[BioC] RNA degradation plot with oligo package GeneFeatureSet objects

James W. MacDonald jmacdon at uw.edu
Fri Aug 16 21:59:19 CEST 2013

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>  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, 12:52 PM
>   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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>   -- James W. MacDonald, M.S.
>   Biostatistician
>   University of Washington
>   Environmental and Occupational Health Sciences
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>   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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