Hello everyone,

I am new to bioinformatics and I am trying to extract gene count using hisat2 pipeline and feature count. But I am getting 0 counts for all genes. Here is the steps that I am taking:

1. I have generated the genome index files using hisat2_build_index command.

2. Then I aligned each RNA seq file against the reference genome using the following command

hisat2 -p 20 -x Genome_basename --no-mixed --no-discordant -k 1 -X 500 --score-min L,0,-0.6 -1 Read1.fq -2 Read2.fq -S sam_directory

1. I converted the sam files to bam files using this command:

samtools view -@ 20 -o sample.bam -b sample.sam

1. I Sorted the sam files based on their position in the reference genome as follows:

samtools sort -@ 20 -m 1G -o sample.sorted.bam sample.bam

1. I ran the feature counts to extract the gene counts.

featureCounts -M -O -p -B -C -T 6 -a .genome.gtf -o sample.counts.txt sample.sorted.bam

Do you have any idea what I am missing so that I am getting 0 counts?