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

Paul Boutros Paul.Boutros at utoronto.ca
Mon Jul 26 07:52:27 CEST 2004

Here's a more recent paper dealing with this in detail:

Nucleic Acids Res. 2004 Jul 8;32(12):e99.
Optimization of probe length and the number of probes per gene for optimal
microarray analysis of gene expression.
Chou CC, Chen CH, Lee TT, Peck K.


> -----Original Message-----
> From: paul.boutros at utoronto.ca [mailto:paul.boutros at utoronto.ca]
> Sent: Friday, July 23, 2004 8:54 AM
> To: bioconductor at stat.math.ethz.ch
> Cc: Michael.Barnes at cchmc.org; Hannah at mpimp-golm.mpg.de
> Subject: Re: [BioC] probe length was:RE: [BioC] GCRMA backgrounds?
> One reference I know is the initial paper introducing the Agilent
> chips by Tim
> Hughes:
> Hughes, T. R., M. Mao, et al. (2001). "Expression profiling using
> microarrays
> fabricated by an ink-jet oligonucleotide synthesizer." Nat
> Biotechnol 19(4):
> 342-7.
> There is a section in the results "impact of oligonucleotide length on
> hybridization properties" that you might want to check out.
> Paul
> Date: Thu, 22 Jul 2004 14:48:28 -0400
> From: "Michael Barnes" <Michael.Barnes at cchmc.org>
> Subject: probe length was:RE: [BioC] GCRMA backgrounds?
> To: <Hannah at mpimp-golm.mpg.de>
> Cc: bioconductor at stat.math.ethz.ch
> Message-ID: <s0ffd3d8.039 at n6mcgw16.cchmc.org>
> Content-Type: text/plain; charset=US-ASCII
> 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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