiIntervalLocalReadShard.java:134)
at org.broadinstitute.hellbender.engine.AssemblyRegionIterator.<init>(AssemblyRegionIterator.java:86)
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:1085)
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)
Using GATK jar /home/huangyi/anaconda/share/gatk4-4.2.6.1-1/gatk-package-4.2.6.1-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 /home/huangyi/anaconda/share/gatk4-4.2.6.1-1/
gatk-package-4.2.6.1-local.jar Mutect2 -R /home/huangyi/data/ref/human_g1k_v37.fasta -I /home/huangyi/data/recal/W00139_recal.bam -I /home/huangyi/data/recal/W00139_2_recal.bam -I /home/huangyi/data/blood_bams/bqsr
/dedup/W00139_b.recal.bam -normal W00139_b --germline-resource /home/huangyi/data/somatic-b37_af-only-gnomad.raw.sites.vcf --panel-of-normals /home/huangyi/data/somatic-b37_Mutect2-exome-panel.vcf --f1r2-tar-gz f1r
2.tar.gz -O /home/huangyi/data/somatic.vcf.gz -
Hi Yi Huang,
Thank you for providing your program log! I appreciate your patience while we worked on this.
It appears that your code is throwing a BufferUnderflowException. This occurs when the program tries to query the index for one of your bam files. The error therefore implies a problem with your bam file indices. I also see a warning in your program log substantiating this:
WARNING: BAM index file /home/huangyi/data/recal/W00139_2_recal.bai is older than BAM /home/huangyi/data/recal/W00139_2_recal.bam
The above suggests that your bam index is out of date. You should re-index all your bam files using --samtoolsindex and try again.
It appears that your VCF indices are also out of date by this line in your program log:
IndexUtils - Index file /home/huangyi/data/somatic-b37_af-only-gnomad.raw.sites.vcf.idx is out of date (index older than input file). Use IndexFeatureFile to make a new index.
You can use GATK’s IndexFeatureFile tool to re-index your VCFs.
I hope this helps! Please let me know if you have had success after implementing these changes. Please do not hesitate to reach out if you have any other questions in the meantime.
Best,
Anthony -
Hi Yi Huang,
We haven't heard from you in a while so we're going to close out this ticket. If you still require assistance, simply respond to this email and we'll be happy to pick up where we left off!
Kind regards,
Anthony
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