STAR and HTSeq command line parameters for 3' mRNA-Seq data

Question

[Ld_60]

Hi everyone,

I am looking for a data analysis pipeline specifically for RNA-Seq data obtained from 3' mRNA-Seq assays (i.e. only the 3' end of the fragmented transcript is sequenced). Particularly, I would like to know which STAR (for the alignment step) and HTSeq (for the quantification step) parameters should be used and to what values should they be set in this case?

Thanks a lot for your help!

Answer 1

[michael.ante]

Hi Ld_60,

I guess it depends on the library prep that was used. If it targets the actual TTS, you'll might need rather an end-to-end alignment rather than splice detection.

If your reads are not directly on the TTS and rather randomly distributed, you'll go very well with the 'ENCODE Options' according to the manual.

I'd check the alignments with RSEQC'S read_distribution, infer_experinent, and geneBodyCoverage analysis tools. There you'll see if your annotation is "too short", which strandedness your libraries have (for HTSEQ), and how your reads are distributed over the genes.

Cheers

Michael