[BioC] LIMMA experimental design affecting modelmatrix

James W. MacDonald jmacdon at med.umich.edu
Thu Dec 10 20:05:01 CET 2009


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:
> [1] 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 Intégrative et des Systèmes
> Université Laval
> 
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-- 
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
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