[BioC] Re: limma and affy (fwd)

[Gordon Smyth]

>Date: Sat, 21 Jun 2003 00:37:37 EDT
>From: Phguardiol at aol.com
>Subject: limma and affy
>
>Sir,
>I m trying to use limma after having extract background and normalize my data
>(HU133A chips from Affy). then I have 3 different groups with duplicates ou
>triplicates and I d like to know what are the genes differentially expressed
>between these 3 groups.
>here is what I tried to do so:
>library(affy)
>library(limma)
>data <- ReadAffy()
>data2<- rma(data)
>design <- c(1,1,1,2,2,3,3,3)
>fit<-lm.series(exprs(data2), design, weights=1/se.exprs(data2)^2)

A couple of problems here. First 'design' has to be the actual design 
matrix, not just a vector of group identifiers. You can calculate the right 
matrix using

    design <- model.matrix(~ -1+factor(c(1,1,1,2,2,3,3,3)))
    colnames(design) <- c("group1", "group2", "group3")

Secondly, 'rma' does not always calculate standard errors. In this case you 
should skip the weights and simply use

   fit <- lm.series(exprs(data2), design)

To make all pairwise comparisons between your three groups you can estimate 
the appropriate contrasts by

   contrast.matrix <- cbind(group2vs1=c(-1,1,0), group3vs2=c(0,-1,1), 
group3vs1=c(-1,0,1))
   fit2 <- contrasts.fit(fit, contrast.matrix)
   eb <- ebayes(fit2)

If you download LIMMA version 1.1.2 then there are some further 
possibilities, for example

   clas <- classifyTests(eb$t, design=design, contrasts=contrast.matrix)

will use F-tests to classify which of the pair-wise differences between the 
groups are significant for each gene.

Gordon

>eb<-ebayes(fit)
>and then I got this message: Error in if (out$df.prior == Inf) out$s2.post <-
>rep(out$s2.prior, length(sigma)) else out$s2.post <- (ifelse(df.residual ==
>:
>         missing value where TRUE/FALSE needed
>I wonder if my design object is correctly define. the way it is constructed
>is that my first 3 chips are representing group 1, the 2 thereafter are
>belonging to group 2, and the last 3 to group 3. should it be something else ?
>thanks for your help
>yours sincerely
>Philippe Guardiola