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).


$ 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.


Does strandedness matter in this alignment? Perhaps I should care about the --nofw/--norc options? Am I missing something here?

  • $\begingroup$ Are you taking transcripts as the reference? --norc might be what you are looking for. $\endgroup$
    – user172818
    May 14, 2019 at 16:34
  • $\begingroup$ I'm using mm10 as my reference (downloaded from Illumina). $\endgroup$ May 14, 2019 at 17:12
  • $\begingroup$ 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. $\endgroup$
    – user172818
    May 14, 2019 at 18:21
  • $\begingroup$ 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," $\endgroup$ May 14, 2019 at 18:38
  • 2
    $\begingroup$ 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 Tophat2 does use bowtie2 for mapping, but it is invoking bowtie2 in a non-standard way and is generally thought to be superseded by star and hisat2 anyway. $\endgroup$
    – user172818
    May 14, 2019 at 18:55

1 Answer 1


I repeat what user172818 says...don't waste your time on bowtie or tophat. Use STAR or HISAT2, or a pseudoaligner like kallisto with a transcriptome.


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