LIMMA experimental design affecting modelmatrix
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We are analysing a cDNA microarray dataset in LIMMA with the following design and we run into "Coefficients not estimable" comments. R = 2.9.0 limma=2.18 We have two groups, "A" and "D" with 6 biological replicates each (indicated in the targets file). We are interested in significant gene expression differences between A and D on average. A given biological replicate of "A" was compared to a biological replicate of "D", with a dye-swap. A1=D1 A2=D2 A3=D3 A4=D4 A5=D5 A6=D6 Therefore, we have six mini-experiments that are not connected one to the other. After normalisation, we use teh following design with the goal of doing a contrast as shown below design <- modelMatrix(targets, ref="D1") design <- cbind(Dye=1, design) fit<-lmFit(MAptip.nba.scale,design) here we get: Coefficients not estimable: D5 A4 D2 D6 D3 And we checked with > is.fullrank(design) and we get:  FALSE Our question is, is our experimental design (non loop, non reference design) with samples not being directly compared on a microarray causing these non estimable coefficients? If so, is there a way that we can keep the information on biological replicates and still use LIMMA? This is the contrast we were planning to use (which of course does not work) cont.matrix <- makeContrasts(AvsD = (A1 + A2 + A3 + A4 + A5 + A6 - D2 - D3 - D4 - D5 - D6)/6, levels=design) fit2 <- contrasts.fit(fit, cont.matrix) Error in contrasts.fit(fit, cont.matrix) : trying to take contrast of non-estimable coefficient In addition: Warning message: In any(contrasts[-est, ]) : coercing argument of type 'double' to logical fit3 <- eBayes(fit2) Thank you Nadia Aubin-Horth Assistant professor Biology Department Institut de Biologie Int?grative et des Syst?mes Universit? Laval
Microarray limma a4 Microarray limma a4 • 889 views
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@james-w-macdonald-5106
Last seen 1 hour ago
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Hi Nadia, Aubin-Horth Nadia wrote: > We are analysing a cDNA microarray dataset in LIMMA with the following > design and we run into "Coefficients not estimable" comments. > > R = 2.9.0 > limma=2.18 > > We have two groups, "A" and "D" with 6 biological replicates each > (indicated in the targets file). We are interested in significant gene > expression differences between A and D on average. A given biological > replicate of "A" was compared to a biological replicate of "D", with a > dye-swap. > A1=D1 > A2=D2 > A3=D3 > A4=D4 > A5=D5 > A6=D6 I don't see a dye-swap here. > > Therefore, we have six mini-experiments that are not connected one to > the other. > > After normalisation, we use teh following design with the goal of doing > a contrast as shown below > > design <- modelMatrix(targets, ref="D1") > design <- cbind(Dye=1, design) Without a dye-swap (and you don't indicate one above), you cannot estimate dye bias. All you can do is compare the two sample types. Since the comparison is intrinsic to the data, all you want to do is compute the mean of the ratios (or log ratios, actually) and then test to see if the mean is different from zero. In this very simple case, you don't have to create a design matrix, as limma will produce one for you. So you just do: fit <- lmFit(MAptip.nba.scale) fit2 <- eBayes(fit) topTable(fit2) FYI, what you are doing is to treat each sample as if it isn't replicated, which is why you are running into problems. Best, Jim > > fit<-lmFit(MAptip.nba.scale,design) > > here we get: > Coefficients not estimable: D5 A4 D2 D6 D3 > > And we checked with > > is.fullrank(design) > and we get: >  FALSE > > Our question is, is our experimental design (non loop, non reference > design) with samples not being directly compared on a microarray causing > these non estimable coefficients? If so, is there a way that we can keep > the information on biological replicates and still use LIMMA? > > This is the contrast we were planning to use (which of course does not > work) > cont.matrix <- makeContrasts(AvsD = (A1 + A2 + A3 + A4 + A5 + A6 > - D2 - D3 - D4 - D5 - D6)/6, levels=design) > fit2 <- contrasts.fit(fit, cont.matrix) > Error in contrasts.fit(fit, cont.matrix) : > trying to take contrast of non-estimable coefficient > In addition: Warning message: > In any(contrasts[-est, ]) : coercing argument of type 'double' to logical > fit3 <- eBayes(fit2) > > > Thank you > > Nadia Aubin-Horth > Assistant professor > Biology Department > Institut de Biologie Int?grative et des Syst?mes > Universit? Laval > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor -- James W. MacDonald, M.S. Biostatistician Douglas Lab University of Michigan Department of Human Genetics 5912 Buhl 1241 E. Catherine St. Ann Arbor MI 48109-5618 734-615-7826 ********************************************************** Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues
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