Question: Remove secondary alignments in RNA-seq analysis?
gravatar for nanoide
20 months ago by
nanoide40 wrote:

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!

hisat2 rna-seq alignment • 1.2k views
ADD COMMENTlink modified 20 months ago • written 20 months ago by nanoide40

Thanks for your answer, I'll keep looking just to be sure I'm proceeding correctly, but what you mentioned is the case.

ADD REPLYlink written 20 months ago by nanoide40
gravatar for Devon Ryan
20 months ago by
Devon Ryan94k
Freiburg, Germany
Devon Ryan94k wrote:

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).

ADD COMMENTlink written 20 months ago by Devon Ryan94k

What if you want to assemble the transcript? Do you still suggest to keep or remove the secondary alignments and the supplementary?

ADD REPLYlink written 20 months ago by rsafavi50

You'll need to keep at least the primary alignments, since that'll break things like stringTie otherwise.

ADD REPLYlink written 20 months ago by Devon Ryan94k
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