Hi Alice, Please check the expression level of these negative control probes. They must be very low. If all the negative control probes are high, you might get a hybridizatin problem; check the stringency control probes. If these low-expressed negative controls are in your Top50 lists, you might try a VST transformation method as reported in http://nar.oxfordjournals.org/cgi/content/abstract/gkm1075v1 Good luck! Simon Lin Northwestern =========================== Date: Fri, 7 Mar 2008 16:48:02 +1300 From: "Johnstone, Alice" <alice.johnstone@esr.cri.nz> Subject: [BioC] What should happen to control probe information in bead level Illumina analyses? To: "bioconductor" <bioconductor at="" stat.math.ethz.ch=""> Message-ID: <92B672A208AB2F419334EE1EC8C58D9D01D13400 at kscmail1.esr.cri.nz> Content-Type: text/plain; charset="us-ascii" Hi again! When running through my analysis of bead summary data that I have created from bead level data (background corrected with normexp, and summarised standard) I produced a topTable list which contains probeIDs that are not found in Illumina's reference file. When I checked the controlprobeprofile.txt there they were! It looks like I haven't got huge DE change in my experiment, but I still wouldn't have expected Illumina's negative controls to make the top 50.. Somethings not right.

Thanks for your reply Simon, Yes the expression level is at the low end of the range so I do not think that this is a hybridisation problem as the original control plots looked fine. I used the VST transformation with a quantile normalisation of the Bead Summary data. My question is whether it is correct to remove the control probe information from the Bead Summary object before fitting the regression model so I don't get any spurious results like I have found, and what is the best way to do this? The expression values for one of the control genes are below, the trt1-control contrast comes up in my topTable results. This only occurs when I do my own bead-level summation, the BeadStudio produced bead summary data does not have the intensity info for this probe. Trt 1 Control Trt 2 7.544901 7.638846 7.531172 7.515447 7.614039 7.581452 7.495771 7.605357 7.555899 7.485725 7.622735 7.588895 7.538404 7.592215 7.516454 Regards Alice -----Original Message----- From: bioconductor-bounces@stat.math.ethz.ch [mailto:bioconductor-bounces at stat.math.ethz.ch] On Behalf Of Simon Lin Sent: Saturday, 8 March 2008 5:11 a.m. To: bioconductor at stat.math.ethz.ch Subject: [BioC] What should happen to control probe information in beadlevel Illumina analyses? Hi Alice, Please check the expression level of these negative control probes. They must be very low. If all the negative control probes are high, you might get a hybridizatin problem; check the stringency control probes. If these low-expressed negative controls are in your Top50 lists, you might try a VST transformation method as reported in http://nar.oxfordjournals.org/cgi/content/abstract/gkm1075v1 Good luck! Simon Lin Northwestern =========================== Date: Fri, 7 Mar 2008 16:48:02 +1300 From: "Johnstone, Alice" <alice.johnstone@esr.cri.nz> Subject: [BioC] What should happen to control probe information in bead level Illumina analyses? To: "bioconductor" <bioconductor at="" stat.math.ethz.ch=""> Message-ID: <92B672A208AB2F419334EE1EC8C58D9D01D13400 at kscmail1.esr.cri.nz> Content-Type: text/plain; charset="us-ascii" Hi again! When running through my analysis of bead summary data that I have created from bead level data (background corrected with normexp, and summarised standard) I produced a topTable list which contains probeIDs that are not found in Illumina's reference file. When I checked the controlprobeprofile.txt there they were! It looks like I haven't got huge DE change in my experiment, but I still wouldn't have expected Illumina's negative controls to make the top 50.. Somethings not right. _______________________________________________ Bioconductor mailing list Bioconductor at stat.math.ethz.ch https://stat.ethz.ch/mailman/listinfo/bioconductor Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor P Think before you print This e-mail transmission and any attachments that accompany it may contain information that is privileged, confidential or otherwise exempt from disclosure under applicable law and is intended solely for the use of the individual(s) to whom it was intended to be addressed. If you have received this e-mail by mistake, or you are not the intended recipient, any disclosure, dissemination, distribution, copying or other use or retention of this communication or its substance is prohibited. If you have received this communication in error, please immediately reply to the author via e-mail that you received this message by mistake and also permanently delete the original and all copies of this e-mail and any attachments from your computer. Thank you.