Read group addition order in RNA-seq short variant discovery (SNPs + Indels)

Question

[iamsmor]

Hello, I’m following the GATK best practices for RNA-seq short variant discovery (SNPs + Indels) and wondering about the correct point to add Read Groups (RGs). Does the order (before/after MarkDuplicates or SplitNCigarReads) matter for RNA-seq variant calling with GATK (HaplotypeCaller)? Any official clarification or reference from the GATK team or papers? Pipeline: HISAT2, AddOrReplaceReadGroups, MarkDuplicates, SplitNCigarReads, BaseRecalibrator, HaplotypeCaller Thank you for any kind of help

[GenoMax]

Ideally you would do this at the beginning of the process as you align the data. Read groups would be tagging the samples with relevant info e.g. what lane they were in, if they ran on multiple flowcells etc (all of this may not be applicable in your case).

See Expected Input section: https://gatk.broadinstitute.org/hc/en-us/articles/360035535912-Data-pre-processing-for-variant-discovery

Answer 1

[iamsmor]

ı didnot do when ı align rnaseq datasets but I did it as soon as I realized I had to do it. ı am following https://gatk.broadinstitute.org/hc/en-us/articles/360035531192-RNAseq-short-variant-discovery-SNPs-Indels but here is not exact information.

[GenoMax]

Do it before you start doing any manipulations after alignments.