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 --fr
and --rf
are relevant only for paired end options. I tried these options as well as not specifying either (default).
E.g.
$ 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_rf_sort.sam
bowtie_default_sort.sam
and bowtie_fr_sort.sam
. They are basically identical excluding the header.
Question:
Does strandedness matter in this alignment? Perhaps I should care about the --nofw/--norc
options? Am I missing something here?
--norc
might be what you are looking for. $\endgroup$