GATK v18.104.22.168 Mutect2 - Attempting to add a read to ActiveRegion out of order w.r.t. error
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
Here is the command:
java -Xmx8g -XX:ParallelGCThreads=4 -Djava.io.tmpdir=./tmp -jar /data/bin/gatk-22.214.171.124/gatk-package-126.96.36.199-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!