[BioC] technical replicates and lmFit error

[Ommen Kloeke, A.E.E. van]

Dear Bioconductor team,

I am working on a 2-colour Agilent dataset which has both biological and technical replicates. A problem which many people already encountered as I could see from the previous posts, so my apologies for bringing it up again.
However, I tried the script given for this situation in the Limma user guide, but my lmFit gives an error if I try this!

My experiment contains three treatments: "AC", "low" and "high" - each has 4 biological replicates and 2 technical replicates as indicated in the attached target file. The main contrasts of interest are "low-AC" and "high-AC". Here's the script I tried:

design = modelMatrix(targets, ref = "AC1")
design = cbind(Dye = 1, design)
colnames(design)
#[1] "Dye"   "AC2"   "AC4"   "AC5"   "high1" "high2" "high4" "high5" "low1"  "low3"  "low4"  "low5"

fit = lmFit(MAbet, design)
cont.matrix = makeContrasts(ACvsLow = (high1+high2+high4+low5-AC2-AC4-AC5)/4,levels = design)
fit2 = contrasts.fit(fit, cont.matrix)
fit2 = eBayes(fit2)
topTable(fit2, adjust = "fdr")

However the LmFit gives an error:
> fit = lmFit(MAbet, design)
Coefficients not estimable: low4 low5
Warning message:
Partial NA coefficients for 43803 probe(s)

I understand this has to do with my design, but I don't know how to fix it:
> design
      Dye AC2 AC4 AC5 high1 high2 high4 high5 low1 low3 low4 low5
[1,]   1   0   0   0     0     0     0     0    0    0    1    0
[2,]   1  -1   0   0     0     0     0     0    0    0    0    1
[3,]   1   0   1   0     0     0     0     0   -1    0    0    0
[4,]   1   0   0   1     0     0     0     0    0   -1    0    0
[5,]   1   0   0   0     0     0     1     0    0    0    0    0
[6,]   1  -1   0   0     0     0     0     1    0    0    0    0
[7,]   1   0   1   0    -1     0     0     0    0    0    0    0
[8,]   1   0   0   1     0    -1     0     0    0    0    0    0
[9,]   1   0   0   0    -1     0     0     0    0    0    1    0
[10,]   1   0   0   0     0    -1     0     0    0    0    0    1
[11,]   1   0   0   0     0     0     1     0   -1    0    0    0
[12,]   1   0   0   0     0     0     0     1    0   -1    0    0
> is.fullrank(design)
[1] FALSE

I completely trust in your expertise! Any help is very welcome and appreciated.

Much obliged and many thanks!

Elaine van Ommen Kloeke



VU university Amsterdam
Department of Ecological Science
room:  H-119
phone: 020-5987217
www.falw.vu.nl/animalecology<http://www.falw.vu.nl/animalecology>

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