Incorrect strand assignment (--libType) in version 4.1.2?

[AnkeB]

Hi rMATS folks,

I'm using rMATS on single-end reverse-stranded RNAseq reads and noticed very few counts assigned to splicing events when setting --libType fr-secondstrand. However, that changes when incorrectly setting --libType fr-firststrand. Then I get high coverage. I started digging and came across the changes you made here: https://github.com/Xinglab/rmats-turbo/pull/126/files#diff-c18340f7c14ac230a459d63c355658e92455261119e4e407770432860418bfe2 Based on this and also on what I see in my data, I'm wondering if the strand assignment is opposite of what it should be? It looks like the strand is switched for reads of forward-stranded libraries and not switched for reads of reverse-stranded libraries, but shouldn't it be the other way around?

Thanks in advance for taking a look!
Best,
Anke.

[kutsc...@gmail.com]

With single end data, rMATS will flip the strand with --libType fr-firststrand and it will not change the strand with --libType fr-secondstrand. The rMATS implementation of --libType is based on TopHat --library-type: http://ccb.jhu.edu/software/tophat/manual.shtml

When I made the recent change for v4.1.2, I used the explanation from the salmon docs: https://salmon.readthedocs.io/en/latest/library_type.html#fraglibtype which says that fr-firststrand corresponds to ISR or SR, and fr-secondstrand corresponds to ISF or SF

Eric

[AnkeB]

Thanks a lot for the explanation, Eric. I misunderstood what --libType fr-firststrand and --libType fr-secondstrand was referring to. I now understand, that it refers to the order of the strands being synthesized (first or second) and e.g. firststrand does not refer to the strand of the event. Thanks for this clarification.

May I suggest that you add this explanation or the link to the salmon docs to the rMATS help pages/function?

Thanks again and best,
Anke.