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@cchmc.org> Subject: probe length was:RE: [BioC] GCRMA backgrounds? To: <hannah@mpimp-golm.mpg.de> Cc: bioconductor@stat.math.ethz.ch Message-ID: <s0ffd3d8.039@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@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@cchmc.org] Sent: Mittwoch, 21. Juli 2004 19:49 To: Matthew Hannah; bioconductor@stat.math.ethz.ch Subject: Re: [BioC] GCRMA backgrounds? What are references for this? Mike >>> "Matthew Hannah" <hannah@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@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

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. Paul > -----Original Message----- > From: paul.boutros@utoronto.ca [mailto:paul.boutros@utoronto.ca] > Sent: Friday, July 23, 2004 8:54 AM > To: bioconductor@stat.math.ethz.ch > Cc: Michael.Barnes@cchmc.org; Hannah@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@cchmc.org> > Subject: probe length was:RE: [BioC] GCRMA backgrounds? > To: <hannah@mpimp-golm.mpg.de> > Cc: bioconductor@stat.math.ethz.ch > Message-ID: <s0ffd3d8.039@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@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@cchmc.org] > Sent: Mittwoch, 21. Juli 2004 19:49 > To: Matthew Hannah; bioconductor@stat.math.ethz.ch > Subject: Re: [BioC] GCRMA backgrounds? > > > What are references for this? > > Mike > > >>> "Matthew Hannah" <hannah@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@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