Hi all!

I am total newcomer in bioinfo - so I apologize in advance for possible naivety of my question ;)

I am incorporating pipeline for exome seq analysis optimized about 2-3 yrs ago by alumn of our lab. It is based on DNA-seq analysis pipeline: https://docs.gdc.cancer.gov/Data/Bioinformatics_Pipelines/DNA_Seq_Variant_Calling_Pipeline/

After few adjustment from gatk3 to gatk4 it seems to work pretty well but problem arises when I come to mutect2 step. We need to operate in tumor-only mode.

The problem is that he is doing only several chromosomes (CHR 3 - CHR5) max and than stops without any errors. He creates output but it is empty. I have no idea what to do.

Importantly when I run in only with panel-of-normals it worked. But I guess it is not enaught references for proper analysis...

./gatk Mutect2 \
-R /mnt/c/NGS2/Homo_sapiens_assembly38.fasta \
-I /mnt/c/NGS2/AML_BWA_47_prnt_reads.bam \
-O /mnt/c/NGS2/AML_BWA_47_MuTect2.vcf \
--panel-of-normals /mnt/c/NGS2/resources_broad_hg38_v0_Mills_and_1000G_gold_standard.indels.hg38.vcf.gz

Do u have any suggestion how should I approach this issue to force him to run whole analysis with proper references?

Looking foward for ur response!

a) GATK version used: gatk-4.2.5.0
b) I am running:

/mnt/c/gatk-4.2.5.0/gatk Mutect2 \
-R /mnt/c/NGS2/Homo_sapiens_assembly38.fasta \
-I /mnt/c/NGS2/AML_BWA_47_prnt_reads.bam \
-O /mnt/c/NGS2/AML_BWA_47_MuTect2_test.vcf \
-L /mnt/c/NGS2/hg38_v0_Mills_and_1000G_gold_standard.indels.hg38.vcf.gz \
-tumor AML \
--germline-resource /mnt/c/NGS2/gnomad.exomes.r2.1.1.sites.liftover_grch38.vcf.bgz \
--panel-of-normals /mnt/c/NGS2/resources_broad_hg38_v0_Mills_and_1000G_gold_standard.indels.hg38.vcf.gz

• c) Entire program log:

nicheworks@NICHE_SERWER:/mnt/c/NGS2$ /mnt/c/gatk-4.2.5.0/gatk Mutect2 \
> -R /mnt/c/NGS2/Homo_sapiens_assembly38.fasta \
> -I /mnt/c/NGS2/AML_BWA_47_prnt_reads.bam \
> -O /mnt/c/NGS2/AML_BWA_47_MuTect2_test.vcf \
> -L /mnt/c/NGS2/hg38_v0_Mills_and_1000G_gold_standard.indels.hg38.vcf.gz \
> -tumor AML \
> --germline-resource /mnt/c/NGS2/gnomad.exomes.r2.1.1.sites.liftover_grch38.vcf.bgz \
> --panel-of-normals /mnt/c/NGS2/resources_broad_hg38_v0_Mills_and_1000G_gold_standard.indels.hg38.vcf.gz
Using GATK jar /mnt/c/gatk-4.2.5.0/gatk-package-4.2.5.0-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 /mnt/c/gatk-4.2.5.0/gatk-package-4.2.5.0-local.jar Mutect2 -R /mnt/c/NGS2/Homo_sapiens_assembly38.fasta -I /mnt/c/NGS2/AML_BWA_47_prnt_reads.bam -O /mnt/c/NGS2/AML_BWA_47_MuTect2_test.vcf -L /mnt/c/NGS2/hg38_v0_Mills_and_1000G_gold_standard.indels.hg38.vcf.gz -tumor AML --germline-resource /mnt/c/NGS2/gnomad.exomes.r2.1.1.sites.liftover_grch38.vcf.bgz --panel-of-normals /mnt/c/NGS2/resources_broad_hg38_v0_Mills_and_1000G_gold_standard.indels.hg38.vcf.gz
12:55:41.211 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/mnt/c/gatk-4.2.5.0/gatk-package-4.2.5.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
Apr 05, 2022 12:55:41 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
12:55:41.339 INFO  Mutect2 - ------------------------------------------------------------
12:55:41.340 INFO  Mutect2 - The Genome Analysis Toolkit (GATK) v4.2.5.0
12:55:41.340 INFO  Mutect2 - For support and documentation go to https://software.broadinstitute.org/gatk/
12:55:41.341 INFO  Mutect2 - Executing as nicheworks@NICHE_SERWER on Linux v4.4.0-19041-Microsoft amd64
12:55:41.342 INFO  Mutect2 - Java runtime: OpenJDK 64-Bit Server VM v11.0.13+8-Ubuntu-0ubuntu1.20.04
12:55:41.343 INFO  Mutect2 - Start Date/Time: April 5, 2022 at 12:55:41 PM CEST
12:55:41.343 INFO  Mutect2 - ------------------------------------------------------------
12:55:41.344 INFO  Mutect2 - ------------------------------------------------------------
12:55:41.345 INFO  Mutect2 - HTSJDK Version: 2.24.1
12:55:41.345 INFO  Mutect2 - Picard Version: 2.25.4
12:55:41.346 INFO  Mutect2 - Built for Spark Version: 2.4.5
12:55:41.347 INFO  Mutect2 - HTSJDK Defaults.COMPRESSION_LEVEL : 2
12:55:41.348 INFO  Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
12:55:41.348 INFO  Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
12:55:41.350 INFO  Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
12:55:41.351 INFO  Mutect2 - Deflater: IntelDeflater
12:55:41.352 INFO  Mutect2 - Inflater: IntelInflater
12:55:41.352 INFO  Mutect2 - GCS max retries/reopens: 20
12:55:41.353 INFO  Mutect2 - Requester pays: disabled
12:55:41.354 INFO  Mutect2 - Initializing engine
12:55:41.674 INFO  FeatureManager - Using codec VCFCodec to read file file:///mnt/c/NGS2/resources_broad_hg38_v0_Mills_and_1000G_gold_standard.indels.hg38.vcf.gz
12:55:41.904 INFO  FeatureManager - Using codec VCFCodec to read file file:///mnt/c/NGS2/gnomad.exomes.r2.1.1.sites.liftover_grch38.vcf.bgz
12:55:41.977 INFO  FeatureManager - Using codec VCFCodec to read file file:///mnt/c/NGS2/hg38_v0_Mills_and_1000G_gold_standard.indels.hg38.vcf.gz
12:55:45.193 WARN  IntelInflater - Zero Bytes Written : 0
12:55:45.435 INFO  IntervalArgumentCollection - Processing 3431504 bp from intervals
12:55:45.911 INFO  Mutect2 - Done initializing engine
12:55:45.964 INFO  NativeLibraryLoader - Loading libgkl_utils.so from jar:file:/mnt/c/gatk-4.2.5.0/gatk-package-4.2.5.0-local.jar!/com/intel/gkl/native/libgkl_utils.so
12:55:45.969 INFO  NativeLibraryLoader - Loading libgkl_pairhmm_omp.so from jar:file:/mnt/c/gatk-4.2.5.0/gatk-package-4.2.5.0-local.jar!/com/intel/gkl/native/libgkl_pairhmm_omp.so
12:55:46.000 INFO  IntelPairHmm - Using CPU-supported AVX-512 instructions
12:55:46.001 INFO  IntelPairHmm - Flush-to-zero (FTZ) is enabled when running PairHMM
12:55:46.003 INFO  IntelPairHmm - Available threads: 24
12:55:46.004 INFO  IntelPairHmm - Requested threads: 4
12:55:46.007 INFO  PairHMM - Using the OpenMP multi-threaded AVX-accelerated native PairHMM implementation
12:55:46.074 INFO  ProgressMeter - Starting traversal
12:55:46.075 INFO  ProgressMeter -        Current Locus  Elapsed Minutes     Regions Processed   Regions/Minute
12:55:56.175 INFO  ProgressMeter -        chr1:11821890              0.2                  3660          21742.6
12:56:06.203 INFO  ProgressMeter -        chr1:24070983              0.3                  9000          26828.3

[...]

13:01:08.944 INFO  ProgressMeter -       chr4:122360812              5.4                351350          65292.7
13:01:18.945 INFO  ProgressMeter -       chr4:184670980              5.5                381670          68796.2
13:01:22.127 INFO  VectorLoglessPairHMM - Time spent in setup for JNI call : 0.0
13:01:22.128 INFO  PairHMM - Total compute time in PairHMM computeLogLikelihoods() : 0.0
13:01:22.129 INFO  SmithWatermanAligner - Total compute time in java Smith-Waterman : 0.00 sec
13:01:22.131 INFO  Mutect2 - Shutting down engine
[April 5, 2022 at 1:01:22 PM CEST] org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2 done. Elapsed time: 5.68 minutes.
Runtime.totalMemory()=19008585728
java.lang.IndexOutOfBoundsException: Index 0 out of bounds for length 0
        at java.base/jdk.internal.util.Preconditions.outOfBounds(Preconditions.java:64)
        at java.base/jdk.internal.util.Preconditions.outOfBoundsCheckIndex(Preconditions.java:70)
        at java.base/jdk.internal.util.Preconditions.checkIndex(Preconditions.java:248)
        at java.base/java.util.Objects.checkIndex(Objects.java:372)
        at java.base/java.util.ArrayList.get(ArrayList.java:459)
        at org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2Engine.isActive(Mutect2Engine.java:427)
        at org.broadinstitute.hellbender.engine.AssemblyRegionIterator.loadNextAssemblyRegion(AssemblyRegionIterator.java:136)
        at org.broadinstitute.hellbender.engine.AssemblyRegionIterator.next(AssemblyRegionIterator.java:112)
        at org.broadinstitute.hellbender.engine.AssemblyRegionIterator.next(AssemblyRegionIterator.java:35)
        at org.broadinstitute.hellbender.engine.AssemblyRegionWalker.processReadShard(AssemblyRegionWalker.java:192)
        at org.broadinstitute.hellbender.engine.AssemblyRegionWalker.traverse(AssemblyRegionWalker.java:173)
        at org.broadinstitute.hellbender.engine.GATKTool.doWork(GATKTool.java:1085)
        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)