We have observed several situations where Mutect 2 has failed to report a known variant from a gene panel based on IDT probes.

The latest example is JAK2 1849G>T Val617Phe GRCh38 chr9:5073770-5073770, from the Horizon Myeloid DNA Reference Standard (nominal variant allele frequency 5.0%).

Mutect2 correctly reports this variant with a 100 ng sample, but omits it with a 25 ng sample.

Varscan2 reports the variant with both sample loadings.

The same behavior happens when b37 is the reference.

From IGV, the T variant in the 25 ng run is present in 47/1627 reads (3%, 26+, 21-).  There do not appear to be any significant nearby other variants. In the successful 100 ng run the variant was observed in 103/2494 reads (4.1%).

The variant does not seem to have been filtered out by post-Mutect2 processing: it is not present in the unfiltered VCF created by Mutect2.

Is there a way to identify the cause of this false negative? A setting that could improve Mutect2 sensitivity?

Can you please provide
a) GATK version used
b) Exact GATK commands used
c) The entire error log if applicable.

##GATKCommandLine=<ID=Mutect2,CommandLine="Mutect2 --f1r2-tar-gz f1r2.tar.gz --tumor-sample HDxMye_25_P --panel-of-normals /gpfs/share/cmoco_sys_dev/nfs/storage/resources/b37/gatk-best-practices/Mutect2-exome-panel.vcf --germline-resource /gpfs/share/cmoco_sys_dev/nfs/storage/resources/b37/gatk-best-practices/af-only-gnomad.raw.sites.vcf --independent-mates true --bam-output bamout.bam --output output.vcf --intervals /gpfs/share/cmoco_sys_dev/nfs/storage/cromwell/cromwell-executions/somatic_snvs_indels/a37f5774-9013-4c15-ac6e-a36c58b76be8/call-VariantCalling/shard-1/VariantCalling/63805b1a-afec-4598-88e4-85512b18d482/call-SplitIntervals/execution/glob-0fc990c5ca95eebc97c4c204e3e303e1/0022-scattered.interval_list --input /gpfs/share/cmoco_sys_dev/nfs/storage/cromwell/cromwell-executions/somatic_snvs_indels/a37f5774-9013-4c15-ac6e-a36c58b76be8/call-DataPreProcessing/data_preprocessing/0f449f93-9d54-4ecb-9db5-0769daea364e/call-BamProcessing/shard-1/bam_processing/31d97dfd-139b-4109-9940-016ea60cccf6/call-ApplyBQSR/execution/HDxMye_25_P@06172020JH_HEME.b37.aligned.duplicates_marked.sorted.recalibrated.bam --reference /gpfs/share/cmoco_sys_dev/nfs/storage/resources/b37/reference/human_g1k_v37_decoy_GOAL+viral.fasta --f1r2-median-mq 50 --f1r2-min-bq 20 --f1r2-max-depth 200 --genotype-pon-sites false --genotype-germline-sites false --af-of-alleles-not-in-resource -1.0 --mitochondria-mode false --tumor-lod-to-emit 3.0 --initial-tumor-lod 2.0 --pcr-snv-qual 40 --pcr-indel-qual 40 --max-population-af 0.01 --downsampling-stride 1 --callable-depth 10 --max-suspicious-reads-per-alignment-start 0 --normal-lod 2.2 --ignore-itr-artifacts false --gvcf-lod-band -2.5 --gvcf-lod-band -2.0 --gvcf-lod-band -1.5 --gvcf-lod-band -1.0 --gvcf-lod-band -0.5 --gvcf-lod-band 0.0 --gvcf-lod-band 0.5 --gvcf-lod-band 1.0 --minimum-allele-fraction 0.0 --disable-adaptive-pruning false --dont-trim-active-regions false --max-extension 25 --padding-around-indels 150 --padding-around-snps 20 --kmer-size 10 --kmer-size 25 --dont-increase-kmer-sizes-for-cycles false --allow-non-unique-kmers-in-ref false --num-pruning-samples 1 --min-dangling-branch-length 4 --recover-all-dangling-branches false --max-num-haplotypes-in-population 128 --min-pruning 2 --adaptive-pruning-initial-error-rate 0.001 --pruning-lod-threshold 2.302585092994046 --max-unpruned-variants 100 --linked-de-bruijn-graph false --debug-assembly false --debug-graph-transformations false --capture-assembly-failure-bam false --error-correct-reads false --kmer-length-for-read-error-correction 25 --min-observations-for-kmer-to-be-solid 20 --likelihood-calculation-engine PairHMM --base-quality-score-threshold 18 --pair-hmm-gap-continuation-penalty 10 --pair-hmm-implementation FASTEST_AVAILABLE --pcr-indel-model CONSERVATIVE --phred-scaled-global-read-mismapping-rate 45 --native-pair-hmm-threads 4 --native-pair-hmm-use-double-precision false --bam-writer-type CALLED_HAPLOTYPES --dont-use-soft-clipped-bases false --min-base-quality-score 10 --smith-waterman JAVA --emit-ref-confidence NONE --max-mnp-distance 1 --force-call-filtered-alleles false --min-assembly-region-size 50 --max-assembly-region-size 300 --assembly-region-padding 100 --max-reads-per-alignment-start 50 --active-probability-threshold 0.002 --max-prob-propagation-distance 50 --force-active false --interval-set-rule UNION --interval-padding 0 --interval-exclusion-padding 0 --interval-merging-rule ALL --read-validation-stringency SILENT --seconds-between-progress-updates 10.0 --disable-sequence-dictionary-validation false --create-output-bam-index true --create-output-bam-md5 false --create-output-variant-index true --create-output-variant-md5 false --lenient false --add-output-sam-program-record true --add-output-vcf-command-line true --cloud-prefetch-buffer 40 --cloud-index-prefetch-buffer -1 --disable-bam-index-caching false --sites-only-vcf-output false --help false --version false --showHidden false --verbosity INFO --QUIET false --use-jdk-deflater false --use-jdk-inflater false --gcs-max-retries 20 --gcs-project-for-requester-pays --disable-tool-default-read-filters false --max-read-length 2147483647 --min-read-length 30 --minimum-mapping-quality 20 --disable-tool-default-annotations false --enable-all-annotations false",Version="4.1.4.1",Date="June 25, 2020 1:33:30 AM UTC">

stderr:

cmoco-sys-dev-web:call-M2$ cat shard-22/execution/stderr
Picked up _JAVA_OPTIONS: -Djava.io.tmpdir=/gpfs/share/cmoco_sys_dev/nfs/storage/cromwell/cromwell-executions/somatic_snvs_indels/a37f5774-9013-4c15-ac6e-a36c58b76be8/call-VariantCalling/shard-1/VariantCalling/63805b1a-afec-4598-88e4-85512b18d482/call-M2/shard-22/tmp.59047bb6
01:33:18.285 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/opt/gatk-4.1.4.1/gatk-package-4.1.4.1-local.jar!/com/intel/gkl/native/libgkl_compression.so
Jun 25, 2020 1:33:19 AM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
01:33:19.322 INFO GetSampleName - ------------------------------------------------------------
01:33:19.323 INFO GetSampleName - The Genome Analysis Toolkit (GATK) v4.1.4.1
01:33:19.323 INFO GetSampleName - For support and documentation go to https://software.broadinstitute.org/gatk/
01:33:19.323 INFO GetSampleName - Executing as cmoco_sys_dev@cubipmcmp006 on Linux v3.10.0-1062.12.1.el7.x86_64 amd64
01:33:19.323 INFO GetSampleName - Java runtime: OpenJDK 64-Bit Server VM v1.8.0_252-b09
01:33:19.323 INFO GetSampleName - Start Date/Time: June 25, 2020 1:33:18 AM UTC
01:33:19.323 INFO GetSampleName - ------------------------------------------------------------
01:33:19.323 INFO GetSampleName - ------------------------------------------------------------
01:33:19.325 INFO GetSampleName - HTSJDK Version: 2.21.0
01:33:19.325 INFO GetSampleName - Picard Version: 2.21.2
01:33:19.325 INFO GetSampleName - HTSJDK Defaults.COMPRESSION_LEVEL : 2
01:33:19.325 INFO GetSampleName - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
01:33:19.325 INFO GetSampleName - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
01:33:19.325 INFO GetSampleName - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
01:33:19.325 INFO GetSampleName - Deflater: IntelDeflater
01:33:19.338 INFO GetSampleName - Inflater: IntelInflater
01:33:19.338 INFO GetSampleName - GCS max retries/reopens: 20
01:33:19.338 INFO GetSampleName - Requester pays: disabled
01:33:19.338 INFO GetSampleName - Initializing engine
01:33:19.798 INFO GetSampleName - Done initializing engine
01:33:19.802 INFO ProgressMeter - Starting traversal
01:33:19.802 INFO ProgressMeter - Current Locus Elapsed Minutes Records Processed Records/Minute
01:33:19.803 INFO ProgressMeter - unmapped 0.0 0 NaN
01:33:19.803 INFO ProgressMeter - Traversal complete. Processed 0 total records in 0.0 minutes.
01:33:19.803 INFO GetSampleName - Shutting down engine
[June 25, 2020 1:33:19 AM UTC] org.broadinstitute.hellbender.tools.GetSampleName done. Elapsed time: 0.03 minutes.
Runtime.totalMemory()=2039545856
Using GATK jar /opt/gatk-4.1.4.1/gatk-package-4.1.4.1-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 -Xmx3000m -jar /opt/gatk-4.1.4.1/gatk-package-4.1.4.1-local.jar GetSampleName -R /gpfs/share/cmoco_sys_dev/nfs/storage/resources/b37/reference/human_g1k_v37_decoy_GOAL+viral.fasta -I /gpfs/share/cmoco_sys_dev/nfs/storage/cromwell/cromwell-executions/somatic_snvs_indels/a37f5774-9013-4c15-ac6e-a36c58b76be8/call-DataPreProcessing/data_preprocessing/0f449f93-9d54-4ecb-9db5-0769daea364e/call-BamProcessing/shard-1/bam_processing/31d97dfd-139b-4109-9940-016ea60cccf6/call-ApplyBQSR/execution/HDxMye_25_P@06172020JH_HEME.b37.aligned.duplicates_marked.sorted.recalibrated.bam -O tumor_name.txt -encode
Picked up _JAVA_OPTIONS: -Djava.io.tmpdir=/gpfs/share/cmoco_sys_dev/nfs/storage/cromwell/cromwell-executions/somatic_snvs_indels/a37f5774-9013-4c15-ac6e-a36c58b76be8/call-VariantCalling/shard-1/VariantCalling/63805b1a-afec-4598-88e4-85512b18d482/call-M2/shard-22/tmp.59047bb6
01:33:24.697 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/opt/gatk-4.1.4.1/gatk-package-4.1.4.1-local.jar!/com/intel/gkl/native/libgkl_compression.so
Jun 25, 2020 1:33:25 AM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
01:33:25.243 INFO Mutect2 - ------------------------------------------------------------
01:33:25.244 INFO Mutect2 - The Genome Analysis Toolkit (GATK) v4.1.4.1
01:33:25.244 INFO Mutect2 - For support and documentation go to https://software.broadinstitute.org/gatk/
01:33:25.244 INFO Mutect2 - Executing as cmoco_sys_dev@cubipmcmp006 on Linux v3.10.0-1062.12.1.el7.x86_64 amd64
01:33:25.244 INFO Mutect2 - Java runtime: OpenJDK 64-Bit Server VM v1.8.0_252-b09
01:33:25.245 INFO Mutect2 - Start Date/Time: June 25, 2020 1:33:24 AM UTC
01:33:25.245 INFO Mutect2 - ------------------------------------------------------------
01:33:25.245 INFO Mutect2 - ------------------------------------------------------------
01:33:25.245 INFO Mutect2 - HTSJDK Version: 2.21.0
01:33:25.245 INFO Mutect2 - Picard Version: 2.21.2
01:33:25.245 INFO Mutect2 - HTSJDK Defaults.COMPRESSION_LEVEL : 2
01:33:25.245 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
01:33:25.246 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
01:33:25.246 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
01:33:25.246 INFO Mutect2 - Deflater: IntelDeflater
01:33:25.246 INFO Mutect2 - Inflater: IntelInflater
01:33:25.246 INFO Mutect2 - GCS max retries/reopens: 20
01:33:25.246 INFO Mutect2 - Requester pays: disabled
01:33:25.246 INFO Mutect2 - Initializing engine
01:33:25.905 INFO FeatureManager - Using codec VCFCodec to read file file:///gpfs/share/cmoco_sys_dev/nfs/storage/resources/b37/gatk-best-practices/Mutect2-exome-panel.vcf
01:33:26.087 INFO FeatureManager - Using codec VCFCodec to read file file:///gpfs/share/cmoco_sys_dev/nfs/storage/resources/b37/gatk-best-practices/af-only-gnomad.raw.sites.vcf
01:33:28.601 INFO FeatureManager - Using codec IntervalListCodec to read file file:///gpfs/share/cmoco_sys_dev/nfs/storage/cromwell/cromwell-executions/somatic_snvs_indels/a37f5774-9013-4c15-ac6e-a36c58b76be8/call-VariantCalling/shard-1/VariantCalling/63805b1a-afec-4598-88e4-85512b18d482/call-SplitIntervals/execution/glob-0fc990c5ca95eebc97c4c204e3e303e1/0022-scattered.interval_list
01:33:28.680 INFO IntervalArgumentCollection - Processing 19606 bp from intervals
01:33:28.691 WARN IndexUtils - Feature file "/gpfs/share/cmoco_sys_dev/nfs/storage/resources/b37/gatk-best-practices/af-only-gnomad.raw.sites.vcf" appears to contain no sequence dictionary. Attempting to retrieve a sequence dictionary from the associated index file
01:33:30.126 WARN IndexUtils - Index file /gpfs/share/cmoco_sys_dev/nfs/storage/resources/b37/gatk-best-practices/af-only-gnomad.raw.sites.vcf.idx is out of date (index older than input file). Use IndexFeatureFile to make a new index.
01:33:30.155 INFO Mutect2 - Done initializing engine
01:33:30.210 INFO NativeLibraryLoader - Loading libgkl_utils.so from jar:file:/opt/gatk-4.1.4.1/gatk-package-4.1.4.1-local.jar!/com/intel/gkl/native/libgkl_utils.so
01:33:30.221 INFO NativeLibraryLoader - Loading libgkl_pairhmm_omp.so from jar:file:/opt/gatk-4.1.4.1/gatk-package-4.1.4.1-local.jar!/com/intel/gkl/native/libgkl_pairhmm_omp.so
01:33:30.286 INFO IntelPairHmm - Flush-to-zero (FTZ) is enabled when running PairHMM
01:33:30.287 INFO IntelPairHmm - Available threads: 1
01:33:30.287 INFO IntelPairHmm - Requested threads: 4
01:33:30.287 WARN IntelPairHmm - Using 1 available threads, but 4 were requested
01:33:30.287 INFO PairHMM - Using the OpenMP multi-threaded AVX-accelerated native PairHMM implementation
01:33:30.555 INFO ProgressMeter - Starting traversal
01:33:30.555 INFO ProgressMeter - Current Locus Elapsed Minutes Regions Processed Regions/Minute
01:33:45.828 INFO ProgressMeter - 8:103301671 0.3 10 39.3
01:34:03.106 INFO ProgressMeter - 8:103326107 0.5 30 55.3
01:34:28.123 INFO ProgressMeter - 8:103354807 1.0 40 41.7
01:35:00.547 INFO ProgressMeter - 8:117868875 1.5 60 40.0
01:35:44.373 INFO ProgressMeter - 8:128750559 2.2 70 31.4
01:36:42.613 INFO ProgressMeter - 9:2039883 3.2 80 25.0
01:37:00.023 INFO ProgressMeter - 9:2073202 3.5 90 25.8
01:37:19.691 INFO ProgressMeter - 9:2086829 3.8 100 26.2
01:37:41.552 INFO ProgressMeter - 9:2186086 4.2 120 28.7
01:37:58.694 INFO ProgressMeter - 9:5073688 4.5 140 31.3
01:38:00.233 INFO Mutect2 - 286972 read(s) filtered by: (((((((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappingQualityNotZeroReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonChimericOriginalAlignmentReadFilter) AND NonZeroReferenceLengthAlignmentReadFilter) AND ReadLengthReadFilter) AND GoodCigarReadFilter) AND WellformedReadFilter)
286972 read(s) filtered by: ((((((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappingQualityNotZeroReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonChimericOriginalAlignmentReadFilter) AND NonZeroReferenceLengthAlignmentReadFilter) AND ReadLengthReadFilter) AND GoodCigarReadFilter)
286972 read(s) filtered by: (((((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappingQualityNotZeroReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonChimericOriginalAlignmentReadFilter) AND NonZeroReferenceLengthAlignmentReadFilter) AND ReadLengthReadFilter)
286432 read(s) filtered by: ((((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappingQualityNotZeroReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonChimericOriginalAlignmentReadFilter) AND NonZeroReferenceLengthAlignmentReadFilter)
286432 read(s) filtered by: (((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappingQualityNotZeroReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonChimericOriginalAlignmentReadFilter)
286432 read(s) filtered by: ((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappingQualityNotZeroReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter)
286432 read(s) filtered by: (((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappingQualityNotZeroReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter)
11653 read(s) filtered by: ((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappingQualityNotZeroReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter)
11653 read(s) filtered by: (((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappingQualityNotZeroReadFilter) AND MappedReadFilter)
11653 read(s) filtered by: ((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappingQualityNotZeroReadFilter)
11653 read(s) filtered by: (MappingQualityReadFilter AND MappingQualityAvailableReadFilter)
11653 read(s) filtered by: MappingQualityReadFilter
274779 read(s) filtered by: NotDuplicateReadFilter
540 read(s) filtered by: ReadLengthReadFilter

01:38:00.233 INFO ProgressMeter - 9:5073688 4.5 149 33.2
01:38:00.234 INFO ProgressMeter - Traversal complete. Processed 149 total regions in 4.5 minutes.
01:38:00.578 INFO VectorLoglessPairHMM - Time spent in setup for JNI call : 0.178354284
01:38:00.579 INFO PairHMM - Total compute time in PairHMM computeLogLikelihoods() : 153.226857488
01:38:00.579 INFO SmithWatermanAligner - Total compute time in java Smith-Waterman : 3.54 sec
01:38:01.677 INFO Mutect2 - Shutting down engine
[June 25, 2020 1:38:01 AM UTC] org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2 done. Elapsed time: 4.62 minutes.
Runtime.totalMemory()=2039545856
Using GATK jar /opt/gatk-4.1.4.1/gatk-package-4.1.4.1-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 -Xmx3000m -jar /opt/gatk-4.1.4.1/gatk-package-4.1.4.1-local.jar Mutect2 --independent-mates true -R /gpfs/share/cmoco_sys_dev/nfs/storage/resources/b37/reference/human_g1k_v37_decoy_GOAL+viral.fasta -I /gpfs/share/cmoco_sys_dev/nfs/storage/cromwell/cromwell-executions/somatic_snvs_indels/a37f5774-9013-4c15-ac6e-a36c58b76be8/call-DataPreProcessing/data_preprocessing/0f449f93-9d54-4ecb-9db5-0769daea364e/call-BamProcessing/shard-1/bam_processing/31d97dfd-139b-4109-9940-016ea60cccf6/call-ApplyBQSR/execution/HDxMye_25_P@06172020JH_HEME.b37.aligned.duplicates_marked.sorted.recalibrated.bam -tumor HDxMye_25_P --germline-resource /gpfs/share/cmoco_sys_dev/nfs/storage/resources/b37/gatk-best-practices/af-only-gnomad.raw.sites.vcf -pon /gpfs/share/cmoco_sys_dev/nfs/storage/resources/b37/gatk-best-practices/Mutect2-exome-panel.vcf -L /gpfs/share/cmoco_sys_dev/nfs/storage/cromwell/cromwell-executions/somatic_snvs_indels/a37f5774-9013-4c15-ac6e-a36c58b76be8/call-VariantCalling/shard-1/VariantCalling/63805b1a-afec-4598-88e4-85512b18d482/call-SplitIntervals/execution/glob-0fc990c5ca95eebc97c4c204e3e303e1/0022-scattered.interval_list -O output.vcf --bam-output bamout.bam --f1r2-tar-gz f1r2.tar.gz
cmoco-sys-dev-web:call-M2$