Setting up my design matrix
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5.0 years ago
marion.ryan ▴ 40

I think I am having an issue with my contrast matrix, in terms of comparing concentrate versus grass fed animals (RMA normalised = eset). I get an output of DE genes, but none of them are differently expressed (Adj P value) and the gene list is different from that of a previous and I believe correct analysis, by an experienced bioinformatition who has now left. Any insight in to this would be greatly appreciated, code below. 1st 7 CEL files =Concentrate 2nd 7 CEL files =Grass

Matrix design

###################################################
design <- model.matrix(~ 0+factor(c(1,1,1,1,1,1,1,2,2,2,2,2,2,2)))
colnames(design) <- c("Concentrate", "Grass")
fit <- lmFit(eset, design)**

###################################################
###Contrast Matrix-new 
###################################################
contrast.matrix <- makeContrasts(Concentrate - Grass, levels=design)
fit2 <- contrasts.fit(fit, contrast.matrix)
fit2 <- eBayes(fit2) 

###################################################
###Output –differentially expressed genes 
###################################################
topTable(fit2,coef=NULL,number=500,genelist=fit2$genes,adjust.method="BH",sort.by="B",resort.by=NULL,p.value=1,lfc=0)
tt=topTable(fit2, number = 150)
write.table(tt, "ConcentratevGrass.txt", sep = "\t")
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1
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It all looks fine to me, aside from the '**' after lmFit. Was there any blocking used in setting up the experiment?

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