I am trying to get read counts from RNA-seq using HTSEq gene counter. I firstly aligned my reads against rRNA using BWA, converted the unaligned files to .fastq using Picard, aligned this fastq file against the MTB genome using BWA and used the .sam file for reads counts using HTSeq gene count. the GTF file was got from MTBdatabase. Unfortunatelly, I am not getting a single feature counted. I believe it has something to do with the sam files. Can anyone help me? it only around 500k genes per sample, just a trainning. I am using Gene counter because I want later user DESeq for diferentially gene expression. Thanks.
the last few lines of the HTseq count output is bellow: . . Rv3920c 0 Rv3921c 0 Rv3922c 0 Rv3923c 0 Rv3924c 0 no_feature 444038 ambiguous 0 too_low_aQual 0 not_aligned 1226003 alignment_not_unique 0 lc@lc:~$