Does Hisat2 automatically align strandedness
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12 weeks ago

I've been using Hisat2 to align some RNA. RNA was prepped using NEB Directional library kit.

When I originally aligned, I did not use the --rna-strandedness option.

I then realised I do want stranded, as part of my reference (which is human genome plus integrated virus) produces proteins on the minus strand only. I was going to realign with the --rna-strandedness option set to RF (which would be correct for that- and most other- Illumina library prep kits) and reads would then get a XS tag.

I noticed looking at the bam file that the reads already have tags however, and the tags seem to align to the strand eg the section that should only produce reads off the minus strand only have tags that are only XS:A:- and there are no XS:A:+. (The other sections which should produce RNA off both strands are a mixture of XS:A:+ and XS:A:-)

I realised the default for single-end reads is unstranded, but when doing paired ends, does Hisat2 automatically detect strandedness and default to RF? I can't see anything about this in the manual but wondered if there had been an update I don't know about?

hisat2 alignment strandedness • 132 views
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