Hi everyone. I'm trying to run Mutect2 on some Illumina sequencing files that were processed as follows (GATK best practices): trimmed with Trimmomatic, mapping with Minimal2, converted to BAM with SAMtools, groups and duplicates added with Picard, Base Recalibrator.

I have a large number of files to analyse. In some cases, Mutect2 seems to work perfectly and I get the message "Tool returned: SUCESS". In other cases, I get the error "Unable to trim uncertain bases without flow order information". I don't understand why this only occurs with certain files. Files that are analysed successfully and files that are not come from the same sample batches and were processed with the identical workflow above.

Thank you for your help!

REQUIRED for all errors and issues:
a) GATK version used: GATK 4.4.0.0

b) Exact command used (dbDir contains database files used in GATK best practices):

gatk Mutect2 \
   -R "$dbDir/chr2_chr4_chr20.fasta" \
   -I "sample1.picard.markedDup.recal.bam"\
   --germline-resource"$dbDir/somatic-hg38_af-only-gnomad.hg38.vcf" \
   -O $resultDir/$sampleName.Mutect.vcf.gz

c) Entire program log:
Using GATK jar /home/mcarta/gatk-4.4.0.0/gatk-package-4.4.0.0-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_level=2 -jar /home/mcarta/gatk-4.4.0.0/gatk-package-4.4.0.0-local.jar Mutect2 -R /home/mcarta/databases/chr2_chr4_chr20.fasta -I 20211027.0-o26424_1_4-CJD13_fr.picard.markedDup.recal.bam --germline-resource /home/mcarta/databases/somatic-hg38_af-only-gnomad.hg38.vcf -O /add/mutect_output/2021-10-19_first_CJD_seq//20211027.0-o26424_1_4-CJD13_fr.Mutect.vcf.gz
15:53:52.396 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/home/mcarta/gatk-4.4.0.0/gatk-package-4.4.0.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
15:53:52.435 INFO  Mutect2 - ------------------------------------------------------------
15:53:52.438 INFO  Mutect2 - The Genome Analysis Toolkit (GATK) v4.4.0.0
15:53:52.438 INFO  Mutect2 - For support and documentation go to https://software.broadinstitute.org/gatk/
15:53:52.439 INFO  Mutect2 - Executing as mcarta@ubuntu22 on Linux v5.15.0-89-generic amd64
15:53:52.439 INFO  Mutect2 - Java runtime: OpenJDK 64-Bit Server VM v17.0.8.1+1-Ubuntu-0ubuntu122.04
15:53:52.439 INFO  Mutect2 - Start Date/Time: January 7, 2024 at 3:53:52 PM UTC
15:53:52.440 INFO  Mutect2 - ------------------------------------------------------------
15:53:52.440 INFO  Mutect2 - ------------------------------------------------------------
15:53:52.441 INFO  Mutect2 - HTSJDK Version: 3.0.5
15:53:52.441 INFO  Mutect2 - Picard Version: 3.0.0
15:53:52.441 INFO  Mutect2 - Built for Spark Version: 3.3.1
15:53:52.442 INFO  Mutect2 - HTSJDK Defaults.COMPRESSION_LEVEL : 2
15:53:52.443 INFO  Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
15:53:52.444 INFO  Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
15:53:52.444 INFO  Mutect2 - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
15:53:52.445 INFO  Mutect2 - Deflater: IntelDeflater
15:53:52.445 INFO  Mutect2 - Inflater: IntelInflater
15:53:52.446 INFO  Mutect2 - GCS max retries/reopens: 20
15:53:52.446 INFO  Mutect2 - Requester pays: disabled
15:53:52.447 INFO  Mutect2 - Initializing engine
15:53:52.619 INFO  FeatureManager - Using codec VCFCodec to read file file:///home/mcarta/databases/somatic-hg38_af-only-gnomad.hg38.vcf
15:53:54.730 INFO  Mutect2 - Done initializing engine
15:53:54.743 INFO  NativeLibraryLoader - Loading libgkl_utils.so from jar:file:/home/mcarta/gatk-4.4.0.0/gatk-package-4.4.0.0-local.jar!/com/intel/gkl/native/libgkl_utils.so
15:53:54.744 INFO  NativeLibraryLoader - Loading libgkl_pairhmm_omp.so from jar:file:/home/mcarta/gatk-4.4.0.0/gatk-package-4.4.0.0-local.jar!/com/intel/gkl/native/libgkl_pairhmm_omp.so
15:53:54.756 INFO  IntelPairHmm - Flush-to-zero (FTZ) is enabled when running PairHMM
15:53:54.757 INFO  IntelPairHmm - Available threads: 2
15:53:54.757 INFO  IntelPairHmm - Requested threads: 4
15:53:54.758 WARN  IntelPairHmm - Using 2 available threads, but 4 were requested
15:53:54.758 INFO  PairHMM - Using the OpenMP multi-threaded AVX-accelerated native PairHMM implementation
15:53:54.787 INFO  ProgressMeter - Starting traversal
15:53:54.788 INFO  ProgressMeter -        Current Locus  Elapsed Minutes     Regions Processed   Regions/Minute
15:53:55.059 INFO  VectorLoglessPairHMM - Time spent in setup for JNI call : 0.0
15:53:55.060 INFO  PairHMM - Total compute time in PairHMM computeLogLikelihoods() : 0.0
15:53:55.060 INFO  SmithWatermanAligner - Total compute time in java Smith-Waterman : 0.00 sec
15:53:55.061 INFO  Mutect2 - Shutting down engine
[January 7, 2024 at 3:53:55 PM UTC] org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2 done. Elapsed time: 0.05 minutes.
Runtime.totalMemory()=822083584
org.broadinstitute.hellbender.exceptions.GATKException: Unable to trim uncertain bases without flow order information
        at org.broadinstitute.hellbender.utils.read.FlowBasedRead.hardClipUncertainBases(FlowBasedRead.java:1039)
        at org.broadinstitute.hellbender.tools.walkers.haplotypecaller.AssemblyBasedCallerUtils.finalizeRegion(AssemblyBasedCallerUtils.java:142)
        at org.broadinstitute.hellbender.tools.walkers.haplotypecaller.AssemblyBasedCallerUtils.assembleReads(AssemblyBasedCallerUtils.java:334)
        at org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2Engine.callRegion(Mutect2Engine.java:262)
        at org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2.apply(Mutect2.java:304)
        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:1098)
        at org.broadinstitute.hellbender.cmdline.CommandLineProgram.runTool(CommandLineProgram.java:149)
        at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMainPostParseArgs(CommandLineProgram.java:198)
        at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:217)
        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)