<p>I have tried to count mapped reads on the human miRNA regions from the miRBAse human GFF</p>

<p>the output was just:</p>

<pre>
__no_feature    70216353
__ambiguous     0
__too_low_aQual 0
__not_aligned   1383340
__alignment_not_unique  0</pre>

<p>So there should be something I do wrong?</p>

<p>I have run it - htseq-count SAMfile Gfffile (SAMfile is from bowtie with option -S)</p>

<p>The GFF is from MirBase:</p>

<pre>
chr1    .    miRNA_primary_transcript    17369    17436    .    -    .    ID=MI0022705;Alias=MI0022705;Name=hsa-mir-6859-1
chr1    .    miRNA    17409    17431    .    -    .    ID=MIMAT0027618;Alias=MIMAT0027618;Name=hsa-miR-6859-5p;Derives_from=MI0022705
chr1    .    miRNA    17369    17391    .    -    .    ID=MIMAT0027619;Alias=MIMAT0027619;Name=hsa-miR-6859-3p;Derives_from=MI0022705
chr1    .    miRNA_primary_transcript    30366    30503    .    +    .    ID=MI0006363;Alias=MI0006363;Name=hsa-mir-1302-2
chr1    .    miRNA    30438    30458    .    +    .    ID=MIMAT0005890;Alias=MIMAT0005890;Name=hsa-miR-1302;Derives_from=MI0006363
chr1    .    miRNA_primary_transcript    187891    187958    .    -    .    ID=MI0026420;Alias=MI0026420;Name=hsa-mir-6859-2
chr1    .    miRNA    187931    187953    .    -    .    ID=MIMAT0027618_1;Alias=MIMAT0027618;Name=hsa-miR-6859-5p;Derives_from=MI0026420
chr1    .    miRNA    187891    187913    .    -    .    ID=MIMAT0027619_1;Alias=MIMAT0027619;Name=hsa-miR-6859-3p;Derives_from=MI0026420
chr1    .    miRNA_primary_transcript    632325    632413    .    -    .    ID=MI0022558;Alias=MI0022558;Name=hsa-mir-6723
chr1    .    miRNA    632382    632403    .    -    .    ID=MIMAT0025855;Alias=MIMAT0025855;Name=hsa-miR-6723-5p;Derives_from=MI0022558</pre>

Did you use the `-t` flag to change the feature type to miRNA_primary_transcript or miRNA? As far as HTSeq would be concerned (without using this flag) you have given it no features to look for.

The default looks for exon in the third column

You should also use the `-I` flag to use 'ID' as the id attribute (default looks in the last column for 'gene_id')

Ensure you read the manuals before using these tools.

http://www-huber.embl.de/users/anders/HTSeq/doc/count.html