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Problems with the normal bam file in mutect2 and getpileupsummaries

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Estel

Good morning,

I'm following the tutorial for the somatic variant calling, and it works fine with the only tumor mode but I tried to use mutect2 with a normal bam sample that I extract from ENCODE and the output has 0 variants, and when I tried it with GetPileupSummaries I haven't had any output. 

The GATK version is v4.2.3

This are the commands that I use it for mutect2:

gatk Mutect2 -R /mnt/beegfs/eferre/bin/index/somatic_var_gen/Homo_sapiens_assembly38.fasta \
-I inputs/bam_files/tumor_H3K27ac.bam -I inputs/bam_files/normal_H3K27ac.bam \
-normal H000H3K27acX1 -pon inputs/files/somatic-hg38_1000g_pon.hg38.vcf \
--germline-resource inputs/files/somatic-hg38_af-only-gnomad.hg38.vcf \
-O outputs/mutect2/somatic_tumor_normal_H3K27ac_m2.vcf \
-bamout outputs/mutect2/tumor_nomral_H3K27ac_m2.bam

And the command for GetPileupSummaries:

gatk GetPileupSummaries -I inputs/bam_files/normal_H3K27ac.bam \
-V inputs/files/somatic-hg38_small_exac_common_3.hg38.vcf \
-L inputs/files/somatic-hg38_small_exac_common_3.hg38.vcf \
-O outputs/estimate_contmaination/normal_H3K27ac_getpileupsummaries.table

And this are the log files:

mutect2:

16:37:42.345 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/opt/software/GATK/4.2.3.0-foss-2021b-Java-11/gatk-package-4.2.3.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
Oct 11, 2023 4:37:42 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
16:37:42.621 INFO  Mutect2 - ------------------------------------------------------------
16:37:42.621 INFO  Mutect2 - The Genome Analysis Toolkit (GATK) v4.2.3.0
16:37:42.622 INFO  Mutect2 - For support and documentation go to https://software.broadinstitute.org/gatk/
16:37:42.622 INFO  Mutect2 - Executing as eferre@c01 on Linux v3.10.0-1160.53.1.el7.x86_64 amd64
16:37:42.623 INFO  Mutect2 - Java runtime: OpenJDK 64-Bit Server VM v11.0.2+9
16:37:42.623 INFO  Mutect2 - Start Date/Time: October 11, 2023 at 4:37:42 PM CEST
16:37:42.623 INFO  Mutect2 - ------------------------------------------------------------
16:37:42.623 INFO  Mutect2 - ------------------------------------------------------------
16:37:42.623 INFO  Mutect2 - HTSJDK Version: 2.24.1
16:37:42.624 INFO  Mutect2 - Picard Version: 2.25.4
16:37:42.624 INFO  Mutect2 - Built for Spark Version: 2.4.5
16:37:42.624 INFO  Mutect2 - HTSJDK Defaults.COMPRESSION_LEVEL : 2
16:37:42.624 INFO  Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
16:37:42.624 INFO  Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
16:37:42.624 INFO  Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
16:37:42.624 INFO  Mutect2 - Deflater: IntelDeflater
16:37:42.624 INFO  Mutect2 - Inflater: IntelInflater
16:37:42.624 INFO  Mutect2 - GCS max retries/reopens: 20
16:37:42.630 INFO  Mutect2 - Requester pays: disabled
16:37:42.630 INFO  Mutect2 - Initializing engine
16:37:43.418 INFO  FeatureManager - Using codec VCFCodec to read file file:///mnt/beegfs/eferre/somatic_variants/inputs/files/somatic-hg38_1000g_pon.hg38.vcf
16:37:43.690 INFO  FeatureManager - Using codec VCFCodec to read file file:///mnt/beegfs/eferre/somatic_variants/inputs/files/somatic-hg38_af-only-gnomad.hg38.vcf
16:37:45.755 INFO  Mutect2 - Done initializing engine
16:37:45.927 INFO  NativeLibraryLoader - Loading libgkl_utils.so from jar:file:/opt/software/GATK/4.2.3.0-foss-2021b-Java-11/gatk-package-4.2.3.0-local.jar!/com/intel/gkl/native/libgkl_utils.so
16:37:45.943 INFO  NativeLibraryLoader - Loading libgkl_pairhmm_omp.so from jar:file:/opt/software/GATK/4.2.3.0-foss-2021b-Java-11/gatk-package-4.2.3.0-local.jar!/com/intel/gkl/native/libgkl_pairhmm_omp.so
16:37:45.976 INFO  IntelPairHmm - Flush-to-zero (FTZ) is enabled when running PairHMM
16:37:45.976 INFO  IntelPairHmm - Available threads: 1
16:37:45.976 INFO  IntelPairHmm - Requested threads: 4
16:37:45.980 WARN  IntelPairHmm - Using 1 available threads, but 4 were requested
16:37:45.980 INFO  PairHMM - Using the OpenMP multi-threaded AVX-accelerated native PairHMM implementation
16:37:46.107 INFO  ProgressMeter - Starting traversal
16:37:46.107 INFO  ProgressMeter -        Current Locus  Elapsed Minutes     Regions Processed   Regions/Minute
16:37:56.108 INFO  ProgressMeter -         chr1:2417701              0.2                  8060          48360.0
16:38:06.109 INFO  ProgressMeter -        chr1:21335701              0.3                 71120         213338.7
16:38:16.110 INFO  ProgressMeter -        chr1:42134701              0.5                140450         280871.9
16:38:26.111 INFO  ProgressMeter -        chr1:66476701              0.7                221590         332360.1
16:38:36.110 INFO  ProgressMeter -        chr1:91850701              0.8                306170         367382.0
16:38:46.110 INFO  ProgressMeter -       chr1:115925701              1.0                386420         386400.7
16:38:56.110 INFO  ProgressMeter -       chr1:153881701              1.2                512940         439644.0
16:39:06.111 INFO  ProgressMeter -       chr1:177029701              1.3                590100         442558.4
16:39:16.111 INFO  ProgressMeter -       chr1:201905701              1.5                673020         448660.1
16:39:26.112 INFO  ProgressMeter -       chr1:225884701              1.7                752950         451747.4
16:39:36.113 INFO  ProgressMeter -           chr2:67201              1.8                830080         452746.2
16:39:46.114 INFO  ProgressMeter -         chr2:9097201              2.0                860180         430064.9
16:39:56.114 INFO  ProgressMeter -        chr2:32137201              2.2                936980         432429.0
16:40:06.114 INFO  ProgressMeter -        chr2:55483201              2.3               1014800         434892.5
16:40:16.114 INFO  ProgressMeter -        chr2:79453201              2.5               1094700         437859.6
16:40:26.114 INFO  ProgressMeter -       chr2:105640201              2.7               1181990         443226.9
16:40:36.114 INFO  ProgressMeter -       chr2:130351201              2.8               1264360         446226.3
16:40:46.114 INFO  ProgressMeter -       chr2:155284201              3.0               1347470         449139.2
16:40:56.114 INFO  ProgressMeter -       chr2:180493201              3.2               1431500         452036.0
16:41:06.114 INFO  ProgressMeter -       chr2:205168201              3.3               1513750         454109.1
16:41:16.115 INFO  ProgressMeter -       chr2:230182201              3.5               1597130         456307.6
16:41:26.115 INFO  ProgressMeter -         chr3:9804601              3.7               1669850         455397.1
16:41:36.116 INFO  ProgressMeter -        chr3:32637601              3.8               1745960         455452.0
16:41:46.115 INFO  ProgressMeter -        chr3:55440601              4.0               1821970         455477.3
16:41:56.115 INFO  ProgressMeter -        chr3:79965601              4.2               1903720         456878.2
16:42:06.116 INFO  ProgressMeter -       chr3:107025601              4.3               1993920         460121.2
16:42:16.116 INFO  ProgressMeter -       chr3:130698601              4.5               2072830         460613.5
16:42:26.116 INFO  ProgressMeter -       chr3:155730601              4.7               2156270         462043.0
16:42:36.116 INFO  ProgressMeter -       chr3:180336601              4.8               2238290         463080.1
16:42:46.116 INFO  ProgressMeter -         chr4:4420801              5.0               2312890         462564.1
16:42:56.117 INFO  ProgressMeter -        chr4:27967801              5.2               2391380         462834.3
16:43:06.116 INFO  ProgressMeter -        chr4:54259801              5.3               2479020         464803.2
16:43:16.116 INFO  ProgressMeter -        chr4:79348801              5.5               2562650         465923.7
16:43:26.116 INFO  ProgressMeter -       chr4:104059801              5.7               2645020         466755.9
16:43:36.116 INFO  ProgressMeter -       chr4:129526801              5.8               2729910         467972.5
16:43:46.117 INFO  ProgressMeter -       chr4:154153801              6.0               2812000         468653.6
16:43:56.117 INFO  ProgressMeter -       chr4:180139801              6.2               2898620         470033.8
16:44:06.117 INFO  ProgressMeter -        chr5:13922101              6.3               2978610         470294.5
16:44:16.118 INFO  ProgressMeter -        chr5:39008101              6.5               3062230         471100.2
16:44:26.118 INFO  ProgressMeter -        chr5:65774101              6.7               3151450         472705.7
16:44:36.153 INFO  ProgressMeter -        chr5:91091101              6.8               3235840         473486.7
16:44:46.151 INFO  ProgressMeter -       chr5:116666101              7.0               3321090         474391.7
16:44:56.152 INFO  ProgressMeter -       chr5:140267101              7.2               3399760         474336.6
16:45:06.152 INFO  ProgressMeter -       chr5:163346101              7.3               3476690         474046.7
16:45:16.151 INFO  ProgressMeter -         chr6:5216701              7.5               3554720         473916.3
16:45:26.151 INFO  ProgressMeter -        chr6:28226701              7.7               3631420         473618.2
16:45:36.152 INFO  ProgressMeter -        chr6:49508701              7.8               3702360         472597.5
16:45:46.151 INFO  ProgressMeter -        chr6:76142701              8.0               3791140         473849.1
16:45:56.151 INFO  ProgressMeter -       chr6:104912701              8.2               3887040         475921.3
16:46:06.152 INFO  ProgressMeter -       chr6:129674701              8.3               3969580         476306.7
16:46:16.159 INFO  ProgressMeter -       chr6:153851701              8.5               4050170         476448.5
16:46:26.153 INFO  ProgressMeter -         chr7:5632501              8.7               4125460         475972.5
16:46:36.154 INFO  ProgressMeter -        chr7:30595501              8.8               4208670         476411.0
16:46:46.154 INFO  ProgressMeter -        chr7:55171501              9.0               4290590         476690.7
16:46:56.154 INFO  ProgressMeter -        chr7:81385501              9.2               4377970         477555.9
16:47:06.156 INFO  ProgressMeter -       chr7:105532501              9.3               4458460         477650.3
16:47:16.156 INFO  ProgressMeter -       chr7:130891501              9.5               4542990         478168.4
16:47:26.156 INFO  ProgressMeter -       chr7:155278501              9.7               4624280         478333.4
16:47:36.156 INFO  ProgressMeter -        chr8:16086301              9.8               4691460         477058.0
16:47:46.223 INFO  ProgressMeter -        chr8:36036301             10.0               4757960         475704.0
16:47:56.223 INFO  ProgressMeter -        chr8:57294301             10.2               4828820         474875.6
16:48:06.223 INFO  ProgressMeter -        chr8:78108301             10.3               4898200         473930.7
16:48:16.224 INFO  ProgressMeter -        chr8:99300301             10.5               4968840         473135.7
16:48:26.231 INFO  ProgressMeter -       chr8:120546301             10.7               5039660         472382.5
16:48:36.226 INFO  ProgressMeter -       chr8:140568301             10.8               5106400         471273.7
16:48:46.226 INFO  ProgressMeter -        chr9:18298501             11.0               5182630         471063.2
16:48:56.227 INFO  ProgressMeter -        chr9:42604501             11.2               5263650         471287.9
16:49:06.227 INFO  ProgressMeter -        chr9:80737501             11.3               5390760         475571.4
16:49:16.229 INFO  ProgressMeter -       chr9:104923501             11.5               5471380         475688.8
16:49:26.229 INFO  ProgressMeter -       chr9:128425501             11.7               5549720         475607.4
16:49:36.229 INFO  ProgressMeter -       chr10:11036701             11.8               5619740         474826.0
16:49:46.230 INFO  ProgressMeter -       chr10:34799701             12.0               5698950         474831.4
16:49:56.234 INFO  ProgressMeter -       chr10:61397701             12.2               5787610         475613.2
16:50:06.232 INFO  ProgressMeter -       chr10:84827701             12.3               5865710         475518.4
16:50:16.231 INFO  ProgressMeter -      chr10:108695701             12.5               5945270         475543.0
16:50:26.232 INFO  ProgressMeter -      chr10:132401701             12.7               6024290         475524.3
16:50:36.231 INFO  ProgressMeter -       chr11:22364101             12.8               6103490         475520.0
16:50:46.231 INFO  ProgressMeter -       chr11:47573101             13.0               6187520         475887.4
16:50:56.231 INFO  ProgressMeter -       chr11:71639101             13.2               6267740         475956.2
16:51:06.232 INFO  ProgressMeter -       chr11:96188101             13.3               6349570         476143.4
16:51:16.232 INFO  ProgressMeter -      chr11:121304101             13.5               6433290         476466.5
16:51:26.232 INFO  ProgressMeter -        chr12:9074401             13.7               6509480         476230.8
16:51:36.232 INFO  ProgressMeter -       chr12:33593401             13.8               6591210         476401.3
16:51:46.234 INFO  ProgressMeter -       chr12:59075401             14.0               6676150         476796.9
16:51:56.232 INFO  ProgressMeter -       chr12:84602401             14.2               6761240         477193.8
16:52:06.232 INFO  ProgressMeter -      chr12:109130401             14.3               6843000         477349.2
16:52:16.232 INFO  ProgressMeter -      chr12:131897401             14.5               6918890         477096.3
16:52:26.233 INFO  ProgressMeter -       chr13:32710801             14.7               7032520         479421.9
16:52:36.232 INFO  ProgressMeter -       chr13:56758801             14.8               7112680         479439.2
16:52:46.232 INFO  ProgressMeter -       chr13:82294801             15.0               7197800         479786.7
16:52:56.232 INFO  ProgressMeter -      chr13:107995801             15.2               7283470         480162.8
16:53:06.232 INFO  ProgressMeter -       chr14:28455301             15.3               7399550         482513.8
16:53:16.232 INFO  ProgressMeter -       chr14:53784301             15.5               7483980         482772.5
16:53:26.232 INFO  ProgressMeter -       chr14:77055301             15.7               7561550         482588.0
16:53:36.233 INFO  ProgressMeter -      chr14:100698301             15.8               7640360         482485.6
16:53:46.233 INFO  ProgressMeter -       chr15:28526401             16.0               7756600         484724.4
16:53:56.233 INFO  ProgressMeter -       chr15:52502401             16.2               7836520         484670.7
16:54:06.233 INFO  ProgressMeter -       chr15:76061401             16.3               7915050         484533.1
16:54:16.234 INFO  ProgressMeter -       chr15:99683401             16.5               7993790         484410.5
16:54:26.234 INFO  ProgressMeter -       chr16:19709101             16.7               8067180         483969.3
16:54:36.234 INFO  ProgressMeter -       chr16:49823101             16.8               8167560         485140.6
16:54:46.235 INFO  ProgressMeter -       chr16:73280101             17.0               8245750         484983.7
16:54:56.235 INFO  ProgressMeter -        chr17:4364701             17.2               8317160         484434.6
16:55:06.235 INFO  ProgressMeter -       chr17:29234701             17.3               8400060         484559.2
16:55:16.237 INFO  ProgressMeter -       chr17:51176701             17.5               8473200         484123.8
16:55:26.235 INFO  ProgressMeter -       chr17:73346701             17.7               8547100         483739.7
16:55:36.241 INFO  ProgressMeter -       chr18:10243201             17.8               8614280         482986.0
16:55:46.235 INFO  ProgressMeter -       chr18:38017201             18.0               8706860         483657.1
16:55:56.235 INFO  ProgressMeter -       chr18:62836201             18.2               8789590         483773.8
16:56:06.235 INFO  ProgressMeter -        chr19:4944901             18.3               8864530         483463.6
16:56:16.236 INFO  ProgressMeter -       chr19:27927901             18.5               8941140         483249.1
16:56:26.235 INFO  ProgressMeter -       chr19:49821901             18.7               9014120         482844.1
16:56:36.236 INFO  ProgressMeter -       chr20:14412001             18.8               9091480         482678.3
16:56:46.239 INFO  ProgressMeter -       chr20:38844001             19.0               9172920         482729.4
16:56:56.239 INFO  ProgressMeter -       chr20:61926001             19.2               9249860         482546.0
16:57:06.239 INFO  ProgressMeter -       chr21:26038801             19.3               9345050         483309.7
16:57:16.239 INFO  ProgressMeter -        chr22:5557801             19.5               9432480         483662.4
16:57:26.239 INFO  ProgressMeter -       chr22:31765801             19.7               9519840         484005.5
16:57:36.239 INFO  ProgressMeter -         chrX:2139301             19.8               9590480         483500.0
16:57:46.239 INFO  ProgressMeter -        chrX:28671301             20.0               9678920         483892.8
16:57:56.239 INFO  ProgressMeter -        chrX:55632301             20.2               9768790         484350.0
16:58:06.240 INFO  ProgressMeter -        chrX:85932301             20.3               9869790         485347.0
16:58:16.240 INFO  ProgressMeter -       chrX:115032301             20.5               9966790         486132.3
16:58:26.240 INFO  ProgressMeter -       chrX:143376301             20.7              10061270         486783.4
16:58:36.240 INFO  ProgressMeter -        chrY:32545201             20.8              10211970         490122.4
16:58:46.316 INFO  ProgressMeter -   chrUn_KI270315v1:1             21.0              10317710         491238.4
16:58:56.337 INFO  ProgressMeter -   chrUn_KI270754v1:1             21.2              10329630         487925.7
16:58:57.408 INFO  Mutect2 - 0 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
0 read(s) filtered by: ReadLengthReadFilter
0 read(s) filtered by: GoodCigarReadFilter
23596217 read(s) filtered by: WellformedReadFilter
23596217 total reads filtered
16:58:57.415 INFO  ProgressMeter -        chrEBV:170701             21.2              10333173         487681.8
16:58:57.415 INFO  ProgressMeter - Traversal complete. Processed 10333173 total regions in 21.2 minutes.
16:58:57.459 INFO  VectorLoglessPairHMM - Time spent in setup for JNI call : 0.0
16:58:57.459 INFO  PairHMM - Total compute time in PairHMM computeLogLikelihoods() : 0.0
16:58:57.459 INFO  SmithWatermanAligner - Total compute time in java Smith-Waterman : 0.00 sec
16:58:57.519 INFO  Mutect2 - Shutting down engine
[October 11, 2023 at 4:58:57 PM CEST] org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2 done. Elapsed time: 21.25 minutes.
Runtime.totalMemory()=2076049408
Tool returned:
SUCCESS

GetPileupSummaries

16:37:44.695 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/opt/software/GATK/4.2.3.0-foss-2021b-Java-11/gatk-package-4.2.3.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
Oct 11, 2023 4:37:44 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
16:37:44.936 INFO  GetPileupSummaries - ------------------------------------------------------------
16:37:44.937 INFO  GetPileupSummaries - The Genome Analysis Toolkit (GATK) v4.2.3.0
16:37:44.940 INFO  GetPileupSummaries - For support and documentation go to https://software.broadinstitute.org/gatk/
16:37:44.940 INFO  GetPileupSummaries - Executing as eferre@c01 on Linux v3.10.0-1160.53.1.el7.x86_64 amd64
16:37:44.940 INFO  GetPileupSummaries - Java runtime: OpenJDK 64-Bit Server VM v11.0.2+9
16:37:44.940 INFO  GetPileupSummaries - Start Date/Time: October 11, 2023 at 4:37:44 PM CEST
16:37:44.941 INFO  GetPileupSummaries - ------------------------------------------------------------
16:37:44.941 INFO  GetPileupSummaries - ------------------------------------------------------------
16:37:44.941 INFO  GetPileupSummaries - HTSJDK Version: 2.24.1
16:37:44.941 INFO  GetPileupSummaries - Picard Version: 2.25.4
16:37:44.941 INFO  GetPileupSummaries - Built for Spark Version: 2.4.5
16:37:44.941 INFO  GetPileupSummaries - HTSJDK Defaults.COMPRESSION_LEVEL : 2
16:37:44.941 INFO  GetPileupSummaries - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
16:37:44.941 INFO  GetPileupSummaries - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
16:37:44.941 INFO  GetPileupSummaries - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
16:37:44.942 INFO  GetPileupSummaries - Deflater: IntelDeflater
16:37:44.942 INFO  GetPileupSummaries - Inflater: IntelInflater
16:37:44.942 INFO  GetPileupSummaries - GCS max retries/reopens: 20
16:37:44.950 INFO  GetPileupSummaries - Requester pays: disabled
16:37:44.950 INFO  GetPileupSummaries - Initializing engine
16:37:45.206 INFO  FeatureManager - Using codec VCFCodec to read file file:///mnt/beegfs/eferre/somatic_variants/inputs/files/somatic-hg38_small_exac_common_3.hg38.vcf
16:37:45.404 INFO  FeatureManager - Using codec VCFCodec to read file file:///mnt/beegfs/eferre/somatic_variants/inputs/files/somatic-hg38_small_exac_common_3.hg38.vcf
16:37:46.189 INFO  IntervalArgumentCollection - Processing 59295 bp from intervals
16:37:46.269 INFO  GetPileupSummaries - Done initializing engine
16:37:46.269 INFO  ProgressMeter - Starting traversal
16:37:46.270 INFO  ProgressMeter -        Current Locus  Elapsed Minutes        Loci Processed      Loci/Minute
16:38:03.108 INFO  GetPileupSummaries - 0 read(s) filtered by: MappingQualityAvailableReadFilter
0 read(s) filtered by: MappingQualityNotZeroReadFilter
0 read(s) filtered by: MappedReadFilter
0 read(s) filtered by: PrimaryLineReadFilter
0 read(s) filtered by: NotDuplicateReadFilter
0 read(s) filtered by: PassesVendorQualityCheckReadFilter
0 read(s) filtered by: NonZeroReferenceLengthAlignmentReadFilter
0 read(s) filtered by: MateOnSameContigOrNoMappedMateReadFilter
0 read(s) filtered by: GoodCigarReadFilter
26442 read(s) filtered by: WellformedReadFilter
26442 total reads filtered
16:38:03.108 INFO  ProgressMeter -             unmapped              0.3                     0              0.0
16:38:03.109 INFO  ProgressMeter - Traversal complete. Processed 0 total loci in 0.3 minutes.
16:38:03.110 INFO  GetPileupSummaries - Shutting down engine
[October 11, 2023 at 4:38:03 PM CEST] org.broadinstitute.hellbender.tools.walkers.contamination.GetPileupSummaries done. Elapsed time: 0.31 minutes.
Runtime.totalMemory()=2076049408
java.util.NoSuchElementException: No value present
        at java.base/java.util.Optional.get(Optional.java:148)
        at org.broadinstitute.hellbender.tools.walkers.contamination.GetPileupSummaries.onTraversalSuccess(GetPileupSummaries.java:210)
        at org.broadinstitute.hellbender.engine.GATKTool.doWork(GATKTool.java:1089)
        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 /opt/software/GATK/4.2.3.0-foss-2021b-Java-11/gatk-package-4.2.3.0-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 -jar /opt/software/GATK/4.2.3.0-foss-2021b-Java-11/gatk-package-4.2.3.0-local.jar GetPileupSummaries -I inputs/bam_files/normal_H3K27ac.bam -V inputs/files/somatic-hg38_small_exac_common_3.hg38.vcf -L inputs/files/somatic-hg38_small_exac_common_3.hg38.vcf -O outputs/estimate_contmaination/normal_H3K27ac_getpileupsummaries.table

I think that the problem is something in the normal bam file, but I extract this bam file from encode (https://www.encodeproject.org/experiments/ENCSR000AUP/), I use this because I need a H3K27ac bcell chip from a human normal sample, we wanted to try this with the normal mached mode.

 

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4 comments

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  • Avatar
    Gökalp Çelik

    Hi Estel

    Do both of the input files have the same SM tag for the sample name? If so that would be the reason why you cannot get any variants from your analysis. Normal and matched tumor bam files should have different sample names for Mutect2 to distinguish the reads coming from them. 

    I hope this helps. 

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  • Avatar
    Estel

    Hi! thanks for responding,I don't know how to find the SM tag in the bam files. This are how to look the alignment reads of both bam files:

    normal bam file:

    D0RRAACXX120419:2:2313:3846:39390       0       chr1    54690   37      36M     *       0       0       TAGGGGCTCTCAAACCTTGTATTTTTCTTTCTTTCT    CCCFFFFFHHHHHJJJIJJHHJJJJJGIJJIIIJJJ   X0:i:1   X1:i:0  MD:Z:36 PG:Z:MarkDuplicates     XG:i:0  NM:i:0  XM:i:0  XO:i:0  XT:A:U

    tumor bam file:

    SN227:490:C9LCAACXX:3:2312:1897:6245    0       chr1    10092   254     50M     *       0       0       AACCCTAACCCTAACCCAACCCTAACCCTAACCCTAACCCTAACCCTAAC      @@@FDFFDFHDFFEHGGGGIGIJJIJJIIGBHIGGGHG>GDEDCHHEEH;      PG:Z:MarkDuplicates     RG:Z:H000H3K27acX1_GGCTAC       NH:i:3  NM:i:0  XT:A:R  md:Z:50

    also when I use getpileupssummaries with the normal file doesn't work may be also for the SM tag, if its the case how can I know this value and change it?

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  • Avatar
    Gökalp Çelik

    Hi Estel

    You need to check the header section of the file using 

    samtools view -H bamfile.bam

    If SM: values are the same you need to change them using AddOrReplaceReadGroups tool or samtools addreplacerg tool. 

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  • Avatar
    Estel

    Thanks, it worked!

     

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