Dear community,

I am performing somatic variant calling using Mutect2 (GATK version 4.1.5) on synthetic cell-free DNA data of a small tumor panel (8kb). Data is deeply sequenced (~50000x) and reads are UMI-consensed and filtered for high-quality reads only (mapping quality >30 and 2+ reads per consensus).

As the data is synthetic. I a priori know of certain variants to be discovered. Some of these, however, are not found, also previous to mutect filtering. When I investigate those missed variants with the --alleles setting providing a vcf with the expected variants, I surprisising get most of the missed variants with the label 'PASS'. DP_tumor and VAF are in the same range as other variants that are detected by mutect.

Does anyone have any suggestion, how to get more clues on what is going on and why I miss these variants?

Regards,

Daniel

#----

# Example variant picked up by forced allele detection

chr7 55259515 . T G . . DP=5838;ECNT=1;MBQ=20,20;MFRL=161,162;MMQ=60,60;MPOS=30;POPAF=7.30;TLOD=94.02 GT:AD:AF:DP:F1R2:F2R1:SB 0/1:5630,68:0.012:5698:3738,46:1892,22:2941,2689,34,34

mutect call:

java -jar /software/opensource/gatk/gatk-4.1.5.0/gatk-package-4.1.5.0-local.jar Mutect2 -R /lib/genomes/human/GRCh37.p5/hs_ref_GRCh37.p5_all_contigs.fa -I filtered_mapped_consensus.bam --germline-resource /data/germlineResource/full/germlineResource_gnomad-r2.0.2_grch37_full.recode.vcf.gz --panel-of-normals /panelOfNormals/pon.vcf.gz -O mutect.vcf -L /data/lib/targetFiles/Panel_codingregion.sorted.bed --genotype-pon-sites true --genotype-germline-sites true --bam-output mutect_assembled.bam --create-output-bam-index --f1r2-tar-gz f1r2.tar.gz --native-pair-hmm-threads 16 -alleles vcfForce/Expected.vcf

stdout:

17:20:03.083 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/software/opensource/gatk/gatk-4.1.5.0/gatk-package-4.1.5.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
Apr 03, 2020 5:20:03 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
17:20:03.299 INFO Mutect2 - ------------------------------------------------------------
17:20:03.300 INFO Mutect2 - The Genome Analysis Toolkit (GATK) v4.1.5.0
17:20:03.300 INFO Mutect2 - For support and documentation go to https://software.broadinstitute.org/gatk/
17:20:03.300 INFO Mutect2 - Executing as daniel@node61 on Linux v3.10.0-514.6.1.el7.x86_64 amd64
17:20:03.300 INFO Mutect2 - Java runtime: Java HotSpot(TM) 64-Bit Server VM v1.8.0_65-b17
17:20:03.300 INFO Mutect2 - Start Date/Time: April 3, 2020 5:20:03 PM CEST
17:20:03.300 INFO Mutect2 - ------------------------------------------------------------
17:20:03.300 INFO Mutect2 - ------------------------------------------------------------
17:20:03.301 INFO Mutect2 - HTSJDK Version: 2.21.2
17:20:03.301 INFO Mutect2 - Picard Version: 2.21.9
17:20:03.301 INFO Mutect2 - HTSJDK Defaults.COMPRESSION_LEVEL : 2
17:20:03.301 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
17:20:03.301 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
17:20:03.301 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
17:20:03.301 INFO Mutect2 - Deflater: IntelDeflater
17:20:03.301 INFO Mutect2 - Inflater: IntelInflater
17:20:03.301 INFO Mutect2 - GCS max retries/reopens: 20
17:20:03.301 INFO Mutect2 - Requester pays: disabled
17:20:03.301 INFO Mutect2 - Initializing engine
17:20:03.669 INFO FeatureManager - Using codec VCFCodec to read file file:///data/panelOfNormals/pon.vcf.gz
17:20:03.755 INFO FeatureManager - Using codec VCFCodec to read file file:///data/research/projects/erik/Mutect2/germlineResource/full/germlineResource_gnomad-r2.0.2_grch37_full.recode.vcf.gz
17:20:03.841 INFO FeatureManager - Using codec VCFCodec to read file file:///data/research/projects/ltg/umi/vcfForce/expected.vcf
17:20:03.911 INFO FeatureManager - Using codec BEDCodec to read file file:///data/lib/targetFiles/Panel.sorted.bed
17:20:03.926 INFO IntervalArgumentCollection - Processing 82278 bp from intervals
17:20:03.932 WARN IndexUtils - Feature file "/data/germlineResource/full/germlineResource_gnomad-r2.0.2_grch37_full.recode.vcf.gz" appears to contain no sequence dictionary. Attempting to retrieve a sequence dictionary from the associated index file
17:20:03.982 WARN IndexUtils - Feature file "/data/research/projects/ltg/umi/vcfForce/SeraCarev2-1.vcf" appears to contain no sequence dictionary. Attempting to retrieve a sequence dictionary from the associated index file
17:20:04.002 INFO Mutect2 - Done initializing engine
17:20:04.029 INFO NativeLibraryLoader - Loading libgkl_utils.so from jar:file:/software/opensource/gatk/gatk-4.1.5.0/gatk-package-4.1.5.0-local.jar!/com/intel/gkl/native/libgkl_utils.so
17:20:04.044 INFO NativeLibraryLoader - Loading libgkl_pairhmm_omp.so from jar:file:/software/opensource/gatk/gatk-4.1.5.0/gatk-package-4.1.5.0-local.jar!/com/intel/gkl/native/libgkl_pairhmm_omp.so
17:20:04.120 INFO IntelPairHmm - Using CPU-supported AVX-512 instructions
17:20:04.120 INFO IntelPairHmm - Flush-to-zero (FTZ) is enabled when running PairHMM
17:20:04.121 INFO IntelPairHmm - Available threads: 16
17:20:04.121 INFO IntelPairHmm - Requested threads: 16
17:20:04.121 INFO PairHMM - Using the OpenMP multi-threaded AVX-accelerated native PairHMM implementation
17:20:04.229 INFO ProgressMeter - Starting traversal
17:20:04.229 INFO ProgressMeter - Current Locus Elapsed Minutes Regions Processed Regions/Minute
17:20:22.798 INFO ProgressMeter - chr1:162745672 0.3 20 64.6
17:20:39.109 INFO ProgressMeter - chr2:29446180 0.6 40 68.8
17:20:49.871 INFO ProgressMeter - chr2:29447264 0.8 50 65.7
17:21:04.288 INFO ProgressMeter - chr2:29448661 1.0 60 59.9
17:21:21.525 INFO ProgressMeter - chr3:178916877 1.3 80 62.1
17:21:32.742 INFO ProgressMeter - chr4:1805631 1.5 100 67.8
17:21:48.644 INFO ProgressMeter - chr4:55146557 1.7 120 69.0
17:22:03.870 INFO ProgressMeter - chr4:55598121 2.0 140 70.2
17:22:19.800 INFO ProgressMeter - chr6:117641377 2.3 160 70.8
17:22:37.368 INFO ProgressMeter - chr6:117644371 2.6 180 70.5
17:22:49.762 INFO ProgressMeter - chr6:117646381 2.8 190 68.9
17:23:07.982 INFO ProgressMeter - chr6:117649053 3.1 210 68.6
17:23:18.522 INFO ProgressMeter - chr6:117650557 3.2 220 67.9
17:23:28.569 INFO ProgressMeter - chr6:117652306 3.4 230 67.5
17:23:39.424 INFO ProgressMeter - chr6:117657275 3.6 260 72.5
17:23:55.398 INFO ProgressMeter - chr7:55224426 3.9 280 72.7
17:24:08.220 INFO ProgressMeter - chr7:55240676 4.1 300 73.8
17:24:19.150 INFO ProgressMeter - chr7:55259346 4.2 310 73.0
17:24:41.120 INFO ProgressMeter - chr7:116340339 4.6 330 71.5
17:25:02.001 INFO ProgressMeter - chr7:116413593 5.0 350 70.5
17:25:16.764 INFO ProgressMeter - chr7:116435806 5.2 370 71.0
17:25:33.125 INFO ProgressMeter - chr8:38275340 5.5 390 71.1
17:25:43.677 INFO ProgressMeter - chr8:38276916 5.7 400 70.7
17:26:00.535 INFO ProgressMeter - chr9:21994396 5.9 420 70.7
17:26:14.222 INFO ProgressMeter - chr10:43606725 6.2 430 69.7
17:26:25.833 INFO ProgressMeter - chr10:43607805 6.4 440 69.2
17:26:38.942 INFO ProgressMeter - chr10:43609752 6.6 450 68.4
17:27:02.990 INFO ProgressMeter - chr10:43611946 7.0 470 67.3
17:27:14.495 INFO ProgressMeter - chr10:89720870 7.2 490 68.3
17:27:25.711 INFO ProgressMeter - chr10:123298121 7.4 510 69.3
17:27:39.729 INFO ProgressMeter - chr15:66729153 7.6 530 69.8
17:27:55.872 INFO ProgressMeter - chr17:7577402 7.9 550 70.0
17:28:14.530 INFO ProgressMeter - chr17:7579212 8.2 560 68.5
17:28:27.364 INFO ProgressMeter - chr17:29508738 8.4 580 69.2
17:28:44.326 INFO ProgressMeter - chr17:29559718 8.7 610 70.4
17:28:57.718 INFO ProgressMeter - chr17:29657523 8.9 630 70.9
17:29:08.451 INFO ProgressMeter - chr17:29679275 9.1 650 71.7
17:29:21.810 INFO ProgressMeter - chr17:37880919 9.3 670 72.1
17:29:36.771 INFO ProgressMeter - chr19:3118905 9.5 690 72.3
17:29:48.186 INFO ProgressMeter - chr19:10610071 9.7 700 71.9
17:29:53.456 INFO Mutect2 - 193 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
2 read(s) filtered by: ReadLengthReadFilter
0 read(s) filtered by: GoodCigarReadFilter
0 read(s) filtered by: WellformedReadFilter
195 total reads filtered
17:29:53.457 INFO ProgressMeter - chrX:47426012 9.8 712 72.5
17:29:53.457 INFO ProgressMeter - Traversal complete. Processed 712 total regions in 9.8 minutes.
17:29:53.780 INFO VectorLoglessPairHMM - Time spent in setup for JNI call : 0.951972232
17:29:53.780 INFO PairHMM - Total compute time in PairHMM computeLogLikelihoods() : 12.689908943
17:29:53.781 INFO SmithWatermanAligner - Total compute time in java Smith-Waterman : 7.55 sec
17:29:58.568 INFO Mutect2 - Shutting down engine
[April 3, 2020 5:29:58 PM CEST] org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2 done. Elapsed time: 9.93 minutes.
Runtime.totalMemory()=10382999552