0
$\begingroup$

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?

$\endgroup$
  • $\begingroup$ Are you taking transcripts as the reference? --norc might be what you are looking for. $\endgroup$ – user172818 May 14 '19 at 16:34
  • $\begingroup$ I'm using mm10 as my reference (downloaded from Illumina). $\endgroup$ – irritable_phd_syndrom May 14 '19 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 '19 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," $\endgroup$ – irritable_phd_syndrom May 14 '19 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 Tophat2 does use bowtie2 for mapping, but it is invoking bowtie2 in a non-standard way and is generally thought to be superseded by star and hisat2 anyway.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 '19 at 18:55
1
$\begingroup$

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.

| improve this answer | |
$\endgroup$

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.