I really hope I can interest some folks in helping me figure this out once and for all. I manage a NGS sequencing core and we process quite a bit of miRNA libraries on our MiSeq. These reads are from mouse. Here is the confusing part:
When I map the reads to the mouse genome (GRCm38) using Bowtie:
bowtie --wrapper basic-0 -n 0 -l 15 -e 99999 -k 200 --best --strata
I get over 30 million mapped reads but I only have a little over 2 million reads.
When I map the reads to the mouse genome using Bowtie2:
bowtie2 --local -p 8 -q --phred33 -D 20 -R 3 -N 0 -L 8 -i S,1,0.50
I get about 1.5 million mapped reads (seemingly much more reasonable).
After mapping I use HtSeq-count to count how many of these mapped reads are miRNAs, obviously the Bowtie mapping renders much better results but how should I interpret this? Is is reasonable to count the same read more than one time. Any thoughts or knowledge on this topic would be helpful. Thanks so much.