Hey folks, I ran into something with differentialabundance and 3′ tag/DGE RNA-seq (QuantSeq, Lexogen 3′ FWD, etc.) and wanted to bring it here first, since I'd like to help fix it rather than just file it and walk away. The DESeq2/limma paths (2.0.0 docs) expect a length-scaled or length-offset gene matrix from nf-core/rnaseq. That's right for full-length libraries, but for 3′ tag data the count per gene is length-independent, so a length offset would bias fold changes by transcript length. Standard handling is CPM via edgeR, no length correction, DESeqDataSetFromMatrix on raw counts. I didn't see this distinction anywhere in the usage/output docs — might just be something I missed. Wherever this lands, I'd like to pitch in: - If it's just a docs gap, I can write the pull request (a warning + guidance for 3′ users on which matrix to supply). - If it's worth a real parameter (e.g. --library_type 3prime|full_length switching the normalization path) — I can put together a first pass and iterate with whoever knows the DESeq2/limma module best. - Either way I have a 3′ DGE dataset I can test against, so I'm not just guessing at the fix. Let me know which direction makes sense, or if I'm missing existing support for this and it's a non-issue.

Yep, I think it's just a docs gap? IIRC the docs just say to either provide the length offset matrix OR the appropriate pre-scaled matrix, in which case no offset accounting is done. So you just need to use the same path that would be used for a pre-scaled matrix (I think). Feel free to make a PR.

opened the PR