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

[A.J. Rossini]

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
>
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>
> 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
>
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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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