I'm comparing several BioC affy methods applied to the same data set. I have noticed a discrepency between mas5(), call.exprs(method="mas"), and call.exprs(method="mas-R"), and a possible similar problem with justPlier. Here is how I generated the data: mydata.A.raw <- ReadAffy() mydata.B.raw <- ReadAffy() mydata.A.ce <- call.exprs(mydata.A.raw,"mas5",sc=500) mydata.B.ce <- call.exprs(mydata.B.raw,"mas5",sc=500) all.exprs.ce <- rbind(exprs(mydata.A.ce),exprs(mydata.B.ce)) mydata.A.cer <- call.exprs(mydata.A.raw,method="mas5-R",sc=500) mydata.B.cer <- call.exprs(mydata.B.raw,method="mas5-R",sc=500) all.exprs.cer <- rbind(exprs(mydata.A.cer),exprs(mydata.B.cer)) mydata.A.mas <- mas5(mydata.A.raw,sc=500) mydata.B.mas <- mas5(mydata.B.raw,sc=500) all.exprs.mas <- rbind(log2(exprs(mydata.A.mas)),log2(exprs(mydata.B.mas))) mydata.A.plr <- justPlier(mydata.A.raw) mydata.B.plr <- justPlier(mydata.B.raw) all.exprs.plr <- rbind(exprs(mydata.A.plr),exprs(mydata.B.plr)) all.exprs <- NULL all.exprs[1] <- list(all.exprs.ce) all.exprs[2] <- list(all.exprs.cer) all.exprs[3] <- list(all.exprs.mas) all.exprs[4] <- list(all.exprs.plr) design <- model.matrix(~ -1+factor(c(rep(1,5),rep(2,5)))) colnames(design) <- c("group1","group2") top.all <- NULL fit2.all <- NULL for( i in 1:length(all.exprs) ){ fit <- lmFit(all.exprs[[i]], design) fit$Amean <- rowMeans(all.exprs[[i]]) cont.matrix <- makeContrasts(compare=group1-group2, levels=design) fit2 <- contrasts.fit(fit, cont.matrix) fit2 <- eBayes(fit2) fit2.all[i] <- list(fit2) top <- topTable(fit2,coef=1,n=nrow(all.exprs[[i]]), genelist=rownames(all.exprs[[i]]), adjust.method="fdr",sort.by="P") o <- order(top$Name) top.all[i] <- list(top[o,]) } Here are the summaries from these four calls: > summary(fit2.all[[1]]$coef[,1]) Min. 1st Qu. Median Mean 3rd Qu. Max. -12.1900 0.1680 0.9118 1.0570 1.8030 10.9100 > summary(fit2.all[[2]]$coef[,1]) Min. 1st Qu. Median Mean 3rd Qu. Max. -9.01800 -0.42810 -0.13710 0.02798 0.26990 7.95600 > summary(fit2.all[[3]]$coef[,1]) Min. 1st Qu. Median Mean 3rd Qu. Max. -13.5100 -1.1550 -0.4108 -0.2666 0.4794 9.5860 summary(fit2.all[[4]]$coef[,1]) Min. 1st Qu. Median Mean 3rd Qu. Max. -19.0700 0.2585 0.9192 1.1290 1.7710 19.2100 Both the call.exprs(method="mas") and justPlier have medians much different than zero. The call to mas5() and call.exprs(method="mas-R") have medians closer to zero, but I would have thought they would be nearly the same. I have tried all the various affy methods on this data set and the call.exprs(method="mas") and justPlier are different than the rest of the methods. Perhaps I am doing something wrong in my use of call.exprs() or justPlier(). I am using HG-U133A/B arrays, and base 2.0.1 datasets 2.0.1 utils 2.0.1 grDevices 2.0.1 graphics 2.0.1 stats 2.0.1 methods 2.0.1 tools 2.0.1 Biobase 1.5.0 reposTools 1.5.1 affy 1.5.8 splines 2.0.1 survival 2.16 genefilter 1.5.0 multtest 1.5.2 siggenes 1.2.11 qvalue 1.1 limma 1.8.18 matchprobes 1.0.12 gcrma 1.1.1 simpleaffy 2.08 vsn 1.5.0 hgu133acdf 1.4.3 hgu133bcdf 1.4.3 plier 0.03 xtable 1.2-4 Thanks, Dick ********************************************************************** ********* Richard P. Beyer, Ph.D. University of Washington Tel.:(206) 616 7378 Env. & Occ. Health Sci. , Box 354695 Fax: (206) 685 4696 4225 Roosevelt Way NE, # 100 Seattle, WA 98105-6099 http://depts.washington.edu/ceeh/ServiceCores/FC5/FC5.html

Please ignore the original posting. Dumb coding mistake. Sorry if someone wasted time being kind enough to try and help. Thanks, Dick ********************************************************************** ********* Richard P. Beyer, Ph.D. University of Washington Tel.:(206) 616 7378 Env. & Occ. Health Sci. , Box 354695 Fax: (206) 685 4696 4225 Roosevelt Way NE, # 100 Seattle, WA 98105-6099 http://depts.washington.edu/ceeh/ServiceCores/FC5/FC5.html