Genome Analysis Toolkit

Jonah Daneshmand · August 30, 2023 01:24

Hello GATK community,

I'm encountering an issue while processing BAM files through an RNA-seq variant calling pipeline using GATK tools. I would greatly appreciate your insights and guidance in resolving this issue.

Issue:

During testing of the pipeline, I'm experiencing unexpected behavior at the apply_bqsr step. Although the split_reads process and base_recalibrator step seem to execute successfully, I encounter issues specifically when applying base quality score recalibration.

Pipeline Overview:

The pipeline follows these steps:

MarkDuplicates
SplitNCigarReads
BaseRecalibrator
ApplyBQSR
HaplotypeCaller
VariantFiltration

Observations:

The mark_duplicates, split_ncigar_reads, and base_recalibrator steps appear to run without any errors.
The split_reads.bam file was indexed it seems by the split_reads step.
However, during the apply_bqsr step, I encounter errors related to premature EOF:
- htsjdk.samtools.util.RuntimeEOFException: Premature EOF. Expected XXX but only received YYY; BinaryCodec in readmode; file: /path/in/container/8751-AM-0002_S1_L005/split_reads.bam

Additional Information:

I'm using GATK version: 4.4.0.0
Docker container: broadinstitute/gatk:latest
BAM files are properly indexed before the apply_bqsr step.
I have ensured that I'm using consistent GTF and genome versions.
I've confirmed that my BAM files are intact and not corrupted.
Each BAM file contains read group information.
The reference genome matches the genome used for the analysis.
This is on cancer RNA-seq data.
I do not have tumor/normal pairs, so I am using the germline approach in GATK because the somatic approach needs tumor/normal pairs; am I correct in this?

Questions:

What could be causing this premature EOF issue during the apply_bqsr step?
The split_reads process and base_recalibrator seem to complete correctly. Why might applying BQSR encounter these errors?
Are there specific considerations when running BQSR in an RNA-seq context that I should be aware of?

Any insights or suggestions you can provide would be incredibly helpful in diagnosing and resolving this issue. If you need any further information or logs, please let me know, and I'll provide them promptly.

Thank you very much for your assistance.

apply_bqsr(split_reads_bam, recalibration_report)
23:16:06.353 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/gatk/gatk-package-4.4.0.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
23:16:06.394 INFO  ApplyBQSR - ------------------------------------------------------------
23:16:06.398 INFO  ApplyBQSR - The Genome Analysis Toolkit (GATK) v4.4.0.0
23:16:06.398 INFO  ApplyBQSR - For support and documentation go to https://software.broadinstitute.org/gatk/
23:16:06.398 INFO  ApplyBQSR - Executing as root@7819211d41d4 on Linux v5.10.16.3-microsoft-standard-WSL2 amd64
23:16:06.398 INFO  ApplyBQSR - Java runtime: OpenJDK 64-Bit Server VM v17.0.6+10-Ubuntu-0ubuntu118.04.1
23:16:06.398 INFO  ApplyBQSR - Start Date/Time: August 29, 2023 at 11:16:06 PM GMT
23:16:06.398 INFO  ApplyBQSR - ------------------------------------------------------------
23:16:06.399 INFO  ApplyBQSR - ------------------------------------------------------------
23:16:06.400 INFO  ApplyBQSR - HTSJDK Version: 3.0.5
23:16:06.400 INFO  ApplyBQSR - Picard Version: 3.0.0
23:16:06.400 INFO  ApplyBQSR - Built for Spark Version: 3.3.1
23:16:06.401 INFO  ApplyBQSR - HTSJDK Defaults.COMPRESSION_LEVEL : 2
23:16:06.401 INFO  ApplyBQSR - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
23:16:06.402 INFO  ApplyBQSR - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
23:16:06.402 INFO  ApplyBQSR - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
23:16:06.402 INFO  ApplyBQSR - Deflater: IntelDeflater
23:16:06.402 INFO  ApplyBQSR - Inflater: IntelInflater
23:16:06.403 INFO  ApplyBQSR - GCS max retries/reopens: 20
23:16:06.403 INFO  ApplyBQSR - Requester pays: disabled
23:16:06.403 INFO  ApplyBQSR - Initializing engine
23:16:06.665 INFO  ApplyBQSR - Done initializing engine
23:16:08.193 INFO  ProgressMeter - Starting traversal
23:16:08.194 INFO  ProgressMeter -        Current Locus  Elapsed Minutes       Reads Processed     Reads/Minute
23:16:08.545 INFO  ApplyBQSR - Shutting down engine
[August 29, 2023 at 11:16:08 PM GMT] org.broadinstitute.hellbender.tools.walkers.bqsr.ApplyBQSR done. Elapsed time: 0.04 minutes.
Runtime.totalMemory()=285212672
htsjdk.samtools.util.RuntimeEOFException: Premature EOF. Expected 315 but only received 161; BinaryCodec in readmode; file: /path/in/container/8751-AM-0002_S1_L005/split_reads.bam
	at htsjdk.samtools.util.BinaryCodec.readBytes(BinaryCodec.java:397)
	at htsjdk.samtools.util.BinaryCodec.readBytes(BinaryCodec.java:380)
	at htsjdk.samtools.BAMRecordCodec.decode(BAMRecordCodec.java:282)
	at htsjdk.samtools.BAMFileReader$BAMFileIterator.getNextRecord(BAMFileReader.java:882)
	at htsjdk.samtools.BAMFileReader$BAMFileIterator.advance(BAMFileReader.java:856)
	at htsjdk.samtools.BAMFileReader$BAMFileIterator.next(BAMFileReader.java:850)
	at htsjdk.samtools.BAMFileReader$BAMFileIterator.next(BAMFileReader.java:818)
	at htsjdk.samtools.SamReader$AssertingIterator.next(SamReader.java:591)
	at htsjdk.samtools.SamReader$AssertingIterator.next(SamReader.java:570)
	at org.broadinstitute.hellbender.utils.iterators.SAMRecordToReadIterator.next(SAMRecordToReadIterator.java:27)
	at org.broadinstitute.hellbender.utils.iterators.SAMRecordToReadIterator.next(SAMRecordToReadIterator.java:13)
	at java.base/java.util.Iterator.forEachRemaining(Iterator.java:133)
	at java.base/java.util.Spliterators$IteratorSpliterator.forEachRemaining(Spliterators.java:1845)
	at java.base/java.util.stream.AbstractPipeline.copyInto(AbstractPipeline.java:509)
	at java.base/java.util.stream.AbstractPipeline.wrapAndCopyInto(AbstractPipeline.java:499)
	at java.base/java.util.stream.ForEachOps$ForEachOp.evaluateSequential(ForEachOps.java:150)
	at java.base/java.util.stream.ForEachOps$ForEachOp$OfRef.evaluateSequential(ForEachOps.java:173)
	at java.base/java.util.stream.AbstractPipeline.evaluate(AbstractPipeline.java:234)
	at java.base/java.util.stream.ReferencePipeline.forEach(ReferencePipeline.java:596)
	at org.broadinstitute.hellbender.engine.ReadWalker.traverse(ReadWalker.java:98)
	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)
Using GATK jar /gatk/gatk-package-4.4.0.0-local.jar
Running:
    java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_sam
tools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /gatk/gatk-package-4.4.0.0-local.jar ApplyBQSR -R /path/to/reference/GRCh38.p14.genome.fa -I /path/in/container/8751-AM-0002_S1_L005/split_reads.bam --bqsr-recal-file /path/in/container/8751-AM-0002_S1_L005/recalibration_report.txt -O /path/in/container/8751-AM-0002_S1_L005/split_reads.bam

Few hours later update: After running ValidateSamFile from picard, it seems that some reads are missing NM tags and that counts as an error. I am adding those using samtools calmd, and will go from there.

Genome Analysis Toolkit

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Issue with RNA-seq Variant Calling Pipeline using GATK

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