HTseq report 80% of reads no feature

Question

[bxia]

Is this number is too large for a mice RNA-seq?

total reads mapped is 12 M from tophat2

__no_feature  10556702
__ambiguous     26522
__too_low_aQual         0
__not_aligned         0
__alignment_not_unique  21173785

[WouterDeCoster]

While it shouldn't make a real difference for this question, you should know that Tophat is no longer the "advisable" tool for RNA-seq analysis. The software is deprecated/ in low maintenance and should be replaced by HISAT2, StringTie and ballgown. See this paper: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. (If you can't get access to that publication, let me know and I'll -cough- help you.) There are also other alternatives, including alignment with STAR and bbmap,...

[harold.smith.tarheel]

rRNA is a frequent source of contamination in RNA-Seq libraries and produces a high percentage of multi-mappers. I suspect that's your problem.

Answer 1

[Devon Ryan]

So 12M initial reads, of which 10.5M didn't overlap exons and there are a LOT of multimappers. I'd say that something went horribly wrong here. Have a look at the alignments in IGV to see if they even make sense.