blocking in limma gives an estimability problem
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@pedro-lopez-romero-1618
Last seen 9.5 years ago
Dear List, I have a problem of estimability using LIMMA that I do not know how to solve. I think that I am using the right model, but I can not solve the problem so I need someone to help me out to figure out what is going on. I have an experiment with paired samples within two different strains. The data is as follows: Array mouse strain treatment 1 1 wt c 2 2 wt c 3 3 wt c 4 1 wt t 5 2 wt t 6 3 wt t 7 4 ko c 8 5 ko c 9 6 ko c 10 4 ko t 11 5 ko t 12 6 ko t It is exactly the same as the example 8.3 in the limma user guide, except that I have two strains, WT and KO with the same paired structure. I am using a linear model with the animal effect as a block effect to consider the pairing of the data, My code is the following: block=factor(c("A1","A2","A3","A1","A2","A3", "A4","A5","A6","A4","A5","A6"), levels=c("A1","A2","A3","A4","A5","A6") ) contrasts(block)=contr.sum(6) ttoEFF=factor(c("wtC","wtC","wtC","wtM","wtM","wtM", "koC","koC","koC","koM","koM","koM"), levels=c("wtC","wtM","koC","koM")) contrasts(ttoEFF)=contr.sum(4) design=model.matrix(~ -1 + ttoEFF + block) colnames(design) =c("wtC","wtM","koC","koM","A1","A2","A3","A4","A5") CM= makeContrasts(wtM-koM,wtC-koC,levels=design) fit=lmFit(eset,design) fit2=contrasts.fit(fit,CM) fit2=eBayes(fit2) DE=decideTests(fit2,method="nestedF",adjust.method="BY",p.value=0.05) >From " fit=lmFit(eset,design)" I get that the coefficient for the animal 5 is not estimable. I am considering the nested nature of the animals to its strain by the codification that I am using for the animals, and I have enough degrees of freedom to estimate the whole set of parameters of the model, so I do not understand why parameter A5 can not be estimated. If I work only in the arrays 1 to 6, considering only the strain WT separately from each other, the things work fine. For arrays 7 to 12 corresponding to the KO strain the things work ok as well , but if I extend the model to include the whole data, the parameter A5 corresponding to the animal 5 is not estimable and I don?t understand why. I want to use the whole data, because I am also interested in the comparisons between lines: CM= makeContrasts(wtC - wtM,koC-koM,levels=design) I will appreciate any help on this. Thank you very much. Pedro.- My session info is: > sessionInfo() Version 2.3.0 (2006-04-24) i386-pc-mingw32 attached base packages: [1] "splines" "tools" "methods" "stats" "graphics" "grDevices" "utils" "datasets" [9] "base" other attached packages: statmod qvalue MASS sma geneplotter affycoretools GOstats "1.2.4" "1.5.0" "7.2-27" "0.5.15" "1.10.0" "1.4.0" "1.6.0" Category hgu95av2 KEGG multtest genefilter survival xtable "1.4.0" "1.12.0" "1.12.0" "1.10.0" "1.10.0" "2.24" "1.3-1" RBGL GO graph Ruuid cluster codelink annotate "1.8.0" "1.12.0" "1.10.0" "1.10.0" "1.10.5" "1.0.0" "1.10.0" limma affy affyio Biobase "2.6.0" "1.10.0" "1.0.0" "1.10.0"
GO hgu95av2 Ruuid multtest affy graph limma RBGL qvalue Category affyio GO hgu95av2 • 1.1k views
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