Strandedness of RNAseq data from SAM alignment flag

Question

[rs.yousaf]

Hi,

I am very new to RNAseq analysis. I have paired end RNAseq data from illumina Hiseq that I aligned using BWA MEM to a virus genome. This generated a BAM file that has alignment flags. I want to distinguish positive strand reads from negative strand as we have RNA from a virus that makes both strands. I have been using the following tags to filter negative and positive reads:

83 and 163 for positive reads 99 and 147 for negative reads

Can someone please confirm if this is correct? Also, how is strandedness determined during library prep. I really struggle to understand this.

Thank you!

Answer 1

[swbarnes2]

Also, how is strandedness determined during library prep.

It depends on the kit. You really need to stop and understand your experiment before you analyze it.