probe length was:RE: [BioC] GCRMA backgrounds?
A.J. Rossini
rossini at blindglobe.net
Tue Aug 3 00:30:43 CEST 2004
My initial theory on large scale gene expression experiments was that
it makes a nifty and expensive version of I-ching. Great fun if you
have a large wad of cash in the bank (or grant, same thing).
My current theory is a cross between that, and the "blind men and the
elephant" parable -- lots of publishable stories in every experiment,
hopefully you'll pick one of them and not the elephant's rear.
best,
-tony
Peter Wilkinson <pwilkinson at videotron.ca> writes:
> 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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--
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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