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Aubin-Horth Nadia
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@aubin-horth-nadia-3844
Last seen 6.8 years ago

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:
[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 Int?grative et des Syst?mes
Universit? Laval