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# HaplotypeCaller 2.4

Posts: 13Member

I am getting the following error. What is the minimum read size to do assembly? 50 basepair too short?

##### ERROR stack trace

java.lang.IllegalStateException: Reads are too small for use in assembly.
at org.broadinstitute.sting.gatk.walkers.haplotypecaller.DeBruijnAssembler.createDeBruijnGraphs(DeBruijnAssembler.java:139)
at org.broadinstitute.sting.gatk.walkers.haplotypecaller.DeBruijnAssembler.runLocalAssembly(DeBruijnAssembler.java:123)
at org.broadinstitute.sting.gatk.walkers.haplotypecaller.HaplotypeCaller.map(HaplotypeCaller.java:483)
at org.broadinstitute.sting.gatk.walkers.haplotypecaller.HaplotypeCaller.map(HaplotypeCaller.java:132)
at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.processActiveRegion(TraverseActiveRegions.java:552)
at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.processActiveRegions(TraverseActiveRegions.java:512)
at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.traverse(TraverseActiveRegions.java:244)
at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.traverse(TraverseActiveRegions.ja
at org.broadinstitute.sting.gatk.executive.LinearMicroScheduler.execute(LinearMicroScheduler.j
at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:283
at org.broadinstitute.sting.gatk.CommandLineExecutable.execute(CommandLineExecutable.java:1
at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:24
at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:15
at org.broadinstitute.sting.gatk.CommandLineGATK.main(CommandLineGATK.java:91)

::::::::::::::

Tagged:

## Answers

• Posts: 13Member

Actually the reads on this sample is 200 bases. It is from a MiSeq run and works fine with version 2.3.

• Posts: 7,205Administrator, GATK Developer admin

Ooh, first bug report for 2.4 already! That was fast.

We'll take a look at it. Could you upload a snippet of your bam where the error occurs? We'll probably need to reproduce this locally to figure out what's going on here.

Geraldine Van der Auwera, PhD

• Posts: 13Member

Let me check a few other things and let this run finish. I will be interested to see if the interval that I am running this on returns any snps/indels or if it exits out.

Speed has improved through by a factor of 4?

• Posts: 7,205Administrator, GATK Developer admin

Sure, no problem. Ryan tells me it's probably a region where lots of reads are getting clipped and resulting in very short leftovers. Before 2.4 the HC would simply have skipped through the region silently, but now it's flipping out unnecessarily. I expect if you look at the interval in question that may confirm his supposition.

Geraldine Van der Auwera, PhD

• Posts: 13Member

It would be nice if the stack trace would give me some idea as to which interval it is flipping out about.

• Posts: 13Member

Any Idea what this one means?

java.lang.ArrayIndexOutOfBoundsException: -1
at org.broadinstitute.sting.gatk.walkers.haplotypecaller.GenotypingEngine.generateVCsFromAlignment(GenotypingEngine.java:675)
at org.broadinstitute.sting.gatk.walkers.haplotypecaller.GenotypingEngine.assignGenotypeLikelihoods(GenotypingEngine.java:140)
at org.broadinstitute.sting.gatk.walkers.haplotypecaller.HaplotypeCaller.map(HaplotypeCaller.java:500)
at org.broadinstitute.sting.gatk.walkers.haplotypecaller.HaplotypeCaller.map(HaplotypeCaller.java:132)
at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.processActiveRegion(TraverseActiveRegions.java:552)
at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.processActiveRegions(TraverseActiveRegions.java:512)
at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.traverse(TraverseActiveRegions.java:244)
at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.traverse(TraverseActiveRegions.java:69)
at org.broadinstitute.sting.gatk.executive.LinearMicroScheduler.execute(LinearMicroScheduler.java:100)
at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:283)
at org.broadinstitute.sting.gatk.CommandLineExecutable.execute(CommandLineExecutable.java:113)
at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:245)
at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:152)
at org.broadinstitute.sting.gatk.CommandLineGATK.main(CommandLineGATK.java:91)

##### ERROR ------------------------------------------------------------------------------------------
• Posts: 13Member

How do I upload?

• Posts: 7,205Administrator, GATK Developer admin

Off-by-one error while taking down the variant context info. Sounds like another edge case that's not being handled properly.

To see which interval HC is choking on, use --debug -- it will print out a lot of info including the interval it's processing.

See the instructions on how to file a bug report here:
http://www.broadinstitute.org/gatk/guide/article?id=1894

Geraldine Van der Auwera, PhD

• Posts: 1Member

For off-by-one error, the following seems to bypass the issue.
In GenotypingEngine.java:675, replace

final byte refByte = ref[refPos-1];
if( BaseUtils.isRegularBase(refByte) ) {
insertionAlleles.add( Allele.create(refByte, true) );
}

with:

// Only create a ref allele if it exists and is a regular base
if (refPos > 0) {
final byte refByte = ref[refPos-1];
if( BaseUtils.isRegularBase(refByte) ) {
insertionAlleles.add( Allele.create(refByte, true) );
}
}
• Posts: 13Member

Is this file in the jar zip file? GenotypingEngine.java
or do I download a different source version of GATK?
Also I was using the Haplotype caller is it still this file?

• Posts: 7,205Administrator, GATK Developer admin

This issue has been fixed in the latest version of GATK (2.4-7).

Geraldine Van der Auwera, PhD

• Posts: 13Member

hrmm. I am using
(version 2.4-7-g5e89f01):

##### ERROR

But still get the error. Is this the most recent version?

I will work on getting an example for you guys. I did not have time in my last attempt unless you tell me this is an old version of 2.4-7

• Posts: 13Member

I have uploaded the snippet of the problem with associated scripts that I was using.
-rw-r--r-- 1 depristo wga 287606741 Mar 15 17:37 upload-minusoneError.zip

• Posts: 13Member

Geraldine,
Did you see I posted the snippet for the error above?
Thanks

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