I need to identify only the somatic mutants in the tumor by normalising the variants in the PBMC DNA(considering it as a germline variation source). I used GenomicsDBImport to call PON. but gatk Mutect2 doesnot take pon from GenomicsDBImport
REQUIRED for all errors and issues:
a) GATK version used: v4.6.1.0
b) Exact command used: to create PON I used
gatk GenomicsDBImport --genomicsdb-workspace-path pon_db --sample-name-map sample_map.txt -L hg19_chrs.list --reader-threads 4
the sample_map.txt contain the path to all the PBMC sample's vcf file along with the index.
it created a directory pon_db and has
ls
callset.json 'chr13$1$115169878' 'chr18$1$78077248' 'chr22$1$51304566' 'chr7$1$159138663' 'chrY$1$59373566'
'chr1$1$249250621' 'chr14$1$107349540' 'chr19$1$59128983' 'chr3$1$198022430' 'chr8$1$146364022' __tiledb_workspace.tdb
'chr10$1$135534747' 'chr15$1$102531392' 'chr2$1$243199373' 'chr4$1$191154276' 'chr9$1$141213431' vcfheader.vcf
'chr11$1$135006516' 'chr16$1$90354753' 'chr20$1$63025520' 'chr5$1$180915260' 'chrM$1$16571' vidmap.json
'chr12$1$133851895' 'chr17$1$81195210' 'chr21$1$48129895' 'chr6$1$171115067' 'chrX$1$155270560'.
But when i am running gatk Mutect2 -R /media/cidmo/051dc678-9fb7-4381-a5cf-164a02a87a37/DSTColon/exome_colon/ref/hg19.fa -I SLT1_recalibrated.bam --panel-of-normals /media/cidmo/051dc678-9fb7-4381-a5cf-164a02a87a37/DSTColon/exome_colon/fastq/alignment/sam/left/sorted_bam/sort/vcf_pon/pon_db/ -O SLT1_output.vcf.gz it ran into error
c) Entire program log:
Using GATK jar /home/cidmo/miniconda3/envs/gatk4/share/gatk4-4.6.1.0-0/gatk-package-4.6.1.0-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 /home/cidmo/miniconda3/envs/gatk4/share/gatk4-4.6.1.0-0/gatk-package-4.6.1.0-local.jar Mutect2 -R /media/cidmo/051dc678-9fb7-4381-a5cf-164a02a87a37/DSTColon/exome_colon/ref/hg19.fa -I SLT1_recalibrated.bam --panel-of-normals /media/cidmo/051dc678-9fb7-4381-a5cf-164a02a87a37/DSTColon/exome_colon/fastq/alignment/sam/left/sorted_bam/sort/vcf_pon/pon_db/ -O SLT1_output.vcf.gz
13:00:11.589 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/home/cidmo/miniconda3/envs/gatk4/share/gatk4-4.6.1.0-0/gatk-package-4.6.1.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
SLF4J(W): Class path contains multiple SLF4J providers.
SLF4J(W): Found provider [org.apache.logging.slf4j.SLF4JServiceProvider@66713605]
SLF4J(W): Found provider [ch.qos.logback.classic.spi.LogbackServiceProvider@2ab09943]
SLF4J(W): See https://www.slf4j.org/codes.html#multiple_bindings for an explanation.
SLF4J(I): Actual provider is of type [org.apache.logging.slf4j.SLF4JServiceProvider@66713605]
13:00:11.761 INFO Mutect2 - ------------------------------------------------------------
13:00:11.765 INFO Mutect2 - The Genome Analysis Toolkit (GATK) v4.6.1.0
13:00:11.765 INFO Mutect2 - For support and documentation go to https://software.broadinstitute.org/gatk/
13:00:11.765 INFO Mutect2 - Executing as cidmo@cidmo-Precision-WorkStation-T3500 on Linux v6.8.0-49-generic amd64
13:00:11.766 INFO Mutect2 - Java runtime: OpenJDK 64-Bit Server VM v17.0.13-internal+0-adhoc..src
13:00:11.766 INFO Mutect2 - Start Date/Time: 16 December 2024 at 1:00:11 pm IST
13:00:11.766 INFO Mutect2 - ------------------------------------------------------------
13:00:11.766 INFO Mutect2 - ------------------------------------------------------------
13:00:11.767 INFO Mutect2 - HTSJDK Version: 4.1.3
13:00:11.767 INFO Mutect2 - Picard Version: 3.3.0
13:00:11.768 INFO Mutect2 - Built for Spark Version: 3.5.0
13:00:11.771 INFO Mutect2 - HTSJDK Defaults.COMPRESSION_LEVEL : 2
13:00:11.772 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
13:00:11.772 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
13:00:11.772 INFO Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
13:00:11.772 INFO Mutect2 - Deflater: IntelDeflater
13:00:11.772 INFO Mutect2 - Inflater: IntelInflater
13:00:11.773 INFO Mutect2 - GCS max retries/reopens: 20
13:00:11.773 INFO Mutect2 - Requester pays: disabled
13:00:11.773 INFO Mutect2 - Initializing engine
13:00:11.992 INFO Mutect2 - Shutting down engine
[16 December 2024 at 1:00:11 pm IST] org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2 done. Elapsed time: 0.01 minutes.
Runtime.totalMemory()=104857600
***********************************************************************
A USER ERROR has occurred: Couldn't read file file:///media/cidmo/051dc678-9fb7-4381-a5cf-164a02a87a37/DSTColon/exome_colon/fastq/alignment/sam/left/sorted_bam/sort/vcf_pon/pon_db/. Error was: It isn't a regular file
***********************************************************************
Set the system property GATK_STACKTRACE_ON_USER_EXCEPTION (--java-options '-DGATK_STACKTRACE_ON_USER_EXCEPTION=true') to print the stack trace.
-
Hi HemaRajan
PoN in Mutect2 is not used as a germline source but a source to exclude sequencing artifacts from Mutect2 calls. If you wish to generate a new germline source for yourself you need to genotype that imported calls and use it as your germline resource file instead of PoN. Mutect2 cannot consume an ungenotyped import. However we highly recommend using our germline resource suggestion as well. You may be able to combine those sites with gnomad-af-only resource for germline to remove possible germline calls from your Mutect2 calls.
Regards.
Please sign in to leave a comment.
1 comment