# bowtie2 options when mapping stranded single end reads

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?

• Are you taking transcripts as the reference? --norc might be what you are looking for. May 14 '19 at 16:34
• I'm using mm10 as my reference (downloaded from Illumina). May 14 '19 at 17:12
• Then you need an RNA-seq mapper, not bowtie2. STAR and HISAT2 should have options to enable strand specific mapping. I am not familiar with those, though. May 14 '19 at 18:21
• Could you please elaborate how bowtie is not an rna-seq mapper? Per their website "Bowtie 2 is often the first step in pipelines for comparative genomics, including for variation calling, ChIP-seq, RNA-seq," May 14 '19 at 18:38
• Bowtie2 is not aware of splicing. For typical RNA-seq applications, you will want to use a splice-aware mapper, such as star and hisat2, which is specifically designed for RNA-seq. Tophat2 does use bowtie2 for mapping, but it is invoking bowtie2 in a non-standard way and is generally thought to be superseded by star and hisat2 anyway. May 14 '19 at 18:55