[BioC] RNA degradation plot with oligo package GeneFeatureSet objects
James W. MacDonald
jmacdon at uw.edu
Sat Aug 17 13:47:38 CEST 2013
That's exactly what I mean.
On 8/16/2013 6:26 PM, heyi xiao wrote:
> Hi Jim,
> You mean, I can’t use makecdfenv package to build CDF package from PGF and CLF files?
> I will definitely check these other plots before decide on quality issue.
> On Fri, 8/16/13, James W. MacDonald<jmacdon at uw.edu> wrote:
> Subject: Re: [BioC] RNA degradation plot with oligo package GeneFeatureSet objects
> To: "heyi xiao"<xiaoheyiyh at yahoo.com>
> Cc: bioconductor at r-project.org
> Date: Friday, August 16, 2013, 3:59 PM
> Hi Heyi,
> No you can't use the affy package for that chip type. As an
> aside, I
> have never found an array that I wanted to exclude based on
> the RNA
> degradation plot that I hadn't already decided to exclude
> based on
> either a PCA plot, a density plot of the raw data, or a NUSE
> or RLE
> plot. In other words, I think there are much better ways to
> find outlier
> plots than the degradation plot.
> On 8/16/2013 3:33 PM, heyi xiao wrote:
> > Hi Jim,
> > Thanks for the informative notes. I really learned
> things about RNA degradation and affy array design!
> > I see you what mean. But I only use RNA degradation as
> a quality assessment tool. I am less interested in estimate
> exactly how much RNA degradation happened in the RNA
> molecules in one sample/array, I am more interested in the
> different degradation patterns seen across different
> samples. Normally degradation curves for different samples
> stack together consistently and nicely. Even with the newer
> generation Affy arrays, an outlier degradation curve always
> suggest some quality issue, mostly likely RNA degradation.
> Such RNA degradation curves together with other quality
> check help me either kick out the problematic samples or
> have them redone.
> > BTW, currently only oligo package seems to work with
> the new Ovine Gene 1.1 ST array, for which I don’t see an
> CDF package in bioconductor as other affy chip types.
> Therefore, I can’t go with affy and other packages which
> provide RNA degradation plots. Can I use makecdfenv package
> to build CDF package from PGF and CLF files?
> > Heyi
> > --------------------------------------------
> > On Fri, 8/16/13, James W. MacDonald<jmacdon at uw.edu>
> > Subject: Re: [BioC] RNA degradation
> plot with oligo package GeneFeatureSet objects
> > To: "heyi xiao"<xiaoheyiyh at yahoo.com>
> > Cc: bioconductor at r-project.org
> > Date: Friday, August 16, 2013, 12:52
> > Hi Heyi,
> > On 8/14/2013 4:47 PM, heyi xiao
> > > 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
> > 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
> > 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
> > >
> > >
> > > Bioconductor mailing list
> > > Bioconductor at r-project.org
> > > https://stat.ethz.ch/mailman/listinfo/bioconductor
> > > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor
> > -- James W. MacDonald, M.S.
> > Biostatistician
> > University of Washington
> > Environmental and Occupational Health
> > 4225 Roosevelt Way NE, # 100
> > Seattle WA 98105-6099
> James W. MacDonald, M.S.
> University of Washington
> Environmental and Occupational Health Sciences
> 4225 Roosevelt Way NE, # 100
> Seattle WA 98105-6099
James W. MacDonald, M.S.
University of Washington
Environmental and Occupational Health Sciences
4225 Roosevelt Way NE, # 100
Seattle WA 98105-6099
More information about the Bioconductor