htseq out zero for every exon

[lazy214]

Hi,

I'm trying to count exons with htseq-count, but it output zero for each exon.

no_feature 25949515
ambiguous 0
too_low_aQual 0
not_aligned 0
alignment_not_unique 8374881

Here is the command I used:
python -m HTSeq.scripts.count --stranded=no --mode=intersection-nonempty -t exon -i exon_id ensembl.human.gtf sample.sam > output

reads are aligned by tophat2, reference gtf file is from ensembl: Homo_sapiens.GRCh37.73.gtf

There are about 68648819 lines in the sam file, as the statistic revealed by htseq
no_feature 25949515
alignment_not_unique 8374881
I wondering where is the other alignments

Would anyone help me solve this problem?

Any help would be appreciated. Thank you!

[Michael.Ante]

Hi lazy214,

ou might have a look at the bioconductor package DEXSeq. It contains some HTSeq-count based scripts to count exon reads. You can simply use it and/or find a solution for your problem there.

Cheers,

Michael

[lazy214]

Thank you for your help!
I have finally found the reason. It is because the reference genome I used is with the prefix "chr" for each chromosome, but the reference gtf is not.
It is a pity that htseq could not check the compatibility of references, and give warnings.