GetPileupSummaries output all 0
I'm running the somatic short variant pipeline and I have all 0s in the output of GetPileupSummaries, I don't understand why this happens, I used this program other times and it worked, I check the quality of the bam input files with validatesamfile but I have no errors, and this happens to both output tables, I don't know what can I do.
GATK version used:
Version 4.2.3.0
Exact command used:
gatk GetPileupSummaries -I bam_files/tumor_1.bam -V ../somatic_variants/inputs/files/somatic-hg38_small_exac_common_3.hg38.vcf -L ../somatic_variants/inputs/files/somatic-hg38_small_exac_common_3.hg38.vcf -O outputs/estimate_contamination/tumor_getpileupsummaries.table gatk GetPileupSummaries -I bam_files/normal_1.bam -V ../somatic_variants/inputs/files/somatic-hg38_small_exac_common_3.hg38.vcf -L ../somatic_variants/inputs/files/somatic-hg38_small_exac_common_3.hg38.vcf -O outputs/estimate_contamination/normal_getpileupsummaries.table
Entire program log:
WARNING: GATK v4.2.3.0 support for Java 11 is in beta state. Use at your own risk. 14:45:01.996 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 23, 2023 2:45:02 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine INFO: Failed to detect whether we are running on Google Compute Engine. 14:45:02.305 INFO GetPileupSummaries - ------------------------------------------------------------ 14:45:02.306 INFO GetPileupSummaries - The Genome Analysis Toolkit (GATK) v4.2.3.0 14:45:02.306 INFO GetPileupSummaries - For support and documentation go to https://software.broadinstitute.org/gatk/ 14:45:02.306 INFO GetPileupSummaries - Executing as eferre@c03 on Linux v3.10.0-1160.53.1.el7.x86_64 amd64 14:45:02.307 INFO GetPileupSummaries - Java runtime: OpenJDK 64-Bit Server VM v11.0.2+9 14:45:02.307 INFO GetPileupSummaries - Start Date/Time: October 23, 2023 at 2:45:01 PM CEST 14:45:02.307 INFO GetPileupSummaries - ------------------------------------------------------------ 14:45:02.307 INFO GetPileupSummaries - ------------------------------------------------------------ 14:45:02.308 INFO GetPileupSummaries - HTSJDK Version: 2.24.1 14:45:02.308 INFO GetPileupSummaries - Picard Version: 2.25.4 14:45:02.308 INFO GetPileupSummaries - Built for Spark Version: 2.4.5 14:45:02.308 INFO GetPileupSummaries - HTSJDK Defaults.COMPRESSION_LEVEL : 2 14:45:02.308 INFO GetPileupSummaries - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false 14:45:02.308 INFO GetPileupSummaries - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true 14:45:02.308 INFO GetPileupSummaries - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false 14:45:02.308 INFO GetPileupSummaries - Deflater: IntelDeflater 14:45:02.308 INFO GetPileupSummaries - Inflater: IntelInflater 14:45:02.308 INFO GetPileupSummaries - GCS max retries/reopens: 20 14:45:02.308 INFO GetPileupSummaries - Requester pays: disabled 14:45:02.308 INFO GetPileupSummaries - Initializing engine 14:45:02.839 INFO FeatureManager - Using codec VCFCodec to read file file:///mnt/beegfs/eferre/bin/somatic_short_variant_discovery/files/somatic-hg38_small_exac_common_3.hg38.vcf 14:45:03.224 INFO FeatureManager - Using codec VCFCodec to read file file:///mnt/beegfs/eferre/bin/somatic_short_variant_discovery/files/somatic-hg38_small_exac_common_3.hg38.vcf 14:45:04.693 INFO IntervalArgumentCollection - Processing 59295 bp from intervals 14:45:04.763 INFO GetPileupSummaries - Done initializing engine 14:45:04.763 INFO ProgressMeter - Starting traversal 14:45:04.763 INFO ProgressMeter - Current Locus Elapsed Minutes Loci Processed Loci/Minute 14:45:23.604 INFO ProgressMeter - chr3:17233681 0.3 3000 9553.6 14:45:35.600 INFO ProgressMeter - chr8:76842668 0.5 8000 15566.2 14:45:46.492 INFO ProgressMeter - chrX:147949906 0.7 18000 25881.9 14:45:46.543 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 113 read(s) filtered by: MateOnSameContigOrNoMappedMateReadFilter 0 read(s) filtered by: GoodCigarReadFilter 0 read(s) filtered by: WellformedReadFilter 113 total reads filtered 14:45:46.545 INFO ProgressMeter - chrX:147949906 0.7 18048 25918.0 14:45:46.545 INFO ProgressMeter - Traversal complete. Processed 18048 total loci in 0.7 minutes. 14:45:46.851 INFO GetPileupSummaries - Shutting down engine [October 23, 2023 at 2:45:46 PM CEST] org.broadinstitute.hellbender.tools.walkers.contamination.GetPileupSummaries done. Elapsed time: 0.75 minutes. Runtime.totalMemory()=2076049408 Tool returned: SUCCESS 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 bam_files/tumor_ATAC0_1.bam -V /mnt/beegfs/eferre/bin/somatic_short_variant_discovery/files/somatic-hg38_small_exac_common_3.hg38.vcf -L /mnt/beegfs/eferre/bin/somatic_short_variant_discovery/files/somatic-hg38_small_exac_common_3.hg38.vcf -O outputs/estimate_contamination/tumor_ATAC_getpileupsummaries.table 14:45:50.067 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 23, 2023 2:45:50 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine INFO: Failed to detect whether we are running on Google Compute Engine. 14:45:50.325 INFO GetPileupSummaries - ------------------------------------------------------------ 14:45:50.326 INFO GetPileupSummaries - The Genome Analysis Toolkit (GATK) v4.2.3.0 14:45:50.326 INFO GetPileupSummaries - For support and documentation go to https://software.broadinstitute.org/gatk/ 14:45:50.326 INFO GetPileupSummaries - Executing as eferre@c03 on Linux v3.10.0-1160.53.1.el7.x86_64 amd64 14:45:50.326 INFO GetPileupSummaries - Java runtime: OpenJDK 64-Bit Server VM v11.0.2+9 14:45:50.327 INFO GetPileupSummaries - Start Date/Time: October 23, 2023 at 2:45:50 PM CEST 14:45:50.327 INFO GetPileupSummaries - ------------------------------------------------------------ 14:45:50.327 INFO GetPileupSummaries - ------------------------------------------------------------ 14:45:50.327 INFO GetPileupSummaries - HTSJDK Version: 2.24.1 14:45:50.327 INFO GetPileupSummaries - Picard Version: 2.25.4 14:45:50.327 INFO GetPileupSummaries - Built for Spark Version: 2.4.5 14:45:50.327 INFO GetPileupSummaries - HTSJDK Defaults.COMPRESSION_LEVEL : 2 14:45:50.327 INFO GetPileupSummaries - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false 14:45:50.328 INFO GetPileupSummaries - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true 14:45:50.328 INFO GetPileupSummaries - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false 14:45:50.328 INFO GetPileupSummaries - Deflater: IntelDeflater 14:45:50.328 INFO GetPileupSummaries - Inflater: IntelInflater 14:45:50.328 INFO GetPileupSummaries - GCS max retries/reopens: 20 14:45:50.328 INFO GetPileupSummaries - Requester pays: disabled 14:45:50.328 INFO GetPileupSummaries - Initializing engine 14:45:50.652 INFO FeatureManager - Using codec VCFCodec to read file file:///mnt/beegfs/eferre/bin/somatic_short_variant_discovery/files/somatic-hg38_small_exac_common_3.hg38.vcf 14:45:50.834 INFO FeatureManager - Using codec VCFCodec to read file file:///mnt/beegfs/eferre/bin/somatic_short_variant_discovery/files/somatic-hg38_small_exac_common_3.hg38.vcf 14:45:51.597 INFO IntervalArgumentCollection - Processing 59295 bp from intervals 14:45:51.684 INFO GetPileupSummaries - Done initializing engine 14:45:51.684 INFO ProgressMeter - Starting traversal 14:45:51.684 INFO ProgressMeter - Current Locus Elapsed Minutes Loci Processed Loci/Minute 14:46:09.691 INFO ProgressMeter - chr6:32522015 0.3 1000 3332.2 14:46:22.204 INFO ProgressMeter - chr12:63617282 0.5 2000 3931.8 14:46:35.022 INFO ProgressMeter - chr22:21388395 0.7 3000 4153.5 14:46:36.260 INFO GetPileupSummaries - 282017 read(s) filtered by: MappingQualityAvailableReadFilter 737 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 0 read(s) filtered by: WellformedReadFilter 282754 total reads filtered 14:46:36.261 INFO ProgressMeter - chr22:21388395 0.7 3147 4235.8 14:46:36.262 INFO ProgressMeter - Traversal complete. Processed 3147 total loci in 0.7 minutes. 14:46:36.322 INFO GetPileupSummaries - Shutting down engine [October 23, 2023 at 2:46:36 PM CEST] org.broadinstitute.hellbender.tools.walkers.contamination.GetPileupSummaries done. Elapsed time: 0.77 minutes. Runtime.totalMemory()=2076049408 Tool returned: SUCCESS
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Hi Estel
Can you try running these commands with the latest GATK version 4.4 and with Java 17. Java 11 has never been officially supported. We recommend Java 1.8 (8) for versions 4.3.0.0 and below and Java 17 is the standard version for GATK 4.4.0.0.
Also we have a few more comments about why this could be happening. It seems that only a small number of reads are getting filtered and the whole process seems to be ending very quickly. This tells us that either your bam files are pretty much empty and there are no usable reads inside due to bad MQ or else, OR those sites that you are using to collect pileup summaries may not be matching to where your reads actually are. Can you check on these possibilities before escalating this issue?
Regards.
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