HaplotypeCaller does not work

Answered

Follow

Roohollah kheiri

• December 13, 2021 11:08

I am using the following command

./gatk --java-options "-Xmx8G" HaplotypeCaller -I /home/kheiri/Urmia/mapping/BAM/mappingBamSorted.bam -R /home/kheiri/Urmia/mapping/ReferenceGenomes/Reference_GenomeSL.fna -O /home/kheiri/Urmia/mapping/HAPLOTYPE.vcf

I receive this text and HaplotypeCaller stops working

Using GATK jar /home/kheiri/Urmia/gatk-4.2.3.0/gatk-package-4.2.3.0-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 -Xmx8G -jar /home/kheiri/Urmia/gatk-4.2.3.0/gatk-package-4.2.3.0-local.jar HaplotypeCaller -I /home/kheiri/Urmia/mapping/BAM/mappingBamSorted.bam -R /home/kheiri/Urmia/mapping/ReferenceGenomes/Reference_GenomeSL.fna -O /home/kheiri/Urmia/mapping/HAPLOTYPE.vcf
14:35:57.675 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/home/kheiri/Urmia/gatk-4.2.3.0/gatk-package-4.2.3.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
Dec 13, 2021 2:35:57 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
14:35:57.817 INFO HaplotypeCaller - ------------------------------------------------------------
14:35:57.818 INFO HaplotypeCaller - The Genome Analysis Toolkit (GATK) v4.2.3.0
14:35:57.818 INFO HaplotypeCaller - For support and documentation go to https://software.broadinstitute.org/gatk/
14:35:57.818 INFO HaplotypeCaller - Executing as kheiri@kheiri-GP60-2OD on Linux v5.11.0-41-generic amd64
14:35:57.818 INFO HaplotypeCaller - Java runtime: OpenJDK 64-Bit Server VM v1.8.0_152-release-1056-b12
14:35:57.818 INFO HaplotypeCaller - Start Date/Time: December 13, 2021 2:35:57 PM IRST
14:35:57.818 INFO HaplotypeCaller - ------------------------------------------------------------
14:35:57.818 INFO HaplotypeCaller - ------------------------------------------------------------
14:35:57.819 INFO HaplotypeCaller - HTSJDK Version: 2.24.1
14:35:57.819 INFO HaplotypeCaller - Picard Version: 2.25.4
14:35:57.819 INFO HaplotypeCaller - Built for Spark Version: 2.4.5
14:35:57.819 INFO HaplotypeCaller - HTSJDK Defaults.COMPRESSION_LEVEL : 2
14:35:57.819 INFO HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
14:35:57.819 INFO HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
14:35:57.819 INFO HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
14:35:57.819 INFO HaplotypeCaller - Deflater: IntelDeflater
14:35:57.819 INFO HaplotypeCaller - Inflater: IntelInflater
14:35:57.820 INFO HaplotypeCaller - GCS max retries/reopens: 20
14:35:57.820 INFO HaplotypeCaller - Requester pays: disabled
14:35:57.820 INFO HaplotypeCaller - Initializing engine
14:35:58.986 INFO HaplotypeCaller - Done initializing engine
14:35:59.191 INFO HaplotypeCallerEngine - Disabling physical phasing, which is supported only for reference-model confidence output
14:35:59.194 INFO HaplotypeCaller - Shutting down engine
[December 13, 2021 2:35:59 PM IRST] org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCaller done. Elapsed time: 0.03 minutes.
Runtime.totalMemory()=700448768
java.lang.IllegalStateException: the sample list cannot be null or empty
at org.broadinstitute.hellbender.utils.Utils.validate(Utils.java:814)
at org.broadinstitute.hellbender.tools.walkers.genotyper.GenotypingEngine.<init>(GenotypingEngine.java:69)
at org.broadinstitute.hellbender.tools.walkers.genotyper.MinimalGenotypingEngine.<init>(MinimalGenotypingEngine.java:46)
at org.broadinstitute.hellbender.tools.walkers.genotyper.MinimalGenotypingEngine.<init>(MinimalGenotypingEngine.java:35)
at org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCallerEngine.initializeActiveRegionEvaluationGenotyperEngine(HaplotypeCallerEngine.java:342)
at org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCallerEngine.initialize(HaplotypeCallerEngine.java:231)
at org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCallerEngine.<init>(HaplotypeCallerEngine.java:188)
at org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCaller.onTraversalStart(HaplotypeCaller.java:259)
at org.broadinstitute.hellbender.engine.GATKTool.doWork(GATKTool.java:1083)
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)

Can anyone help me to solve this problem?

0

• 

  Genevieve Brandt (she/her)

  • December 16, 2021 18:53

  Hi Roohollah kheiri,

  Thanks for writing into the forum so we can help to get HaplotypeCaller working for you! The error message with helpful information is here:

  java.lang.IllegalStateException: the sample list cannot be null or empty

  I think there is a problem in your input bam. Either you do not have read groups properly added, or the bam is not formatted correctly. Take a look at these resources:

  1. Validate that the bam file has correct formatting with the tool ValidateSamFile.
  2. Learn more about read groups & how to fix them.

  I hope this helps, please let me know if you have further questions.

  Best,

  Genevieve

  1

  Comment actions Permalink
• 

  Daniel Castañeda Mogollón

  • April 03, 2022 02:06

  Hello! I would like to follow up on this as I am getting the same error as well. Here's what I've done so far:

  Alignment of raw .fastq to a reference:

  bwa -mem 3d7_dhps.fasta R1.fastq R2.fastq > S13_dhps.sam

  Conversion from sam to bam:

  samtools view -b S13_dhps.sam > S13_dhps.bam

  Sorting:

  samtools sort S13_dhps.bam > S13_dhps_sorted.bam

  Marking duplicates:

  picard MarkDuplicates -I S13_dhps_sorted.bam -O S13_marked_duplicates.bam -M S13_marked_duplicates_summary.txt

  Validating sam file:

  picard ValidateSamFile -I S13_dhps.bam -MODE SUMMARY

  Then I get this output, which I believe is the cause of my error message when I try running the HaplotypeCaller:

  ## HISTOGRAM java.lang.String
  
  Error Type Count
  
  ERROR:MISSING_READ_GROUP 1
  
  WARNING:RECORD_MISSING_READ_GROUP 48808
  
  

  For referencing I did this:

  samtools faidx 3d7_dhps.fasta

  And for the dictionary:

  gatk CreateSequenceDictionary -R 3d7_dhps.fasta

   

  I am a bit lost and desperate! Thanks for your help!

  0

  Comment actions Permalink
• 

  Genevieve Brandt (she/her)

  • April 04, 2022 21:08

  Daniel Castañeda Mogollón your issue looks different from this original post. It looks like you are having a problem with your read groups. If the linked article does not solve your issue, feel free to make a new post.

  0

  Comment actions Permalink
• 

  Daniel Castañeda Mogollón

  • April 05, 2022 05:27

  Yes! That fixed the issue! Thanks a lot!

  0

  Comment actions Permalink
• 

  Genevieve Brandt (she/her)

  • April 05, 2022 20:15

  Great, glad to hear it!

  0

  Comment actions Permalink

Please sign in to leave a comment.