probe length was:RE: [BioC] GCRMA backgrounds?

Mon Aug 2 22:15:38 CEST 2004

I have looked at this issue somewhat and you know there is no real answer 
to that question. We simply do not know enough about what is going on.

I have seen data, where if you find significant genes on one platform and 
then select significant genes on another (same sample run on multiple 
platforms), and you draw a VENN diagram, there is little in common between 
the platforms. Then when RT PCR is all the lists seem to be validating. So 
the different technologies seem to detect different genes differently in 
some magical mysterious way. I think we just do not understand the kinetics.

The whole issue makes me and want to throw microarrays of the top floor of 
our institute consume a more than suitable amount of migraine medication, 
then chase is down with a more than suitable amount of alcoholic bevvy.

Peter

At 02:56 PM 7/22/2004, A.J. Rossini wrote:

>When we were looking at Agilent vs. Affy, we heard points from both
>(read: 20-ish-mers vs. 70-ish-mers).  Unfortunately, it is hard to get
>solid data for comparisons.  We are looking at some comparative data
>on those two and another platform, but interpreting the results is a bit
>(to put it mildly) tricky.
>
>best,
>-tony
>
>
>
>"Michael Barnes" <Michael.Barnes at cchmc.org> writes:
>
> > I wasn't trying to be difficult and I hope you didn't take it that way.
> >
> >
> > Simply I am currently in need of information regarding what probe
> > length is best and I thought following up your comment might be a way to
> > find references.  Of course, Affy says 25-mers are best.  And there must
> > be an optimal length for the reasons you explained.  However, I wonder
> > what is the evidence that 25-mers are best as opposed to, say 20-mers,
> > 30-mers, 50-mers, 70-mers or anything else.  Hopefully there are some
> > suggestions and references out there that could help me.
> >
> > On a related question...  Affy claims 25-mers, yet they synthesize
> > their oligos on the chips.  We all know reactions are not perfect so
> > there must be some amount of synthesis failure.  Does anyone have a feel
> > for the percentage of complete/incomplete oligos on an affy feature?
> > And are the short oligos prevented from binding to your sample in some
> > way?
> >
> > BTW:  If you can find ANYTHING on the Affy site, more power to you:)
> >
> > Mike
> >
> >
> >>>> "Matthew  Hannah" <Hannah at mpimp-golm.mpg.de> 07/22/04 03:29AM >>>
> > I should have said it was just a logical guess.
> >
> > What I meant was that if you had 2 homologous genes, obviously it
> > is going to be harder to avoid homologous regions if you need to find
> > 50bp versus 25bp? But this is refering to cross-hybridisation between
> > PM and related sequences, I don't know how it would affect non-specific
> >
> > binding of PM to non-complementary sequences (am I right to distinguish
> >
> > these?). I should have said 'less-' rather than non-homologous, or
> > just
> > dropped the 'non-' in the initial post. Also this would only apply
> > where
> > there were related sequences present, but then different probe-lengths
> >
> > for different sequences wouldn't be ideal.
> >
> > Also while we're on logic another reason to consider is that with
> > 11-20
> > probesets per mRNA, for short mRNAs there is already some overlap,
> > this
> > would be worse for longer probes, making them less independent. It
> > would
> > also extend the probed region further from the 3' end from where
> > labelling
> > occurs and so efficiency may be reduced?
> >
> > If you need a reference I'm sure the affy website or some of their
> > publications
> > would have something.
> >
> > Sorry for any confusion.
> >
> > Matt
> >
> > -----Original Message-----
> > From: Michael Barnes [mailto:Michael.Barnes at cchmc.org]
> > Sent: Mittwoch, 21. Juli 2004 19:49
> > To: Matthew Hannah; bioconductor at stat.math.ethz.ch
> > Subject: Re: [BioC] GCRMA backgrounds?
> >
> >
> > What are references for this?
> >
> > Mike
> >
> >>>> "Matthew  Hannah" <Hannah at mpimp-golm.mpg.de> 07/21/04 12:45PM >>>
> >
> > As for the 25mers, the obvious thing to take into account is that
> > as you increase in length it is more likely that non-homologous
> > probes will bind as it would be more difficult to find sequences
> > that are gene specific.
> >
> > HTH,
> > Matt
> >
> > _______________________________________________
> > Bioconductor mailing list
> > Bioconductor at stat.math.ethz.ch
> > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor
> >
> > Hi,
> >
> >       I've been using GCRMA and the new speedier version (1.1)
> > gives different values than the older slower version (1.0).
> >
> >       Looking through the bioconductor mails suggests that
> > a few other people identified a similar problem, related to a
> > background not being subtracted. Hopefully people are on the case,
> > but this problem seems to have been around since April. I've been
> > plugging GCRMA to my colleagues, who are now starting to use it,
> > so I hope the problem can be sorted out.
> >
> >       On a different note, what technical limitations stop
> > Affymetrix going for much longer probes than 25 bases? The work
> > of Naef and Magnasco, and Wu and Irizarry, highlight the
> > limitations of Affy technology due to cross-hybridisation, when
> > there are only 25 bases. Pushing upwards to 50 bases will reduce CH,
> > but what other factors then come in?
> >
> >       My understanding is that the Affy SNP chips have 25 base
> > oligos. What is stopping these chips from also having
> > cross-hybridisation issues?
> >
> >       Best wishes,
> >               Harry
> >
> > _______________________________________________
> > Bioconductor mailing list
> > Bioconductor at stat.math.ethz.ch
> > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor
> >
>
>--
>Anthony Rossini                     Research Associate Professor
>rossini at u.washington.edu            http://www.analytics.washington.edu/
>Biomedical and Health Informatics   University of Washington
>Biostatistics, SCHARP/HVTN          Fred Hutchinson Cancer Research Center
>UW (Tu/Th/F): 206-616-7630 FAX=206-543-3461 | Voicemail is unreliable
>FHCRC  (M/W): 206-667-7025 FAX=206-667-4812 | use Email
>
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