I'm trying to map RNA-Seq reads generated using the
NEB Ultra Directional kit is first strand reversed protocal. I'm using bowtie-2.3.5_1 to align against the mus musculuous (mm10) genome. The reads are single ended.
It seems that
--rf are relevant only for paired end options. I tried these options as well as not specifying either (default).
$ bowtie2 --fr \ --threads 20 \ --very-sensitive \ -x /reference/mus_musculus/mm10/ucsc_assmebly/illumina_download/Sequence/Bowtie2Index/genome \ -U trimmed_out/my_trimmed.fq.gz \ -S bowtie_fr_1.sam $ samtools sort -n -o bowtie_fr_sort_1.sam bowtie_def_1.sam
I then compared the output of
bowtie_fr_sort.sam. They are basically identical excluding the header.
Does strandedness matter in this alignment? Perhaps I should care about the
--nofw/--norc options? Am I missing something here?