Errors While Running Printreads Walker Of Gatk

Question

[Kssr]

I ran BaseRecalibrator on some of my realigned bam files, there were no errors reported while running it and I was to able to generate the "recal.grp" files. However, while running the PrintReads walker to generate the recalibrated BAM files, I get the following error message for some of the BAMs.

##### ERROR MESSAGE: Exception when processing alignment for BAM index WTCHG_35305_101:4:1105:10332:129428#TGTTAACT 2/2 100b aligned read.

So, I tried to validate my realigned BAM as well as the original BAM (before realignment) using Picard's ValidateSAMFile and I get the following:

original BAM: Mate unmapped flag does not match read unmapped flag of mate, Mate alignment does not match alignment start of mate, Mate negative strand flag does not match read negative strand flag of mate

Aditionally these errors on realigned BAMs: Mate reference index (MRNM) does not match reference index of mate, Mate not found for paired read

Do I need to worry about initial alignments? I read on the forums that using -rf MateSameStrand Filter should help me work around this, what exactly does this filter do? Any other approaches/ideas to solve this problem would be appreciated.

Answer 1

[Jorge Amigo]

from the ValidateSAMFile output you can see that you've got a problem with the reads' pairing. you should be able to solve it by running a FixMateInformation step before the 2 BSQR steps.

Answer 2

[Kssr]

I ran Picard's FixMateInformation step, and revalidated my BAM. However, I still get the following error: Mate not found for paired read. Any ideas on how to fix this?

[Jorge Amigo]

this should be a comment to my answer, not an answer itself. and no, if FixMateInformation doesn't get rid of those errors, I don't know how you would be able to proceed in any other way than removing those unpaired reads if they bother you in your downstream analysis.