Unable to trim uncertain bases without flow order information
Answered
Hi all,
I am using GATK to call my variants (WGS) and am getting the error "Unable to trim uncertain bases without flow order information" when I run HaplotypeCaller. I added the read groups and validated my .bam files and there are no errors. However, when I run gat HaplotypleCaller I get "Unable to trim uncertain bases without flow order information". I cannot find any information regarding this message.
REQUIRED for all errors and issues:
a) GATK version used: gatk-4.3.0.0
b) Exact command used:
#!/bin/bash
#SBATCH --job-name=vatiants
#SBATCH --ntasks=1
#SBATCH --time=124:10:00
#SBATCH --mem=14000
#SBATCH --mail-type=BEGIN,END
/mnt/bandit/users/lsanz/Software/gatk.tool/gatk-4.3.0.0/gatk --java-options "-Xmx8g" HaplotypeCaller \
-R /mnt/bandit/users/lsanz/Projects/mapping/ncbi_dataset/data/GCF_000001405.26/GCF_000001405.26_GRCh38_genomic.fna \
-I RG_sorted_RCH_001.bam \
-O RCH_001.vcf.gz \
-bamout out_RCH_001.bam
c) Entire program log:
Using GATK jar /mnt/bandit/users/lsanz/Software/gatk.tool/gatk-4.3.0.0/gatk-package-4.3.0.0-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_l
evel=2 -Xmx8g -jar /mnt/bandit/users/lsanz/Software/gatk.tool/gatk-4.3.0.0/gatk-package-4.3.0.0-local.jar HaplotypeCaller -R /mnt/bandit/users/lsanz/Projects
/mapping/ncbi_dataset/data/GCF_000001405.26/GCF_000001405.26_GRCh38_genomic.fna -I RG_sorted_RCH_001.bam -O RCH_001.vcf.gz -bamout out_RCH_001.bam
11:49:48.083 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/mnt/bandit/users/lsanz/Software/gatk.tool/gatk-4.3.0.0/gatk-package-4.3
.0.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
11:49:48.234 INFO HaplotypeCaller - ------------------------------------------------------------
11:49:48.235 INFO HaplotypeCaller - The Genome Analysis Toolkit (GATK) v4.3.0.0
11:49:48.235 INFO HaplotypeCaller - For support and documentation go to https://software.broadinstitute.org/gatk/
11:49:48.235 INFO HaplotypeCaller - Executing as lsanz@scn02.svi.edu.au on Linux v4.18.0-372.19.1.el8_6.x86_64 amd64
11:49:48.235 INFO HaplotypeCaller - Java runtime: OpenJDK 64-Bit Server VM v1.8.0_352-b08
11:49:48.235 INFO HaplotypeCaller - Start Date/Time: 21 December 2022 11:49:48 AM
11:49:48.235 INFO HaplotypeCaller - ------------------------------------------------------------
11:49:48.235 INFO HaplotypeCaller - ------------------------------------------------------------
11:49:48.236 INFO HaplotypeCaller - HTSJDK Version: 3.0.1
11:49:48.236 INFO HaplotypeCaller - Picard Version: 2.27.5
11:49:48.236 INFO HaplotypeCaller - Built for Spark Version: 2.4.5
11:49:48.236 INFO HaplotypeCaller - HTSJDK Defaults.COMPRESSION_LEVEL : 2
11:49:48.236 INFO HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
11:49:48.236 INFO HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
11:49:48.236 INFO HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
11:49:48.236 INFO HaplotypeCaller - Deflater: IntelDeflater
11:49:48.236 INFO HaplotypeCaller - Inflater: IntelInflater
11:49:48.238 INFO HaplotypeCaller - GCS max retries/reopens: 20
11:49:48.238 INFO HaplotypeCaller - Requester pays: disabled
11:49:48.238 INFO HaplotypeCaller - Initializing engine
11:49:48.791 INFO HaplotypeCaller - Done initializing engine
11:49:48.825 INFO HaplotypeCallerEngine - Disabling physical phasing, which is supported only for reference-model confidence output
11:49:48.890 INFO NativeLibraryLoader - Loading libgkl_utils.so from jar:file:/mnt/bandit/users/lsanz/Software/gatk.tool/gatk-4.3.0.0/gatk-package-4.3.0.0-local.jar!/com/intel/gkl/native/libgkl_utils.so
11:49:48.892 INFO NativeLibraryLoader - Loading libgkl_pairhmm_omp.so from jar:file:/mnt/bandit/users/lsanz/Software/gatk.tool/gatk-4.3.0.0/gatk-package-4.3.0.0-local.jar!/com/intel/gkl/native/libgkl_pairhmm_omp.so
11:49:48.920 INFO IntelPairHmm - Using CPU-supported AVX-512 instructions
11:49:48.920 INFO IntelPairHmm - Flush-to-zero (FTZ) is enabled when running PairHMM
11:49:48.921 INFO IntelPairHmm - Available threads: 2
11:49:48.921 INFO IntelPairHmm - Requested threads: 4
11:49:48.921 WARN IntelPairHmm - Using 2 available threads, but 4 were requested
11:49:48.921 INFO PairHMM - Using the OpenMP multi-threaded AVX-accelerated native PairHMM implementation
11:49:48.959 INFO ProgressMeter - Starting traversal
11:49:48.959 INFO ProgressMeter - Current Locus Elapsed Minutes Regions Processed Regions/Minute
11:49:49.209 INFO VectorLoglessPairHMM - Time spent in setup for JNI call : 0.0
11:49:49.209 INFO PairHMM - Total compute time in PairHMM computeLogLikelihoods() : 0.0
11:49:49.210 INFO SmithWatermanAligner - Total compute time in java Smith-Waterman : 0.00 sec
11:49:49.280 INFO HaplotypeCaller - Shutting down engine
[21 December 2022 11:49:49 AM] org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCaller done. Elapsed time: 0.02 minutes.
Runtime.totalMemory()=446693376
org.broadinstitute.hellbender.exceptions.GATKException: Unable to trim uncertain bases without flow order information
at org.broadinstitute.hellbender.utils.read.FlowBasedRead.hardClipUncertainBases(FlowBasedRead.java:1035)
at org.broadinstitute.hellbender.tools.walkers.haplotypecaller.AssemblyBasedCallerUtils.finalizeRegion(AssemblyBasedCallerUtils.java:140)
at org.broadinstitute.hellbender.tools.walkers.haplotypecaller.AssemblyBasedCallerUtils.assembleReads(AssemblyBasedCallerUtils.java:332)
at org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCallerEngine.callRegion(HaplotypeCallerEngine.java:632)
at org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCaller.apply(HaplotypeCaller.java:271)
at org.broadinstitute.hellbender.engine.AssemblyRegionWalker.processReadShard(AssemblyRegionWalker.java:200)
at org.broadinstitute.hellbender.engine.AssemblyRegionWalker.traverse(AssemblyRegionWalker.java:173)
at org.broadinstitute.hellbender.engine.GATKTool.doWork(GATKTool.java:1095)
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)
Thanks
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Hi,
Did you ever solve this problem? I am having the same issue. I am trying to run HaplotypeCaller with two input BAMs, one from an Illumina sequencing experiment and one from an Oxford Nanopore run. I have assigned read groups to both and validated both BAM files. Full error message is:
org.broadinstitute.hellbender.exceptions.GATKException: Unable to trim uncertain bases without flow order information
at org.broadinstitute.hellbender.utils.read.FlowBasedRead.hardClipUncertainBases(FlowBasedRead.java:1035)
at org.broadinstitute.hellbender.tools.walkers.haplotypecaller.AssemblyBasedCallerUtils.finalizeRegion(AssemblyBasedCallerUtils.java:140)
at org.broadinstitute.hellbender.tools.walkers.haplotypecaller.AssemblyBasedCallerUtils.assembleReads(AssemblyBasedCallerUtils.java:332)
at org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCallerEngine.callRegion(HaplotypeCallerEngine.java:632)
at org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCaller.apply(HaplotypeCaller.java:271)
at org.broadinstitute.hellbender.engine.AssemblyRegionWalker.processReadShard(AssemblyRegionWalker.java:200)
at org.broadinstitute.hellbender.engine.AssemblyRegionWalker.traverse(AssemblyRegionWalker.java:173)
at org.broadinstitute.hellbender.engine.GATKTool.doWork(GATKTool.java:1095)
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)
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Hi Allison,
Those bam files had been generated using minimap2. I repeated the mapping to reference with bwa (https://github.com/lh3/bwa) and the problem got solved.
I hope it helps.
Laura
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Hi Laura,
Thank you so much! I used Minimap2 as well. I appreciate the help.
Best,
Allison
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I had the same issue with Minimap2.
It seems that it is the TP:A:P field that Minimap2 outputs that is causing trouble. For some reason, it triggers hardclipping in GATK, which then fails for some other reason.
I solved it by removing the TP:A:P field from the output (piping the output through a hacky cut -f 16 --complement thing).
I'm not sure if triggering hardclipping because there is a "TP:A:P" field in GATK is a bug or not, but it seems to be caused by this line which defines TP as a flow field:
Then, "isFlow" returns True:
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Hi, I'm having the same issue, and I'm using minimap2 is there any solution for the variant calling with gatk, or should I perform the align with bwa.
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Hi Debora,
I rolled back to GATK version 4.2.6.1 and that worked for me with the minimap2 reads.
Allison
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It worked thanks
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Hello All, Thank you for bringing this to our attention. We hope to fix this issue in the next GATK release. We are tracking the issue here: https://github.com/broadinstitute/gatk/issues/8335.
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