[BioC] RNA degradation plot with oligo package GeneFeatureSet objects
xiaoheyiyh at yahoo.com
Sat Aug 17 00:26:41 CEST 2013
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
Cc: bioconductor at r-project.org
Date: Friday, August 16, 2013, 3:59 PM
No you can't use the affy package for that chip type. As an
have never found an array that I wanted to exclude based on
degradation plot that I hadn't already decided to exclude
either a PCA plot, a density plot of the raw data, or a NUSE
plot. In other words, I think there are much better ways to
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
[[elided Yahoo spam]]
> 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?
> On Fri, 8/16/13, James W. MacDonald<jmacdon at uw.edu>
> Subject: Re: [BioC] RNA degradation
plot with oligo package GeneFeatureSet objects
> 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
> 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
> 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
> 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
> In addition, with the newer generation
of Affy arrays, we
> can collapse the probes into different
> on what we are trying to measure
(e.g., you can try to
> measure expression at the exon level
or the transcript
> 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.
> > Heyi
> > Bioconductor mailing list
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> -- James W. MacDonald, M.S.
> 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
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