mtDNA calling problems
Answered
Hi, i am hojinlee, a graduate student at Korea university.
i am doing mtDNA variant calling using this guideline and hg38 version bam file. (https://sites.google.com/a/broadinstitute.org/legacy-gatk-forum-discussions/announcements/23598-new-mitochondrial-analysis-with-mutect2)
but i can not move on next steps, because of the following problem. and one more thing, this step is for making both of a normal mitochondrial fasta file and shifted one?
thank you.
hojinlee
REQUIRED for all errors and issues:
a) GATK version used: 4.1.9.0-GCCcore-10.2.0-Java-1.8
b) Exact command used: module load GATK/4.1.9.0-GCCcore-10.2.0-Java-1.8; gatk SamToFastq -I thy_P1_MT_reverted.bam -FASTQ thy_P1_MT.fastq
c) Entire program log:
[jc2545@c13n04 P1]$ module load GATK/4.1.9.0-GCCcore-10.2.0-Java-1.8; gatk SamToFastq I=thy_P1_MT_reverted.bam FASTQ=thy_P1_MT.fastq
Using GATK jar /gpfs/ycga/apps/hpc/software/GATK/4.1.9.0-GCCcore-10.2.0-Java-1.8/gatk-package-4.1.9.0-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 /gpfs/ycga/apps/hpc/software/GATK/4.1.9.0-GCCcore-10.2.0-Java-1.8/gatk-package-4.1.9.0-local.jar SamToFastq I=thy_P1_MT_reverted.bam FASTQ=thy_P1_MT.fastq
INFO 2023-03-16 00:15:31 SamToFastq
********** NOTE: Picard's command line syntax is changing.
**********
********** For more information, please see:
**********
********** The command line looks like this in the new syntax:
**********
********** SamToFastq -I thy_P1_MT_reverted.bam -FASTQ thy_P1_MT.fastq
**********
00:15:31.518 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/gpfs/ycga/apps/hpc/software/GATK/4.1.9.0-GCCcore-10.2.0-Java-1.8/gatk-package-4.1.9.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
[Thu Mar 16 00:15:31 EDT 2023] SamToFastq INPUT=thy_P1_MT_reverted.bam FASTQ=thy_P1_MT.fastq OUTPUT_PER_RG=false COMPRESS_OUTPUTS_PER_RG=false RG_TAG=PU RE_REVERSE=true INTERLEAVE=false INCLUDE_NON_PF_READS=false CLIPPING_MIN_LENGTH=0 READ1_TRIM=0 READ2_TRIM=0 INCLUDE_NON_PRIMARY_ALIGNMENTS=false VERBOSITY=INFO QUIET=false VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=2 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false GA4GH_CLIENT_SECRETS=client_secrets.json USE_JDK_DEFLATER=false USE_JDK_INFLATER=false
Mar 16, 2023 12:15:31 AM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
[Thu Mar 16 00:15:31 EDT 2023] Executing as jc2545@c13n04.ruddle.hpc.yale.internal on Linux 3.10.0-1160.76.1.el7.x86_64 amd64; Java HotSpot(TM) 64-Bit Server VM 1.8.0_212-b10; Deflater: Intel; Inflater: Intel; Provider GCS is available; Picard version: 4.1.9.0
[Thu Mar 16 00:15:31 EDT 2023] picard.sam.SamToFastq done. Elapsed time: 0.00 minutes.
Runtime.totalMemory()=2174746624
To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp
htsjdk.samtools.SAMFormatException: SAM validation error: ERROR::INVALID_FLAG_SUPPLEMENTARY_ALIGNMENT:Record 47, Read name A01056:234:H2YWVDSX3:3:1122:27814:30780, Supplementary alignment flag should not be set for unmapped read.
at htsjdk.samtools.SAMUtils.processValidationErrors(SAMUtils.java:457)
at htsjdk.samtools.BAMFileReader$BAMFileIterator.advance(BAMFileReader.java:849)
at htsjdk.samtools.BAMFileReader$BAMFileIterator.next(BAMFileReader.java:834)
at htsjdk.samtools.BAMFileReader$BAMFileIterator.next(BAMFileReader.java:802)
at htsjdk.samtools.SamReader$AssertingIterator.next(SamReader.java:574)
at htsjdk.samtools.SamReader$AssertingIterator.next(SamReader.java:553)
at picard.sam.SamToFastq.doWork(SamToFastq.java:205)
at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:301)
at org.broadinstitute.hellbender.cmdline.PicardCommandLineProgramExecutor.instanceMain(PicardCommandLineProgramExecutor.java:37)
at org.broadinstitute.hellbender.Main.runCommandLineProgram(Main.java:160)
at org.broadinstitute.hellbender.Main.mainEntry(Main.java:203)
at org.broadinstitute.hellbender.Main.main(Main.java:289)
[jc2545@c13n04 P1]$ module load GATK/4.1.9.0-GCCcore-10.2.0-Java-1.8; gatk SamToFastq -I thy_P1_MT_reverted.bam -FASTQ thy_P1_MT.fastq
Using GATK jar /gpfs/ycga/apps/hpc/software/GATK/4.1.9.0-GCCcore-10.2.0-Java-1.8/gatk-package-4.1.9.0-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 /gpfs/ycga/apps/hpc/software/GATK/4.1.9.0-GCCcore-10.2.0-Java-1.8/gatk-package-4.1.9.0-local.jar SamToFastq -I thy_P1_MT_reverted.bam -FASTQ thy_P1_MT.fastq
00:15:51.171 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/gpfs/ycga/apps/hpc/software/GATK/4.1.9.0-GCCcore-10.2.0-Java-1.8/gatk-package-4.1.9.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
[Thu Mar 16 00:15:51 EDT 2023] SamToFastq --INPUT thy_P1_MT_reverted.bam --FASTQ thy_P1_MT.fastq --OUTPUT_PER_RG false --COMPRESS_OUTPUTS_PER_RG false --RG_TAG PU --RE_REVERSE true --INTERLEAVE false --INCLUDE_NON_PF_READS false --CLIPPING_MIN_LENGTH 0 --READ1_TRIM 0 --READ2_TRIM 0 --INCLUDE_NON_PRIMARY_ALIGNMENTS false --VERBOSITY INFO --QUIET false --VALIDATION_STRINGENCY STRICT --COMPRESSION_LEVEL 2 --MAX_RECORDS_IN_RAM 500000 --CREATE_INDEX false --CREATE_MD5_FILE false --GA4GH_CLIENT_SECRETS client_secrets.json --help false --version false --showHidden false --USE_JDK_DEFLATER false --USE_JDK_INFLATER false
Mar 16, 2023 12:15:51 AM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
[Thu Mar 16 00:15:51 EDT 2023] Executing as jc2545@c13n04.ruddle.hpc.yale.internal on Linux 3.10.0-1160.76.1.el7.x86_64 amd64; Java HotSpot(TM) 64-Bit Server VM 1.8.0_212-b10; Deflater: Intel; Inflater: Intel; Provider GCS is available; Picard version: Version:4.1.9.0
[Thu Mar 16 00:15:51 EDT 2023] picard.sam.SamToFastq done. Elapsed time: 0.00 minutes.
Runtime.totalMemory()=2175270912
To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp
htsjdk.samtools.SAMFormatException: SAM validation error: ERROR::INVALID_FLAG_SUPPLEMENTARY_ALIGNMENT:Record 47, Read name A01056:234:H2YWVDSX3:3:1122:27814:30780, Supplementary alignment flag should not be set for unmapped read.
at htsjdk.samtools.SAMUtils.processValidationErrors(SAMUtils.java:457)
at htsjdk.samtools.BAMFileReader$BAMFileIterator.advance(BAMFileReader.java:849)
at htsjdk.samtools.BAMFileReader$BAMFileIterator.next(BAMFileReader.java:834)
at htsjdk.samtools.BAMFileReader$BAMFileIterator.next(BAMFileReader.java:802)
at htsjdk.samtools.SamReader$AssertingIterator.next(SamReader.java:574)
at htsjdk.samtools.SamReader$AssertingIterator.next(SamReader.java:553)
at picard.sam.SamToFastq.doWork(SamToFastq.java:205)
at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:301)
at org.broadinstitute.hellbender.cmdline.PicardCommandLineProgramExecutor.instanceMain(PicardCommandLineProgramExecutor.java:37)
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)
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please answer about my question :)
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It looks like your input bam has some validation errors. Can you try running the tool ValidateSamFile and see whether it reports any problems with your bam?
It may be possible to bypass this validation check by running with VALIDATION_STRINGENCY=LENIENT, though this is not recommended.
Regards,
David
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thank you david!
could you wait a couple of weeks?
i am doing test slim pipeline provided by terra, which is not dependent the problematic step. after that, i want to come back this problem.
Best,
Hojin Lee
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