[BioC] RNA degradation plot with oligo package GeneFeatureSet objects
cstrato at aon.at
Fri Aug 16 22:32:49 CEST 2013
Yes, xps should work with the Ovine Gene 1.1 ST arrays, since it does
work with the Human, Mouse and Rat Gene 1.1 ST arrays. These arrays use
the PGF-files from Affymetrix and 'xps/examples/script4schemes.R' shows
you how to create the 'scheme' files for xps.
On 8/16/13 9:36 PM, heyi xiao wrote:
> Hi Christian,
> Thanks for the suggestion and sharing of degradation plot experience. Does xps work with the Ovine Gene 1.1 ST array? I don’t have a CDF package.
> On Fri, 8/16/13, cstrato <cstrato at aon.at> wrote:
> Subject: Re: [BioC] RNA degradation plot with oligo package GeneFeatureSet objects
> To: "James W. MacDonald" <jmacdon at uw.edu>, "heyi xiao" <xiaoheyiyh at yahoo.com>
> Cc: bioconductor at r-project.org
> Date: Friday, August 16, 2013, 2:01 PM
> Dear Heyi,
> Function 'plotAffyRNAdeg()' of package 'xps' does allow you
> to plot RNA
> degradation plots for Whole Genome and Exon arrays, see
> Cahpter 5.4.1 of
> vignette 'xps.pdf'.
> I have done this for a couple of HuGene array data, and the
> results look
> interestingly. Especially there is a difference when you
> compare RNA
> degradation plots from frozen tissues vs paraffin embedded
> However, I am not sure how to interpret the results.
> Best regards,
> C.h.r.i.s.t.i.a.n S.t.r.a.t.o.w.a
> e.m.a.i.l: cstrato at aon.at
> On 8/16/13 6:52 PM, James W. MacDonald wrote:
> > Hi Heyi,
> > On 8/14/2013 4:47 PM, heyi xiao wrote:
> >> Hi all,
> >> In affy package, I can use AffyRNAdeg and
> plotAffyRNAdeg to plot and
> >> check RNA degradation. Is there any way to do so in
> oligo package for
> >> GeneFeatureSet,which is equivalent to AffyBatch in
> affy package. I
> >> look at the GeneFeatureSet and AffyBatch, they
> quite similar. But not
> >> sure what can be done here. I can either modify
> AffyRNAdeg and
> >> plotAffyRNAdeg functions to fit them for
> GeneFeatureSet, or I can
> >> convert GeneFeatureSet to AffyBatch and use the
> affy package
> >> degradation functions. Any suggestions would be
> highly appreciated.
> > While I suppose you could hypothetically do the
> conversion, I wonder if
> > it makes conceptual sense.
> > The 3'-biased Affy arrays were all based off an
> oligo-dT primer that was
> > used to convert mRNA to cDNA, so the reverse
> transcription proceeded
> > from the 3' end of the mRNA, always. In this case you
> can wonder about
> > two things. First, how far did the RT step proceed? Did
> you in general
> > get good RT all the way to the most 5' of the probes in
> the probesets?
> > Second, since we were using the polyA tail at the 3'
> end, by definition
> > the mRNA wasn't degraded from the 3' end. However, it
> might have had
> > more or less extensive degradation from the 5' end, so
> the RT may have
> > gone to completion, but the degradation had proceeded
> past the most 5'
> > probes.
> > So both things are confounded, as we cannot distinguish
> RT that didn't
> > proceed too far from highly degraded mRNA, but no
> matter. What we could
> > do is say how much signal we were getting from the more
> 5' probes, and
> > decide if we wanted to do something about that (like
> only use the first
> > 8 probes or whatever).
> > For the newer generation of Affy arrays, we use a
> random primer, so the
> > RT step proceeds from a random point in the transcript
> and proceeds
> > towards the 5' end (at least I think it is still
> directional). Since the
> > RT no longer starts from one end of the transcript, it
> is no longer
> > clear what differential amounts of probe signal would
> actually signify.
> > In addition, with the newer generation of Affy arrays,
> we can collapse
> > the probes into different probesets, depending on what
> we are trying to
> > measure (e.g., you can try to measure expression at the
> exon level or
> > the transcript level).
> > I think trying to do this would be more difficult than
> it would be
> > worth, especially given that I don't know what you
> would do if you were
> > to decide there had been degradation.
> > Best,
> > Jim
> >> Heyi
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