[BioC] RNA degradation plot with oligo package GeneFeatureSet objects

James W. MacDonald jmacdon at uw.edu
Sat Aug 17 13:47:38 CEST 2013


Hi Heyi,

That's exactly what I mean.

Best,

Jim

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.
> Thanks!
> 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, 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.
>
>
>   Best,
>
>   Jim
>
>
>
>   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
>   >    4225 Roosevelt Way NE, # 100
>   >    Seattle WA 98105-6099
>   >
>   >
>
>   -- 
>   James W. MacDonald, M.S.
>   Biostatistician
>   University of Washington
>   Environmental and Occupational Health Sciences
>   4225 Roosevelt Way NE, # 100
>   Seattle WA 98105-6099
>
>

-- 
James W. MacDonald, M.S.
Biostatistician
University of Washington
Environmental and Occupational Health Sciences
4225 Roosevelt Way NE, # 100
Seattle WA 98105-6099



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