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Problem with recalibrated bam files

astrandastrand New YorkMember


I recently re-generated my realigned bam files (realigned.bam) from the GATK Best Practices, but I encountered an error when I validated the bam files using the validation tool (ValidateSamFile.jar) in Picard:

ERROR: Record 188823, Read name HWI-ST1329:286:C2DT8ACXX:2:1303:11866:8561, Mate alignment does not match alignment start of mate
ERROR: Record 188823, Read name HWI-ST1329:286:C2DT8ACXX:2:1303:11866:8561, Mate negative strand flag does not match read negative strand flag of mate


What exactly is going on? Where in the pipeline could this have occurred? I do not have the intermediary files so I cannot diagnose this accurately. Before I started the GATK Best Practices, my bam files had no problems; when I validated them I did not get any error messages. Does this mean that I have to regenerate the files again and start from the very beginning?

I tried to use the Picard tool FixMateInformation.jar, but then I got an error message regarding the index files. I tried deleting the bam index for one bam file and then recreating it to see if there was a problem with the indexes, but doing so did not resolve the issue. So it seems that something went wrong with the bam files at some earlier step.

Has this error been encountered before? Not sure how to proceed next, except restart everything.



  • SheilaSheila Broad InstituteMember, Broadie, Moderator admin

    Hi Alva,

    What version of GATK are you using? I think this was an issue that was fixed in one of the recent nightly builds. Can you try using the latest nightly build and let us know if the error still persists?


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