[khmer] orientation of SE reads

C. Titus Brown ctb at msu.edu
Tue Oct 1 20:06:24 PDT 2013

On Wed, Oct 02, 2013 at 11:01:42AM +0800, bioques wrote:
> Thanks for the quick response! 
> Unfortunately, I do deal with strand-specific dataset right now. So in this case, I suppose I need to RC the s2_se reads? Would you please tell me what other reasons bother you that this protocol would not work for strand-specific dataset?

Hi Nate,

then you need to present the reads to Trinity in whatever orientation it
requires -- I don't have any experience with this, except with their demo
data set.

The reason why I worry about the protocol more generally is that none of
the QC procedures or abundance-filtering or digital normalization code
were built for single-stranded.  This is one reason why Trinity has built
their own in silico normalization procedure.  I'm unsure of what the effect
would be.  Note that none of the code will *mess up* the orientation of
the reads, though; it just might have unpredictable effects (I would bet
that the filtering would be too stringent, is all).

We will add a caveat at the top of the protocols.


> At 2013-10-02 10:48:55,"C. Titus Brown" <ctb at msu.edu> wrote:
> >On Wed, Oct 02, 2013 at 09:12:01AM +0800, bioques wrote:
> >> Hi Professor Brown,
> >> 
> >> I tried to follow the protocol you kindly offer here (https://github.com/ged-lab/khmer-protocols/tree/master/mrnaseq). It really a great help to newbie like me for RNA-Seq analysis. But I got one question about the step after trimming the adapter with Trimmomatic (document in 1-quality.txt). You suggest to combine the SE reads s1_se and s2_se together. Would this hurt the orientation of the reads when doing the assembly (I am going to use Trinity to do the assembly)? I wonder if I should convert the s2_se reads to its reverse complement before combine the two files together?
> >> 
> >> Hope to hear from you and thanks in advance!
> >
> >Hi Nate,
> >
> >no need to RC the s2_se reads, unless you are dealing with single-strand
> >mRNAseq (in which case I'm not sure this protocol works for other reasons).
> >Basic dsDNA sequencing reads do not have any particular orientation unless
> >you're doing something tricky with mate pairs or single-strand mRNAseq.
> >
> >cheers,
> >--titus
> >-- 
> >C. Titus Brown, ctb at msu.edu

C. Titus Brown, ctb at msu.edu

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