Hi, I use HTSeq to map my reads to annotation and output as gtf file, but I didn't see any genes with count more than 0...
What could be the potential problem?
Well, I would ask any question with some background and basic information, for instance:
Looks like most of reads not mapped uniquely, is that caused by the alignment?
I used Hisat2 to map the reads.
Could you be a little more specific on this? What were the alignment stats reported by hisat2? What is in the bottom lines of the htseq output? (__no_feature and __ambigious, etc)
The more information you give us, the easier we can track down your problem.
The corresponding mapping results:
4197952 (16.42%) aligned 0 times
16784289 (60.57%) aligned exactly 1 time
5352503 (22.15%) aligned > 1 times
Check out whether your annotation gtf file and reference genome file are from the same source. Avoid using these files from different sources (e.g., USCS gtf file and Ensemble reference genome).
I download the annotation and genome index from igenomes
Following with your previous question, there could be a problem with annotation format. Could you post first 2 - 3 lines of your annotation file and the command you used ?