Hello, so I'm mapping reads from a RNA-seq library to a reference genome using hisat2 with default parameters. Of my total output mapped reads, when checking with samtools flagstat/stats I'm getting a significant amount of secondary alignments (SAM flag 0x100) I've looking on how to deal with these. I have read a good practice article and it says I should keep them, but others keep only uniquely mapped reads and recommend to remove them. Any thoughts? Tools in downstream analysis, for example htseq-count are going to be able to manage them correctly right? I would lose too many reads by removing them, so it is correct if I kept them?
Thank you for time!
1 answer
Since it sounds like you'll be extracting counts with featureCounts or htseq-count then you can leave the secondary alignments in, as both of the aforementioned programs will ignore secondary alignments (and their associated primary alignments).
You'll need to keep at least the primary alignments, since that'll break things like stringTie otherwise.
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Thanks for your answer, I'll keep looking just to be sure I'm proceeding correctly, but what you mentioned is the case.