Hello, I found that the question was already answered, but the solution proposed did not work for me. Could you please help to resolve an issue with limma I faced. Please see the attachment. I have a set of arrays for 4 groups of patients (Atype) taken at two time points (Visit). Every patient was analyzed twice, so they are paired and I cannot ignore this. The questions are 1) what genes are differentially expressed between time points in every group; 2) what genes are differentially expressed between disease types at V1 and V3. I make a design matrix this way: atype<-factor(c("lco","bri","bri","bri","con","con","con","lco","mild" ,"mild","mild","lco","con","con","lco","bri","bri","bri","con","con"," con","lco","mild","mild","mild","lco","con","con")) patient<-factor(c("x1","x8","x10","x14","x17","x18","x19","x20","x24", "x26","x27","x29","x31","x38","x1","x8","x10","x14","x17","x18","x19", "x20","x24","x26","x27","x29","x31","x38")) visit<-factor(c("v1","v1","v1","v1","v1","v1","v1","v1","v1","v1","v1" ,"v1","v1","v1","v3","v3","v3","v3","v3","v3","v3","v3","v3","v3","v3" ,"v3","v3","v3")) fact<-paste(visit,atype,sep=".") fact<-as.factor(fact) design<-model.matrix(~0+fact+patient) colnames(design)<-c(levels(fact),colnames(design[,9:21])) cont<-makeContrasts(Mild.Bri.V1=v1.mild-v1.bri,Mild.Bri.V3=v3.mild-v3. bri,Mild.Bri.V1.V3=(v1.mild-v3.mild)-(v1.bri-v3.bri),Mild.Con.V1=v1.mi ld-v1.con,Mild.Con.V3=v3.mild-v3.con,Mild.Con.V1.V3=(v1.mild-v3.mild)- (v1.con-v3.con),Mild.Lco.V1=v1.mild-v1.lco,Mild.Lco.V3=v3.mild-v3.lco, Mild.Lco.V1.V3=(v1.mild-v3.mild)-(v1.lco-v3.lco),Bri.Con.V1=v1.bri-v1. con,Bri.Con.V3=v3.bri-v3.con,Bri.Con.V1.V3=(v1.bri-v3.bri)-(v1.con-v3. con),Bri.Lco.V1=v1.bri-v1.lco,Bri.Lco.V3=v3.bri-v3.lco,Bri.Lco.V1.V3=( v1.bri-v3.bri)-(v1.lco-v3.lco),Lco.Con.V1=v1.lco-v1.con,Lco.Con.V3=v3. lco-v3.con,Lco.Con.V1.V3=(v1.lco-v3.lco)-(v1.con-v3.con),levels=(desig n)) fit<-lmFit(eset.norm,design) # Coefficients not estimable: patientx27 patientx38 patientx8 fit2<-eBayes(contrasts.fit(fit,cont)) In my opinion, the design matrix looks all right, so does it mean there is a bug in limma? Thanks Best wishes, Maxim -------------- next part -------------- An embedded and charset-unspecified text was scrubbed... Name: targets.txt URL: <https: stat.ethz.ch="" pipermail="" bioconductor="" attachments="" 20100317="" 3820d214="" attachment.txt="">

Hello, I found that the question was already answered, but the solution proposed did not work for me. Could you please help to resolve an issue with limma I faced. Please see the attachment. I have a set of arrays for 4 groups of patients (Atype) taken at two time points (Visit). Every patient was analyzed twice, so they are paired and I cannot ignore this. The questions are 1) what genes are differentially expressed between time points in every group; 2) what genes are differentially expressed between disease types at V1 and V3. I make a design matrix this way: atype<-factor(c("lco","bri","bri","bri","con","con","con","lco","mild" ,"mild","mild","lco","con","con","lco","bri","bri","bri","con","con"," con","lco","mild","mild","mild","lco","con","con")) patient<-factor(c("x1","x8","x10","x14","x17","x18","x19","x20","x24", "x26","x27","x29","x31","x38","x1","x8","x10","x14","x17","x18","x19", "x20","x24","x26","x27","x29","x31","x38")) visit<-factor(c("v1","v1","v1","v1","v1","v1","v1","v1","v1","v1","v1" ,"v1","v1","v1","v3","v3","v3","v3","v3","v3","v3","v3","v3","v3","v3" ,"v3","v3","v3")) fact<-paste(visit,atype,sep=".") fact<-as.factor(fact) design<-model.matrix(~0+fact+patient) colnames(design)<-c(levels(fact),colnames(design[,9:21])) cont<-makeContrasts(Mild.Bri.V1=v1.mild-v1.bri,Mild.Bri.V3=v3.mild-v3. bri,Mild.Bri.V1.V3=(v1.mild-v3.mild)-(v1.bri-v3.bri),Mild.Con.V1=v1.mi ld-v1.con,Mild.Con.V3=v3.mild-v3.con,Mild.Con.V1.V3=(v1.mild-v3.mild)- (v1.con-v3.con),Mild.Lco.V1=v1.mild-v1.lco,Mild.Lco.V3=v3.mild-v3.lco, Mild.Lco.V1.V3=(v1.mild-v3.mild)-(v1.lco-v3.lco),Bri.Con.V1=v1.bri-v1. con,Bri.Con.V3=v3.bri-v3.con,Bri.Con.V1.V3=(v1.bri-v3.bri)-(v1.con-v3. con),Bri.Lco.V1=v1.bri-v1.lco,Bri.Lco.V3=v3.bri-v3.lco,Bri.Lco.V1.V3=( v1.bri-v3.bri)-(v1.lco-v3.lco),Lco.Con.V1=v1.lco-v1.con,Lco.Con.V3=v3. lco-v3.con,Lco.Con.V1.V3=(v1.lco-v3.lco)-(v1.con-v3.con),levels=(desig n)) fit<-lmFit(eset.norm,design) # Coefficients not estimable: patientx27 patientx38 patientx8 fit2<-eBayes(contrasts.fit(fit,cont)) In my opinion, the design matrix looks all right, so does it mean there is a bug in limma? Thanks Best wishes, Maxim -------------- next part -------------- An embedded and charset-unspecified text was scrubbed... Name: targets.txt URL: <https: stat.ethz.ch="" pipermail="" bioconductor="" attachments="" 20100317="" e0adce95="" attachment.txt="">