# HTSeq-count is returning 0 for every gene, instead of expression value

(Ported from stack overflow)

I'm trying to summarize gene count using htseq-count; and it's returning 0 counts at every gene. I'm not sure what I'm doing wrong; I think it has to do with the gene flag I'm using. I've tried using the GTF for Arabidopsis TAIR 10: which I got from: ftp://ftp.ensemblgenomes.org/pub/release-46/plants/gtf/arabidopsis_thaliana/Arabidopsis_thaliana.TAIR10.46.gtf.gz as well as the GFF file available on Araport.

I'm using various BAM files I know to have gene expression values.

I've tried both locally and on galaxy The local commands I've tried so far are:

python3 -m HTSeq.scripts.count --idattr=gene_id #bam #gtf > summary.txt
python3 -m HTSeq.scripts.count -i gene_id #bam #gff > summary.txt
python3 -m HTSeq.scripts.count -i locus_tag #bam #gff > summary.txt
python3 -m HTSeq.scripts.count -i ID #bam #gff > summary.txt
python3 -m HTSeq.scripts.count -i SeqID #bam #gff > summary.txt
python3 -m HTSeq.scripts.count -i Parent #bam #gff > summary.txt


I've done the same on galaxy; inputing the -i value in.

Any help as to where I'm going wrong would be great.

Thanks!

• hey @sdshinghal, check a few things. 1. ensure bam file is ok (you can look at it), 2. like the answer below, check your gtf matches the reference genome Mar 6 '20 at 14:09

Maybe the contig names in your BAM files don't match those in the GFF file? Ensembl uses contig names like 1, some other reference sources use chr1.