GATK v4.1.0.0 Mutect2 - Attempting to add a read to ActiveRegion out of order w.r.t. error

pongorlorincpongorlorinc Member
edited February 6 in Ask the GATK team

Dear GATK,

I am trying to analyze a set of genome sequencing files from normal and tumor samples. The HaplotypeCaller mutation calling worked, but I keep getting an error with Mutect2.

This is the error message (removed the actual read names):
java.lang.IllegalArgumentException: Attempting to add a read to ActiveRegion out of order w.r.t. other reads: lastRead chr22:16050117-16050216 at 16050117 attempting to add chr22:16050044-16050143 at 16050044

The problem is, that the two reads are not from one BAM file, they are from two separate BAM files. (Both BAM files have indexes, and germline mutation calling worked well).

The GATK v3.8 Mutect2 seems to work well.

The read group is present in both BAM files. They are "tumor_T" and normal_N".

Here are a few key parameters:
java version 1.8.0_181
GATK: gatk-4.1.0.0/gatk-package-4.1.0.0-local.jar

Here is the command:
java -Xmx8g -XX:ParallelGCThreads=4 -Djava.io.tmpdir=./tmp -jar /data/bin/gatk-4.1.0.0/gatk-package-4.1.0.0-local.jar Mutect2 -R /data/genome/GRCh37-lite.mod.fa -I tumor_T.main.re.reheader.bam -I normal_N.main.re.reheader.bam -tumor tumor_T -normal normal_N -L chr22 -O ./mutect2.chr22.raw.vcf

1) Do you have any suggestions why this problem might come up?

2) Is there any difference between mutect2 in version 3.8 and 4.1 in terms of accuracy? If they are similar, then I can perform the analysis with version 3.8.

Thanks in advance!

Best Answer

  • shleeshlee Cambridge admin
    Accepted Answer

    Hi @pongorlorinc,

    @AdelaideR asked I look into your question. The error:

    Attempting to add a read to ActiveRegion out of order w.r.t. other reads:
    [...] chr22:16050117-16050216 at 16050117 attempting to add 
    [...] chr22:16050044-16050143 at 16050044
    

    stems from this code. My guess is that your reads are out of order, i.e. not coordinate-sorted. Can you try sorting with SortSam and see if this solves the issue?

    The tool will assemble tumor and normal reads jointly. This is expected.

    The GATK asks you use GATK4's Mutect2 and discontinue use of GATK3 MuTect2. Blog#10911 outlines some differences between the two versions to help you get oriented if you are not already familiar. In all, GATK4 Mutect2 should increase the sensitivity of calling and also offer more filtering options to increase precision in a manner that balances sensitivity. Blog#1137 gives links to various Mutect2 resources that you may find of interest.

Answers

  • shleeshlee CambridgeMember, Broadie, Moderator admin
    Accepted Answer

    Hi @pongorlorinc,

    @AdelaideR asked I look into your question. The error:

    Attempting to add a read to ActiveRegion out of order w.r.t. other reads:
    [...] chr22:16050117-16050216 at 16050117 attempting to add 
    [...] chr22:16050044-16050143 at 16050044
    

    stems from this code. My guess is that your reads are out of order, i.e. not coordinate-sorted. Can you try sorting with SortSam and see if this solves the issue?

    The tool will assemble tumor and normal reads jointly. This is expected.

    The GATK asks you use GATK4's Mutect2 and discontinue use of GATK3 MuTect2. Blog#10911 outlines some differences between the two versions to help you get oriented if you are not already familiar. In all, GATK4 Mutect2 should increase the sensitivity of calling and also offer more filtering options to increase precision in a manner that balances sensitivity. Blog#1137 gives links to various Mutect2 resources that you may find of interest.

  • Thank you @shlee

    The sorting solved the problem!

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