I think the problem is with lmFit(). The merged eset definitely contain different data for each samples, since the MDS plot shows data points scattered around the origin. But after feeding this eset to lmFit(), here's what I get: ***************************************************** > fit An object of class "MArrayLM" $coefficients CTL EXPvsCTL 1007_s_at 7.045851 1.487227e-15 1053_at 5.498793 7.281216e-16 117_at 5.047563 -8.262373e-16 121_at 4.350444 4.131187e-16 1255_g_at 2.257968 -2.780886e-16 22272 more rows ... $rank [1] 2 $assign NULL $qr $qr CTL EXPvsCTL [1,] -6.0000000 -4.33333333 [2,] 0.1666667 -2.68741925 [3,] 0.1666667 0.08859624 [4,] 0.1666667 0.08859624 [5,] 0.1666667 0.08859624 31 more rows ... $qraux [1] 1.166667 1.088596 $pivot [1] 1 2 $tol [1] 1e-07 $rank [1] 2 $df.residual [1] 34 34 34 34 34 22272 more elements ... $sigma 1007_s_at 1053_at 117_at 121_at 1255_g_at 0.954188915 0.174833541 1.082247491 0.346694394 0.001783684 22272 more elements ... $cov.coefficients CTL EXPvsCTL CTL 0.1 -0.1000000 EXPvsCTL -0.1 0.1384615 $stdev.unscaled CTL EXPvsCTL 1007_s_at 0.3162278 0.3721042 1053_at 0.3162278 0.3721042 117_at 0.3162278 0.3721042 121_at 0.3162278 0.3721042 1255_g_at 0.3162278 0.3721042 22272 more rows ... $pivot [1] 1 2 $Amean 1007_s_at 1053_at 117_at 121_at 1255_g_at 7.045851 5.498793 5.047563 4.350444 2.257968 22272 more elements ... $method [1] "ls" $design CTL EXPvsCTL [1,] 1 1 [2,] 1 1 [3,] 1 1 [4,] 1 1 [5,] 1 1 31 more rows ... ***************************************************** For some reason fit$stdev.unscaled gives the same values for every probe. On Fri, Jun 7, 2013 at 12:52 PM, Ed Siefker wrote: > I am trying to compare find differentially expressed genes between > appendix and colon tumor samples, which have been arrayed > on different platforms. Namely hgu133a2 and hgu133plus2. > hgu133a2 is a subset of hgu133plus2, and Bioconductor provides > a package, inSilicoMerging, that's supposed to do this, so I > thought it would be straight forward. > > First read in my CEL files and normalize them: > > > targets_hsu <- readTargets("Hsu-targets.txt") > > targets_kai <- readTargets("kaiser-targets.txt") > > ab_hsu <- ReadAffy(filenames=targets_hsu$FileName) > > ab_kai <- ReadAffy(filenames=targets_kai$FileName) > > eset_hsu <- gcrma(ab_hsu) > > eset_kai <- gcrma(ab_kai) > > So far so good. Now I merge the esets with inSilicoMerging: > > library(inSilicoMerging) > > eset <- merge(list(eset_hsu,eset_kai),method="COMBAT") > INSILICOMERGING: Run COMBAT... > INSILICOMERGING: => Found 2 batches > INSILICOMERGING: => Found 0 covariate(s) > > dim(eset_hsu) > Features Samples > 22277 26 > > dim(eset_kai) > Features Samples > 54675 10 > > dim(eset) > Features Samples > 22277 36 > > This looks like it worked. I used plotMDS(), and the data are > nicely intermixed as one would hope. Now I need to do DE > analysis with Limma. Hsu (the first 26 samples) are experimental > and Kai (the last 10 samples) are control. So I create a design > matrix like this: > > > > design <- cbind(CTL=1, EXPvsCTL=c(rep(1,26),rep(0,10))) > > fit <- lmFit(eset, design) > > fit <- eBayes(fit) > > tt<-topTable(fit, coef="EXPvsCTL",number=100000) > > head(tt,n=3) > logFC AveExpr t P.Value adj.P.Val B > 1007_s_at 1.487227e-15 7.045851 4.223110e-15 1 1 -6.235399 > 1053_at 7.281216e-16 5.498793 1.127582e-14 1 1 -6.235399 > 117_at -8.262373e-16 5.047563 -2.068570e-15 1 1 -6.235399 > > tail(tt,n=3) > logFC AveExpr t P.Value adj.P.Val > AFFX-TrpnX-3_at -3.953675e-16 2.257998 -1.507504e-13 1 1 > AFFX-TrpnX-5_at 1.024821e-16 2.257637 4.062598e-14 1 1 > AFFX-TrpnX-M_at 1.024821e-16 2.257637 4.062598e-14 1 1 > B > AFFX-TrpnX-3_at -6.235399 > AFFX-TrpnX-5_at -6.235399 > AFFX-TrpnX-M_at -6.235399 > > > As you can see, the p values and B statistics are the same for every > probe. Clearly > something is wrong here. Did I do something wrong? Is this sort of thing > expected > when you merge datasets like this? Any nudges in the right direction > would be > appreciated. > -Ed > [[alternative HTML version deleted]]