Hi Arnar, Wei Shi and I have checked out our own HT-12 v4 data, and we do not see the bimodal distribution that you have observed. So we think that this might be a property of your data rather than a property of HT-12 v4 BeadChips in general. Note that we are now recommending the neqc() function of the limma package for background correcting and normalizing Illumina arrays. This is a modication of the normexp algorithm to use negative control probe estimates. See ?neqc and the Illumina case study in the limma User's Guide. This function will not be very sensitive to the shape of the distribution of the negative controls. Best wishes Gordon ------ ORIGINAL MESSAGE ------- [BioC] Distribution of negative controls on Illumina HT12-v4 chips (double background?) Arnar Flatberg arnar.flatberg at gmail.com Tue Feb 22 15:46:45 CET 2011 Hi list, We have recently seen an odd distribution of signal intensities using the new version 4 HT-12 chips. Specifically, the distribution of negative controls looks like a mixture of two gaussians. This invalidates the common assumption of normal distributed background used in both the detection calls and some error-model based normalizations, e.g. norm- exp in limma. We did not have this type of distribution in version 3 of HT12, nor in any other chip type from Illumina. Has anyone else seen this particular issue, and specifically on the HT-12 v4 chip? Below is a link to density plots of the negative control probes of 6 experiments I've compared. The top plots are from a lab in Oslo on the HT-12 v4, at hte bottom are two experiments from our lab using HT12 v4 and a comparison of a Rat v1 chip from our lab. Figure: http://oi52.tinypic.com/tz05v.jpg <%20http://oi52.tinypic.com/tz05v.jpg> Thanks, Arnar ---------------------- Arnar Flatberg Data scientist Trondheim Genome Resource Center St. Olav's hospital, Norwegian University of Science and Technology Trondheim Norway ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:4}}

Hi Arnar, Gordon et al., we also have data on an increasing number of these chips, and so far I see no evidence for a bimodal distribution. Ina ----- Original Message ----- From: "Gordon K Smyth" <smyth@wehi.edu.au> To: "arnar flatberg" <arnar.flatberg at="" gmail.com=""> Cc: "Bioconductor mailing list" <bioconductor at="" r-project.org=""> Sent: Saturday, February 26, 2011 11:39:20 PM Subject: [BioC] Distribution of negative controls on Illumina HT12-v4 chips Hi Arnar, Wei Shi and I have checked out our own HT-12 v4 data, and we do not see the bimodal distribution that you have observed. So we think that this might be a property of your data rather than a property of HT-12 v4 BeadChips in general. Note that we are now recommending the neqc() function of the limma package for background correcting and normalizing Illumina arrays. This is a modication of the normexp algorithm to use negative control probe estimates. See ?neqc and the Illumina case study in the limma User's Guide. This function will not be very sensitive to the shape of the distribution of the negative controls. Best wishes Gordon ------ ORIGINAL MESSAGE ------- [BioC] Distribution of negative controls on Illumina HT12-v4 chips (double background?) Arnar Flatberg arnar.flatberg at gmail.com Tue Feb 22 15:46:45 CET 2011 Hi list, We have recently seen an odd distribution of signal intensities using the new version 4 HT-12 chips. Specifically, the distribution of negative controls looks like a mixture of two gaussians. This invalidates the common assumption of normal distributed background used in both the detection calls and some error-model based normalizations, e.g. norm- exp in limma. We did not have this type of distribution in version 3 of HT12, nor in any other chip type from Illumina. Has anyone else seen this particular issue, and specifically on the HT-12 v4 chip? Below is a link to density plots of the negative control probes of 6 experiments I've compared. The top plots are from a lab in Oslo on the HT-12 v4, at hte bottom are two experiments from our lab using HT12 v4 and a comparison of a Rat v1 chip from our lab. Figure: http://oi52.tinypic.com/tz05v.jpg <%20http://oi52.tinypic.com/tz05v.jpg> Thanks, Arnar ---------------------- Arnar Flatberg Data scientist Trondheim Genome Resource Center St. Olav's hospital, Norwegian University of Science and Technology Trondheim Norway ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:8}}