GATK HaplotypeCaller Error

Question

[sahar.salimi92]

Hi, I run all the steps below to run GATK

gatk HaplotypeCaller -R chrX.fa -I ERR188044_chrX.bam -O ERR188044_chrX.vcf -ERC GVCF

But I got the following error

Using GATK jar /home/sahar/apps/gatk-4.1.9.0/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 /home/sahar/apps/gatk-4.1.9.0/gatk-package-4.1.9.0-local.jar HaplotypeCaller -R chrX.fa -I ERR188044_chrX.bam -O ERR188044_chrX.vcf -ERC GVCF
23:14:55.161 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/home/sahar/apps/gatk-4.1.9.0/gatk-package-4.1.9.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
Feb 18, 2021 11:15:03 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
23:15:03.691 INFO  HaplotypeCaller - ------------------------------------------------------------
23:15:03.700 INFO  HaplotypeCaller - The Genome Analysis Toolkit (GATK) v4.1.9.0
23:15:03.703 INFO  HaplotypeCaller - For support and documentation go to https://software.broadinstitute.org/gatk/
23:15:03.704 INFO  HaplotypeCaller - Executing as sahar@sahar-VirtualBox on Linux v5.8.0-43-generic amd64
23:15:03.711 INFO  HaplotypeCaller - Java runtime: Java HotSpot(TM) 64-Bit Server VM v15.0.2+7-27
23:15:03.712 INFO  HaplotypeCaller - Start Date/Time: February 18, 2021 at 11:14:54 PM IRST
23:15:03.712 INFO  HaplotypeCaller - ------------------------------------------------------------
23:15:03.712 INFO  HaplotypeCaller - ------------------------------------------------------------
23:15:03.733 INFO  HaplotypeCaller - HTSJDK Version: 2.23.0
23:15:03.733 INFO  HaplotypeCaller - Picard Version: 2.23.3
23:15:03.734 INFO  HaplotypeCaller - HTSJDK Defaults.COMPRESSION_LEVEL : 2
23:15:03.734 INFO  HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
23:15:03.739 INFO  HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
23:15:03.739 INFO  HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
23:15:03.739 INFO  HaplotypeCaller - Deflater: IntelDeflater
23:15:03.748 INFO  HaplotypeCaller - Inflater: IntelInflater
23:15:03.748 INFO  HaplotypeCaller - GCS max retries/reopens: 20
23:15:03.749 INFO  HaplotypeCaller - Requester pays: disabled
23:15:03.749 INFO  HaplotypeCaller - Initializing engine
23:15:03.775 INFO  HaplotypeCaller - Shutting down engine
[February 18, 2021 at 11:15:03 PM IRST] org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCaller done. Elapsed time: 0.15 minutes.
Runtime.totalMemory()=62980096
***********************************************************************

A USER ERROR has occurred: Fasta dict file file:///home/sahar/project/chrX/chrX_data/gatk/chrX.dict for reference file:///home/sahar/project/chrX/chrX_data/gatk/chrX.fa does not exist. Please see http://gatkforums.broadinstitute.org/discussion/1601/how-can-i-prepare-a-fasta-file-to-use-as-reference for help creating it.

***********************************************************************
Set the system property GATK_STACKTRACE_ON_USER_EXCEPTION (--java-options '-DGATK_STACKTRACE_ON_USER_EXCEPTION=true') to print the stack trace.

What should I do to fix this error?

The GATK version 4.1.9.0, The python version 3.8.5, The java version java-8-openjdk-amd64 and java-15-oracle which is set to java-8-openjdk-amd64.

Thank you for your help.

Answer 1

[Pierre Lindenbaum]

the answer to your problem is quite clear to me:

A USER ERROR has occurred: Fasta dict file file:///home/sahar/project/chrX/chrX_data/gatk/chrX.dict for reference file:///home/sahar/project/chrX/chrX_data/gatk/chrX.fa does not exist. Please see http://gatkforums.broadinstitute.org/discussion/1601/how-can-i-prepare-a-fasta-file-to-use-as-reference for help creating it.

[sahar.salimi92]

I have both orders

samtools dict chrX.fa 
@HD VN:1.0  SO:unsorted
@SQ SN:chrX LN:156040895    M5:49527016a48497d9d1cbd8e4a9049bd3 UR:file:///home/sahar/project/chrX/chrX_data/gatk/chrX.fa

and

samtools faidx chrX.fa

I run the same error again.

[GenoMax]

You need to create a sequence dictionary: https://gatk.broadinstitute.org/hc/en-us/articles/360036729911-CreateSequenceDictionary-Picard-

[Pierre Lindenbaum]

samtools dict output must be written in a file chrX.dict

[sahar.salimi92]

I executed the command like this,

gatk HaplotypeCaller -R chrX.dict -I ERR188044_chrX.bam -O ERR188044_chrX.vcf -ERC GVCF

but it still has the same error.

[Pierre Lindenbaum]

gatk HaplotypeCaller -R chrX.fa -I ERR188044_chrX.bam -O ERR188044_chrX.vcf -ERC GVCF

with an existing chrX.dict in the same directory of chrX.fa

[sahar.salimi92]

By executing this command,

gatk HaplotypeCaller -R chrX.fa -I ERR188044_chrX.bam -O ERR188044_chrX.vcf -ERC GVCF

I encountered this error.

A USER ERROR has occurred: Argument emit-ref-confidence has a bad value: Can only be used in single sample mode currently. Use the --sample-name argument to run on a single sample out of a multi-sample BAM file.

[Pierre Lindenbaum]

there are several samples in the lines @RG SM:xxx in the header of the bam file.

what it the output of

samtools view -H ERR188044_chrX.bam | grep '^RG'

how many SM: do you see ?

Only one sample is allowed in GVCF mode...

[sahar.salimi92]

There is only one item inside the ERR188044_chrX.bam file.

Data is related to the ftp://ftp.ccb.jhu.edu/pub/RNAseq_protocol link.

[Pierre Lindenbaum]

can I see the output of the samtools view command above please

[sahar.salimi92]

Sorry, answered late, becuase I can only send 5 posts in 6 hours. I did not see the output, but I used the

samtools view -Sb ERR188044_chrX.sam > ERR188044_chrX.bam

command to convert the sam file to bam. And I answered the previous question according to the bam file created.