[BioC] probe length was:RE: [BioC] GCRMA backgrounds?
paul.boutros at utoronto.ca
paul.boutros at utoronto.ca
Fri Jul 23 14:54:16 CEST 2004
One reference I know is the initial paper introducing the Agilent chips by Tim
Hughes, T. R., M. Mao, et al. (2001). "Expression profiling using microarrays
fabricated by an ink-jet oligonucleotide synthesizer." Nat Biotechnol 19(4):
There is a section in the results "impact of oligonucleotide length on
hybridization properties" that you might want to check out.
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
BTW: If you can find ANYTHING on the Affy site, more power to you:)
>>> "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
dropped the 'non-' in the initial post. Also this would only apply
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
probesets per mRNA, for short mRNAs there is already some overlap,
would be worse for longer probes, making them less independent. It
also extend the probed region further from the 3' end from where
occurs and so efficiency may be reduced?
If you need a reference I'm sure the affy website or some of their
would have something.
Sorry for any confusion.
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?
>>> "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.
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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
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