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Funcotator maf output problems on Varscan2 vcf files

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A. Brink

Dear GATK team, 

When I run Funcotator (v4.1.4.1) on VCF files that are produced using Samtools mpileup + Varscan2 I obtain errors when I want MAF output. However, running Funcotator with VCF output on the same varscan2 input VCF files works fine. Furthermore, I don't have problems with obtaining MAF and VCF output of Funcotator with Mutect2 VCF files.

A USER ERROR has occurred: Allelic Depth (AD field) for Variant[chr1:120509038_C*->T] Does not contain both a REF and an ALT value (only one value is present)!

It appears that Funcotator expects 2 values in the AD field, but varscan2 outputs only 1 value in its AD field. I don't understand why Funcotator with VCF output works fine with this AD field, but MAF output doesn't. Any ideas?

Using GATK jar /home/AB/Packages/GATK/v4.1.4.1/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 -jar /home/AB/Packages/GATK/v4.1.4.1/gatk-package-4.1.4.1-local.jar Funcotator --variant vcf/varscan/Sample_varscan.vcf -L /home/AB/Necessary_files/OID46455_hg19_26sep2018_capture_targets.bed --reference /net/nfs/PAT/data/ref/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa --ref-version hg19 --data-sources-path /home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s --output funcotator/varscan/Sample_varscan_funcotator.maf --output-file-format MAF

14:14:07.909 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/home/AB/Packages/GATK/v4.1.4.1/gatk-package-4.1.4.1-local.jar!/com/intel/gkl/native/libgkl_compression.so

Jan 23, 2020 2:14:08 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine

INFO: Failed to detect whether we are running on Google Compute Engine.

14:14:08.118 INFO Funcotator - ------------------------------------------------------------

14:14:08.118 INFO Funcotator - The Genome Analysis Toolkit (GATK) v4.1.4.1

14:14:08.118 INFO Funcotator - For support and documentation go to https://software.broadinstitute.org/gatk/

14:14:08.119 INFO Funcotator - Executing as AB@zeus.vumc.nl on Linux v3.10.0-693.17.1.el7.x86_64 amd64

14:14:08.119 INFO Funcotator - Java runtime: Java HotSpot(TM) 64-Bit Server VM v1.8.0_73-b02

14:14:08.119 INFO Funcotator - Start Date/Time: January 23, 2020 2:14:07 PM CET

14:14:08.119 INFO Funcotator - ------------------------------------------------------------

14:14:08.119 INFO Funcotator - ------------------------------------------------------------

14:14:08.119 INFO Funcotator - HTSJDK Version: 2.21.0

14:14:08.119 INFO Funcotator - Picard Version: 2.21.2

14:14:08.119 INFO Funcotator - HTSJDK Defaults.COMPRESSION_LEVEL : 2

14:14:08.120 INFO Funcotator - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false

14:14:08.120 INFO Funcotator - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true

14:14:08.120 INFO Funcotator - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false

14:14:08.120 INFO Funcotator - Deflater: IntelDeflater

14:14:08.120 INFO Funcotator - Inflater: IntelInflater

14:14:08.120 INFO Funcotator - GCS max retries/reopens: 20

14:14:08.120 INFO Funcotator - Requester pays: disabled

14:14:08.120 INFO Funcotator - Initializing engine

14:14:08.496 INFO FeatureManager - Using codec VCFCodec to read file file:///home/AB/Samples/vcf/varscan/Sample_varscan.vcf

14:14:08.537 WARN IndexUtils - Feature file "/home/AB/Samples/vcf/varscan/Sample_varscan.vcf" appears to contain no sequence dictionary. Attempting to retrieve a sequence dictionary from the associated index file

14:14:08.553 INFO FeatureManager - Using codec BEDCodec to read file file:///home/AB/Necessary_files/OID46455_hg19_26sep2018_capture_targets.bed

14:14:08.569 INFO IntervalArgumentCollection - Processing 122092 bp from intervals

14:14:08.573 WARN IndexUtils - Feature file "/home/AB/Samples/vcf/varscan/Sample_varscan.vcf" appears to contain no sequence dictionary. Attempting to retrieve a sequence dictionary from the associated index file

14:14:08.582 INFO Funcotator - Done initializing engine

14:14:08.583 INFO Funcotator - Validating Sequence Dictionaries...

14:14:08.583 WARN IndexUtils - Feature file "/home/AB/Samples/vcf/varscan/Sample_varscan.vcf" appears to contain no sequence dictionary. Attempting to retrieve a sequence dictionary from the associated index file

14:14:08.590 WARN IndexUtils - Feature file "/home/AB/Samples/vcf/varscan/Sample_varscan.vcf" appears to contain no sequence dictionary. Attempting to retrieve a sequence dictionary from the associated index file

14:14:08.597 INFO Funcotator - Processing user transcripts/defaults/overrides...

14:14:08.598 INFO Funcotator - Initializing data sources...

14:14:08.599 INFO DataSourceUtils - Initializing data sources from directory: /home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s

14:14:08.600 INFO DataSourceUtils - Data sources version: 1.6.2019124s

14:14:08.600 INFO DataSourceUtils - Data sources source: ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/funcotator/funcotator_dataSources.v1.6.20190124s.tar.gz

14:14:08.600 INFO DataSourceUtils - Data sources alternate source: gs://broad-public-datasets/funcotator/funcotator_dataSources.v1.6.20190124s.tar.gz

14:14:08.605 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/Cosmic.db -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/cosmic/hg19/Cosmic.db

14:14:08.606 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/hgnc_download_Nov302017.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/hgnc/hg19/hgnc_download_Nov302017.tsv

14:14:08.607 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/cosmic_fusion.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/cosmic_fusion/hg19/cosmic_fusion.tsv

14:14:10.101 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/chr1_a_bed.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/chr1_a_bed/hg19/chr1_a_bed.tsv

14:14:10.103 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/cosmic_tissue.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/cosmic_tissue/hg19/cosmic_tissue.tsv

14:14:10.104 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/oreganno.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/oreganno/hg19/oreganno.tsv

14:14:10.106 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/gencode_xrefseq_v75_37.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/gencode_xrefseq/hg19/gencode_xrefseq_v75_37.tsv

14:14:10.107 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/gencode_xhgnc_v75_37.hg19.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/gencode_xhgnc/hg19/gencode_xhgnc_v75_37.hg19.tsv

14:14:10.108 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/simple_uniprot_Dec012014.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/simple_uniprot/hg19/simple_uniprot_Dec012014.tsv

14:14:10.110 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/Familial_Cancer_Genes.no_dupes.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/familial/hg19/Familial_Cancer_Genes.no_dupes.tsv

14:14:10.111 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/dnaRepairGenes.20180524T145835.csv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/dna_repair_genes/hg19/dnaRepairGenes.20180524T145835.csv

14:14:10.112 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/chr1_b_bed.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/chr1_b_bed/hg19/chr1_b_bed.tsv

14:14:10.114 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/gencode.v19.annotation.REORDERED.gtf -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/gencode/hg19/gencode.v19.annotation.REORDERED.gtf

14:14:10.114 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/gencode.v19.pc_transcripts.fa -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/gencode/hg19/gencode.v19.pc_transcripts.fa

14:14:10.115 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/CancerGeneCensus_Table_1_full_2012-03-15.txt -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/cancer_gene_census/hg19/CancerGeneCensus_Table_1_full_2012-03-15.txt

14:14:10.117 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/hg19_All_20170710.vcf.gz -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/dbsnp/hg19/hg19_All_20170710.vcf.gz

14:14:10.118 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/achilles_lineage_results.import.txt -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/achilles/hg19/achilles_lineage_results.import.txt

14:14:10.119 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/clinvar_hgmd.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/clinvar/hg19/clinvar_hgmd.tsv

14:14:11.361 INFO Funcotator - Finalizing data sources (this step can be long if data sources are cloud-based)...

14:14:11.365 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/Cosmic.db -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/cosmic/hg19/Cosmic.db

14:14:11.464 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/hgnc_download_Nov302017.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/hgnc/hg19/hgnc_download_Nov302017.tsv

14:14:11.702 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/cosmic_fusion.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/cosmic_fusion/hg19/cosmic_fusion.tsv

14:14:11.704 INFO DataSourceUtils - Setting lookahead cache for data source: gnomAD_genome : 100000

14:14:18.774 INFO FeatureManager - Using codec VCFCodec to read file gs://broad-public-datasets/funcotator/gnomAD_2.1_VCF_INFO_AF_Only/hg19/gnomad.genomes.r2.1.sites.INFO_ANNOTATIONS_FIXED.vcf.bgz

14:14:33.466 INFO FeatureManager - Using codec VCFCodec to read file gs://broad-public-datasets/funcotator/gnomAD_2.1_VCF_INFO_AF_Only/hg19/gnomad.genomes.r2.1.sites.INFO_ANNOTATIONS_FIXED.vcf.bgz

14:14:36.324 INFO DataSourceUtils - Setting lookahead cache for data source: chr1_a_bed : 100000

14:14:36.330 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/chr1_a_bed.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/chr1_a_bed/hg19/chr1_a_bed.tsv

14:14:36.331 INFO FeatureManager - Using codec XsvLocatableTableCodec to read file file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/chr1_a_bed/hg19/chr1_a_bed.config

14:14:36.339 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/chr1_a_bed.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/chr1_a_bed/hg19/chr1_a_bed.tsv

14:14:36.340 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/chr1_a_bed.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/chr1_a_bed/hg19/chr1_a_bed.tsv

WARNING 2020-01-23 14:14:36 AsciiLineReader Creating an indexable source for an AsciiFeatureCodec using a stream that is neither a PositionalBufferedStream nor a BlockCompressedInputStream

14:14:36.344 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/cosmic_tissue.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/cosmic_tissue/hg19/cosmic_tissue.tsv

14:14:36.374 INFO DataSourceUtils - Setting lookahead cache for data source: Oreganno : 100000

14:14:36.376 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/oreganno.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/oreganno/hg19/oreganno.tsv

14:14:36.376 INFO FeatureManager - Using codec XsvLocatableTableCodec to read file file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/oreganno/hg19/oreganno.config

14:14:36.398 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/oreganno.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/oreganno/hg19/oreganno.tsv

14:14:36.398 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/oreganno.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/oreganno/hg19/oreganno.tsv

WARNING 2020-01-23 14:14:36 AsciiLineReader Creating an indexable source for an AsciiFeatureCodec using a stream that is neither a PositionalBufferedStream nor a BlockCompressedInputStream

14:14:36.400 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/gencode_xrefseq_v75_37.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/gencode_xrefseq/hg19/gencode_xrefseq_v75_37.tsv

14:14:36.513 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/gencode_xhgnc_v75_37.hg19.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/gencode_xhgnc/hg19/gencode_xhgnc_v75_37.hg19.tsv

14:14:37.480 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/simple_uniprot_Dec012014.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/simple_uniprot/hg19/simple_uniprot_Dec012014.tsv

14:14:37.521 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/Familial_Cancer_Genes.no_dupes.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/familial/hg19/Familial_Cancer_Genes.no_dupes.tsv

14:14:37.522 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/dnaRepairGenes.20180524T145835.csv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/dna_repair_genes/hg19/dnaRepairGenes.20180524T145835.csv

14:14:37.523 INFO DataSourceUtils - Setting lookahead cache for data source: chr1_b_bed : 100000

14:14:37.524 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/chr1_b_bed.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/chr1_b_bed/hg19/chr1_b_bed.tsv

14:14:37.524 INFO FeatureManager - Using codec XsvLocatableTableCodec to read file file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/chr1_b_bed/hg19/chr1_b_bed.config

14:14:37.525 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/chr1_b_bed.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/chr1_b_bed/hg19/chr1_b_bed.tsv

14:14:37.526 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/chr1_b_bed.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/chr1_b_bed/hg19/chr1_b_bed.tsv

WARNING 2020-01-23 14:14:37 AsciiLineReader Creating an indexable source for an AsciiFeatureCodec using a stream that is neither a PositionalBufferedStream nor a BlockCompressedInputStream

14:14:37.527 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/gencode.v19.annotation.REORDERED.gtf -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/gencode/hg19/gencode.v19.annotation.REORDERED.gtf

14:14:37.527 INFO DataSourceUtils - Setting lookahead cache for data source: Gencode : 100000

14:14:37.528 INFO FeatureManager - Using codec GencodeGtfCodec to read file file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/gencode/hg19/gencode.v19.annotation.REORDERED.gtf

14:14:37.534 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/gencode.v19.pc_transcripts.fa -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/gencode/hg19/gencode.v19.pc_transcripts.fa

14:14:41.700 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/CancerGeneCensus_Table_1_full_2012-03-15.txt -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/cancer_gene_census/hg19/CancerGeneCensus_Table_1_full_2012-03-15.txt

14:14:41.703 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/hg19_All_20170710.vcf.gz -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/dbsnp/hg19/hg19_All_20170710.vcf.gz

14:14:41.703 INFO DataSourceUtils - Setting lookahead cache for data source: dbSNP : 100000

14:14:41.705 INFO FeatureManager - Using codec VCFCodec to read file file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/dbsnp/hg19/hg19_All_20170710.vcf.gz

14:14:41.750 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/hg19_All_20170710.vcf.gz -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/dbsnp/hg19/hg19_All_20170710.vcf.gz

14:14:41.799 INFO FeatureManager - Using codec VCFCodec to read file file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/dbsnp/hg19/hg19_All_20170710.vcf.gz

14:14:41.857 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/achilles_lineage_results.import.txt -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/achilles/hg19/achilles_lineage_results.import.txt

14:14:41.858 INFO DataSourceUtils - Setting lookahead cache for data source: ClinVar : 100000

14:14:41.861 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/clinvar_hgmd.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/clinvar/hg19/clinvar_hgmd.tsv

14:14:41.861 INFO FeatureManager - Using codec XsvLocatableTableCodec to read file file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/clinvar/hg19/clinvar_hgmd.config

14:14:41.919 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/clinvar_hgmd.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/clinvar/hg19/clinvar_hgmd.tsv

14:14:41.919 INFO DataSourceUtils - Resolved data source file path: file:///home/AB/Samples/clinvar_hgmd.tsv -> file:///home/AB/Necessary_files/GATK/funcotator_dataSources.v1.6.20190124s/clinvar/hg19/clinvar_hgmd.tsv

WARNING 2020-01-23 14:14:41 AsciiLineReader Creating an indexable source for an AsciiFeatureCodec using a stream that is neither a PositionalBufferedStream nor a BlockCompressedInputStream

14:14:41.921 INFO DataSourceUtils - Setting lookahead cache for data source: gnomAD_exome : 100000

14:14:48.779 INFO FeatureManager - Using codec VCFCodec to read file gs://broad-public-datasets/funcotator/gnomAD_2.1_VCF_INFO_AF_Only/hg19/gnomad.exomes.r2.1.sites.INFO_ANNOTATIONS_FIXED.vcf.bgz

14:15:02.120 INFO FeatureManager - Using codec VCFCodec to read file gs://broad-public-datasets/funcotator/gnomAD_2.1_VCF_INFO_AF_Only/hg19/gnomad.exomes.r2.1.sites.INFO_ANNOTATIONS_FIXED.vcf.bgz

14:15:04.222 INFO Funcotator - Initializing Funcotator Engine...

14:15:04.222 WARN IndexUtils - Feature file "/home/AB/Samples/vcf/varscan/Sample_varscan.vcf" appears to contain no sequence dictionary. Attempting to retrieve a sequence dictionary from the associated index file

14:15:04.234 INFO Funcotator - Creating a MAF file for output: file:/home/AB/Samples/funcotator/varscan/Sample_varscan_funcotator.maf

14:15:04.251 INFO ProgressMeter - Starting traversal

14:15:04.251 INFO ProgressMeter - Current Locus Elapsed Minutes Variants Processed Variants/Minute

14:15:09.256 INFO VcfFuncotationFactory - dbSNP 9606_b150 cache hits/total: 0/0

14:15:09.256 INFO VcfFuncotationFactory - gnomAD_exome 2.1 cache hits/total: 0/0

14:15:09.256 INFO VcfFuncotationFactory - gnomAD_genome 2.1 cache hits/total: 0/0

14:15:09.282 INFO Funcotator - Shutting down engine

[January 23, 2020 2:15:11 PM CET] org.broadinstitute.hellbender.tools.funcotator.Funcotator done. Elapsed time: 1.06 minutes.

Runtime.totalMemory()=3515875328

***********************************************************************

A USER ERROR has occurred: Allelic Depth (AD field) for Variant[chr1:120509038_C*->T] Does not contain both a REF and an ALT value (only one value is present)!

***********************************************************************

 

 

 

 

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10 comments

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  • Avatar
    Bhanu Gandham

    Hi,

    Sorry about the delay in getting back to you. Our lead funcotator developer is doing a workshop in Costa Rica to help community outreach.

    Here is the suggestion with regards to your question: The MAF specification expects a tumor/normal pair and in this instance, this requirement is manifesting as requiring a ref (normal) and alt (tumor) allelic depth.  This is not a funcotator issue, but rather a limitation on funcotator due to the specification of MAF files.  This does not occur with VCF output because VCF files do not have the same requirements as MAF files.  This cannot be worked around in the funcotator code, but adding in dummy values for the missing AD field in the data coming from Varscan2 will fix this issue and allow MAF files to be created.

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  • Avatar
    Egmidio Medina

    Hello,

    I am also interested in running Funcotator on VarScan2 outputs. Are there any new updates regarding when/if Funcotator will allow VarScan2 inputs?

    Thank you.

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    Bhanu Gandham

    Hi Egmidio Medina

     

    Funcotator currently does not support MAF files. You could convert MAF to VCF files and then annotate with Funcotator.

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    Bhanu Gandham

    We do we have a ticket open for adding maf reading to Funcotator but this will take sometime. You can follow its progress here: https://github.com/broadinstitute/gatk/issues/3703

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    Siddharth Prakash

    Hi Bhanu,

    I am running Funcotator on Mutect2 outputs and encountered a similar problem. Funcotator starts normally but then abruptly exits with this error:

    06:26:13.703 INFO Funcotator - Creating a VCF file for output: file:/corral-secure/uth/Sex-Chromosome-Loss/bamFiles/14047.303211.annotated.vcf.gz
    06:26:13.736 INFO ProgressMeter - Starting traversal
    06:26:13.737 INFO ProgressMeter - Current Locus Elapsed Minutes Variants Processed Variants/Minute
    06:26:15.468 INFO VcfFuncotationFactory - dbSNP 9606_b150 cache hits/total: 0/0
    06:26:15.468 INFO VcfFuncotationFactory - gnomAD_exome 2.1 cache hits/total: 0/0
    06:26:15.472 INFO Funcotator - Shutting down engine
    [July 19, 2020 6:26:16 AM CDT] org.broadinstitute.hellbender.tools.funcotator.Funcotator done. Elapsed time: 0.32 minutes.
    Runtime.totalMemory()=1669332992
    java.lang.NullPointerException

    However, Funcotator creates MAF files without errors. What can I do? Should I convert the MAF output to VCF?

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    Genevieve Brandt (she/her)

    Hi Siddharth Prakash, could you please provide the GATK version you are using, your command, and the entire error log?

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    Siddharth Prakash

    GATK 4.1.8.0

    java -Xmx4G -jar ${GATK_DIR}/gatk-package-4.1.8.0-local.jar Funcotator -R ${REF_DIR}/hs37d5.fa -V ${BAM_DIR}/14047.303211.filtered.vcf.gz -O ${BAM_DIR}/14047.303211.annotated.vcf.gz --output-file-format VCF --data-sources-path ${REF_DIR}/funcotator_dataSources.v1.6.20190124s/ --ref-version hg19

    where

    GATK_DIR="/work/03437/sprakash/lonestar/apps/gatk"

    REF_DIR="/work/03437/sprakash/lonestar/references"

    BAM_DIR="/corral-secure/uth/Sex-Chromosome-Loss/bamFiles"

     

    10:24:36.331 INFO Funcotator - Creating a VCF file for output: file:/corral-secure/uth/Sex-Chromosome-Loss/bamFiles/14047.303211.annotated.vcf.gz
    10:24:36.363 INFO ProgressMeter - Starting traversal
    10:24:36.363 INFO ProgressMeter - Current Locus Elapsed Minutes Variants Processed Variants/Minute
    10:24:37.879 INFO VcfFuncotationFactory - dbSNP 9606_b150 cache hits/total: 0/0
    10:24:37.879 INFO VcfFuncotationFactory - gnomAD_exome 2.1 cache hits/total: 0/0
    10:24:37.882 INFO Funcotator - Shutting down engine
    [July 20, 2020 10:24:39 AM CDT] org.broadinstitute.hellbender.tools.funcotator.Funcotator done. Elapsed time: 0.31 minutes.
    Runtime.totalMemory()=1749024768
    java.lang.NullPointerException
    at htsjdk.tribble.index.tabix.TabixIndexCreator.finalizeFeature(TabixIndexCreator.java:106)
    at htsjdk.tribble.index.tabix.TabixIndexCreator.finalizeIndex(TabixIndexCreator.java:129)
    at htsjdk.variant.variantcontext.writer.IndexingVariantContextWriter.close(IndexingVariantContextWriter.java:177)
    at htsjdk.variant.variantcontext.writer.VCFWriter.close(VCFWriter.java:233)
    at org.broadinstitute.hellbender.tools.funcotator.vcfOutput.VcfOutputRenderer.close(VcfOutputRenderer.java:137)
    at org.broadinstitute.hellbender.tools.funcotator.Funcotator.closeTool(Funcotator.java:890)
    at org.broadinstitute.hellbender.engine.GATKTool.doWork(GATKTool.java:1053)
    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:163)
    at org.broadinstitute.hellbender.Main.mainEntry(Main.java:206)
    at org.broadinstitute.hellbender.Main.main(Main.java:292)

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    Genevieve Brandt (she/her)

    Hi Siddharth Prakash, it looks like there is an error regarding your index file. Here is a similar example on the forum that may help you with this problem:

    https://gatk.broadinstitute.org/hc/en-us/community/posts/360062293851-Mutect2-issue-with-long-scaffolds-non-human-mouse-

    If you cannot solve it, please start a new post since it is not the same as the one above.

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    Siddharth Prakash

    Thanks Genevieve,

    Outputting as uncompressed .vcf worked.

     

    -Siddharth

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    Genevieve Brandt (she/her)

    Great! Thanks for the update.

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