GATK Muteck2 missing call
Answered
a) GATK version used: v4.2.6.1
b) Exact command used:
docker run --rm -v $(PWD):/marco broadinstitute/gatk gatk Mutect2 -R /marco/hs37d5.fa -I /marco/C6216_BM_IonCode_0129.bam -tumor sample_name -L 12:12020000-12030000 --force-active true -bamout /marco/C6216_BM_IonCode_0129_selectedALLgvcf.bam --bam-writer-type ALL_PO
SSIBLE_HAPLOTYPES -O /marco/single_sample.bpvcf.gz --emit-ref-confidence BP_RESOLUTION
c) Entire program log:
08:46:51.623 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/gatk/gatk-package-4.2.6.1-local.jar!/com/intel/gkl/native/libgkl_compression.so
08:46:52.203 INFO Mutect2 - ------------------------------------------------------------
08:46:52.204 INFO Mutect2 - The Genome Analysis Toolkit (GATK) v4.2.6.1
08:46:52.205 INFO Mutect2 - For support and documentation go to https://software.broadinstitute.org/gatk/
08:46:52.206 INFO Mutect2 - Executing as root@3730d5ed5f76 on Linux v5.10.124-linuxkit amd64
08:46:52.206 INFO Mutect2 - Java runtime: OpenJDK 64-Bit Server VM v1.8.0_242-8u242-b08-0ubuntu3~18.04-b08
08:46:52.207 INFO Mutect2 - Start Date/Time: September 23, 2022 8:46:51 AM GMT
08:46:52.207 INFO Mutect2 - ------------------------------------------------------------
08:46:52.208 INFO Mutect2 - ------------------------------------------------------------
08:46:52.212 INFO Mutect2 - HTSJDK Version: 2.24.1
08:46:52.212 INFO Mutect2 - Picard Version: 2.27.1
08:46:52.213 INFO Mutect2 - Built for Spark Version: 2.4.5
08:46:52.213 INFO Mutect2 - HTSJDK Defaults.COMPRESSION_LEVEL : 2
08:46:52.214 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
08:46:52.214 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
08:46:52.214 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
08:46:52.215 INFO Mutect2 - Deflater: IntelDeflater
08:46:52.215 INFO Mutect2 - Inflater: IntelInflater
08:46:52.215 INFO Mutect2 - GCS max retries/reopens: 20
08:46:52.215 INFO Mutect2 - Requester pays: disabled
08:46:52.216 INFO Mutect2 - Initializing engine
08:46:54.194 INFO IntervalArgumentCollection - Processing 10001 bp from intervals
08:46:54.298 INFO Mutect2 - Done initializing engine
08:46:54.824 INFO NativeLibraryLoader - Loading libgkl_utils.so from jar:file:/gatk/gatk-package-4.2.6.1-local.jar!/com/intel/gkl/native/libgkl_utils.so
08:46:54.849 INFO PairHMM - OpenMP multi-threaded AVX-accelerated native PairHMM implementation is not supported
08:46:54.850 WARN PairHMM - ***WARNING: Machine does not have the AVX instruction set support needed for the accelerated AVX PairHmm. Falling back to the MUCH slower LOGLESS_CACHING implementation!
08:46:55.050 WARN Mutect2 - Note that the Mutect2 reference confidence mode is in BETA -- the likelihoods model and output format are subject to change in subsequent versions.
08:46:55.215 INFO ProgressMeter - Starting traversal
08:46:55.218 INFO ProgressMeter - Current Locus Elapsed Minutes Regions Processed Regions/Minute
08:47:07.122 INFO ProgressMeter - 12:12022463 0.2 10 50.4
08:47:08.212 INFO Mutect2 - 1 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
3 read(s) filtered by: ReadLengthReadFilter
0 read(s) filtered by: GoodCigarReadFilter
0 read(s) filtered by: WellformedReadFilter
4 total reads filtered
08:47:08.214 INFO ProgressMeter - 12:12028214 0.2 35 161.6
08:47:08.215 INFO ProgressMeter - Traversal complete. Processed 35 total regions in 0.2 minutes.
08:47:08.349 INFO PairHMM - Total compute time in PairHMM computeLogLikelihoods() : 0.904647708
08:47:08.350 INFO SmithWatermanAligner - Total compute time in java Smith-Waterman : 0.29 sec
08:47:08.543 INFO Mutect2 - Shutting down engine
[September 23, 2022 8:47:08 AM GMT] org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2 done. Elapsed time: 0.28 minutes.
Runtime.totalMemory()=370671616
Tool returned:
SUCCESS
Using GATK jar /gatk/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 /gatk/gatk-package-4.2.6.1-local.jar Mutect2 -R /marco/hs37d5.fa -I /marco/C6216_BM_IonCode_0129.bam
-tumor sample_name -L 12:12020000-12030000 --force-active true -bamout /marco/C6216_BM_IonCode_0129_selectedALLgvcf.bam --bam-writer-type ALL_POSSIBLE_HAPLOTYPES -O /marco/single_sample.bpvcf.gz --emit-ref-confidence BP_RESOLUTION
I am looking for ETV6 gene mutation in chr12:12022496. I can see the variant in bam file and also in Mutect2 bamout file. Below you can see a screenshot of IGV. Above you have the original bam file, below the bamout file produced by Mutect2.
I can also see the variat in GVCF file (highlighted in yellow). Even if it is not called, the AD shows the presence of alt nucleotide and the TLOD is 61.
Could you, please, explain me why this variant is not called?
I already ready the post about "Mutect2 do not call an expected variant" and it did not helped.
Thank you,
marco
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Hi Marco Chiapello,
Thank you for writing to the GATK forum! I hope that we can help you sort this out.
I brought the issue you encountered to our developers and received some feedback.
The team first noticed that you are running Mutect2 in gvcf-mode, which they created for rare instances of variant-calling in mitochondria. Even then, our users mostly use gvcf-mode for edge cases. Secondly, you are running the tool without a Panel of Normals (PoN). We strongly recommend running Mutect2 with a PoN containing at least 50 samples.
That said, there are a couple of action items before we can move forward with diagnosing your problem.
- Please confirm that the command line you included is the entire command you used when running the tool.
- Please try re-running Mutect2 again in regular vcf-mode.
If you are still having trouble after re-running, please respond with your full command line, and I will bring it back to our developers to figure out our next steps.
Best,
Anthony
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Hi Marco Chiapello,
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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