Question: HTseq report 80% of reads no feature
gravatar for bxia
3.3 years ago by
bxia150 wrote:

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
rna-seq • 686 views
ADD COMMENTlink modified 3.3 years ago by Devon Ryan96k • written 3.3 years ago by bxia150

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,...

ADD REPLYlink written 3.3 years ago by WouterDeCoster44k

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.

ADD REPLYlink written 3.2 years ago by harold.smith.tarheel4.6k
gravatar for Devon Ryan
3.3 years ago by
Devon Ryan96k
Freiburg, Germany
Devon Ryan96k wrote:

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.

ADD COMMENTlink written 3.3 years ago by Devon Ryan96k
Please log in to add an answer.


Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1159 users visited in the last hour