I know this question has been posted before but I could not find answers to increase my counts for RNA-seq reads. I have done RNA-seq in Illumina Nextseq. I prepared libraries with NEBNext Ultra II Directional library kit. I used bowtie2 to map my reads to the reference genome. It is a bacterial genome. I have more than 95 % alignment rates in my samples. I converted sam file to bam file and name sorted. Then, I used ht-seq count to get the RNA-seq counts. here is my command line:
-o eo8_FP1_2_counts.txt htseq-count -f bam -t gene -i ID --stranded=reverse eo8_FP1_2_trimmed_sorted.bam ../Lplantarum.gff3 __no_feature 3587262 __ambiguous 39161 __too_low_aQual 15356 __not_aligned 35528 __alignment_not_unique 0
This is for a sample which has 5128080 (100.00%) were paired and 99.24% overall alignment rate. It seems like I am losing half of my reads.
What should I do to improve my ht-seq counts?