Hi all,

I am trying to perform an RNAseq analysis. I have created an index using HISAT2, aligned my paired-end reads to the index, and am now trying to count reads using HTseq. Unfortunately, when counting, HTseq yields the following result.

__no_feature    6280013
__ambiguous 0
__too_low_aQual 4434361
__not_aligned   4758201
__alignment_not_unique  3366408

What I have done to troubleshoot this.

Create new index using different file (transcriptome instead of genome) Use .gff or .gtf file for counting. Sort .sam file prior to counting. Ensure --stranded=no and --nonunique=all

These changes have not yielded correctly counting output.

At this point I am not sure what to do. I imagine there is something wrong with the way I set my index up and potentially not matching the .gtf file, however, I have downloaded these from the same source so I am not sure. Thanks

Have you checked if the cromosome names are represented the same way in both bam files and the gtf/gff? For example if in bams the chromosomes are named as chr1,chr2... and in the gtf file as 1,2,3 migh produce a problem in counting.