SAM validation error: RG ID not found in header

Question

[Synanth]

Hi,

I'm trying to edit LIONS to utilize more updated software for TE analysis. One of the steps was alignment through Tophat2, which I replaced with HiSAT2. I used the '--un' parameter on HiSAT2 to produce two output files: accepted_hits and unmapped. These two files are then sorted and merged with samtools.

A bit downstream I am converting the merged bam to wig, and am encountering a slew of errors resembling the following:

Ignoring SAM validation error: ERROR::READ_GROUP_NOT_FOUND:Record [X], Read name [XX], RG ID on SAMRecord not found in header: accepted_hits

Looking at the header, I don't see any @RG fields:

 @PG ID:hisat2   PN:hisat2   VN:2.2.1    CL:"[dir]hisat2-align-s --wrapper basic-0 -p 8 --rna-strandness FR --secondary -x [dir] [dir] --passthrough --read-lengths 101"
 @PG ID:samtools PN:samtools PP:hisat2   VN:1.15.1   CL:samtools sort -o accepted_hits.bam accepted_hits.sam
 @PG ID:samtools-510607B0    PN:samtools VN:1.15.1   CL:samtools sort -o unmapped.bam unmapped.sam
 @PG ID:samtools.1   PN:samtools PP:samtools VN:1.15.1   CL:[dir]samtools merge -r me_cfs.bam accepted_hits.bam unmapped.bam
 @PG ID:samtools.2   PN:samtools PP:samtools.1   VN:1.15.1   CL:samtools view -h me_cfs.bam

It looks like bam2wig still runs, just ignores the errors, but it makes navigating the output an absolute pain. Any ideas on how I modify the header such that the @RG:Z: IDs (accepted_hits && unmapped) are represented?

Thank you so much for any assistance,

Synanth

Answer 1

[GenoMax]

Read groups is a concept that GATK software from Broad institute uses: https://gatk.broadinstitute.org/hc/en-us/articles/360035890671-Read-groups

You can add RG to your file if they are needed using picard: https://gatk.broadinstitute.org/hc/en-us/articles/360037226472-AddOrReplaceReadGroups-Picard-

[Synanth]

Thank you so much for the resources!