Hi, List: I have a question regarding paired samples test in limma package. Here is what I got ####W and B are race ####T and N are tumor and normal(paired by same Samples), respectively ####Samples are individual patients: tar$AccNum######## #### > library(limma); > tar<-readTargets("Test_Desc.txt"); > mydata<-read.delim("TestData.txt",as.is=TRUE,row.names=1); > mydata<-mydata[,tar$mRNASampleNames]; > tar$Type<-gsub("Tumor","T",tar$Type) > tar$Type<-gsub("Normal","N",tar$Type) > group1<-paste(tar$RACE,tar$Type,sep="."); > group<-factor(group1, levels=c( "W.T","W.N","B.T","B.N")) > Samples<-factor(tar$AccNum); ####first option to set up the design matrix######### > design<-model.matrix(~Samples+group); > colnames(design)<-sub("group","",colnames(design)); > colnames(design)<-sub("Samples","",colnames(design)); > colnames(design)[1] <- "Intercept"; > con.matrix<-makeContrasts(AA.T_AA.N=B.T-B.N,EA.T_EA.N=-W.N,levels=de sign); > lmFit(mydata,design)->fit1; Coefficients not estimable: B.N Warning message: Partial NA coefficients for 26804 probe(s) > contrasts.fit(fit1, con.matrix)->fit2 Error in contrasts.fit(fit1, con.matrix) : trying to take contrast of non-estimable coefficient ################it did not work!!! not sure why??######### ##########then I tried the 2nd option to set up the design matrix#### > design<-model.matrix(~-1+group+Samples); > colnames(design)<-sub("group","",colnames(design)); > colnames(design)<-sub("Samples","",colnames(design)); > con.matrix<-makeContrasts(AA.T_AA.N=B.T-B.N,EA.T_EA.N=W.T-W.N,levels =design); > lmFit(mydata,design)->fit1; Coefficients not estimable: S14810 Warning message: Partial NA coefficients for 26804 probe(s) > contrasts.fit(fit1, con.matrix)->fit2 > eBayes(fit2)->fit3 > #######it works somehow in terms of generating lists, but leave an warning message when I did the fit1 using > lmFit(mydata,design)->fit1 (...Coefficients not estimable: S14810) I am sure why is like this? Using some other dataset, I didn't see such warning at all. It seems dataset specific, I am not sure why there is warning here and whether the list come out is good or not. Could someone please have some suggestions? Thanks in advance! Myi