Dear list members,

though I am aware of each of the single design models, I'm not quite sure if I got the design for the following setting correct:

The experiment consists of 24 single-color samples in total obtained from 6 animals. From each animal two time points (prior to treatment and post treatment) exist. In addition each sample was obtained in a technical duplicate. 3 of the animals were treated with a reagent, the other three with a control. The matrix is provided below.

Now what I would like to do is a classic calculation of time, treatment and time x treatment effects ... under consideration of 1) sample pairing and 2) technical replication.

My first (simple) approach neglecting treatment is as follows:
-----------------------------
Line 1 > biolrep <- pData$Replicate
Line 2 > timep <- factor(pData$Timepoint, levels=c("Pre", "Post"))
Line 3 > treat <- factor(pData$Treatment, levels=c("reagent","control"))
Line 4 > sibship <- factor(pData$Animal)
Line 5 > design <- model.matrix(~sibship+timep) # I will first skip treatment here for simplicity

Line 6 > corfit<-duplicateCorrelation(eset,design, ndups=1, block=biolrep) # account for tech-reps.
Line 7 > fit <- lmFit(eset, design, block=biolrep, cor=corfit$consensus) #include it in fit

Line 8 > fit <- lmFit(eset, design)
Line 9 > fit <- eBayes(fit)
Line 10 > topTable(fit, adjust = "BH", coef="timepPost")
--------------------------------

Once I incorporate the treatment instead of the time

Line 5 > design <- model.matrix(~sibship+treat)

... I get an error:

>> Partial NA coefficients for 96 probe(s)

Note: Our array has 96 probes in total so actually all probe coefficients are NA.


If I remove the animal source info  and swap line 5 by:

Line 5 > design <- model.matrix(~treat)

... then everything is fine. Probably because of the overlap of the groups (all samples of each animal are either part of the treatment or the non treatment group).


So my question is: Is there some design model where I can incorporate time AND treatment under consideration of sample pairing and technical replication at the same time?


Thanks for the help

Benjamin






Sample Animal Replicate Timepoint Treatment
1 Animal 1 1 Pre control
2 Animal 1 1 Pre control
3 Animal 1 2 Post control
4 Animal 1 2 Post control
5 Animal 2 3 Pre reagent
6 Animal 2 3 Pre reagent
7 Animal 2 4 Post reagent
8 Animal 2 4 Post reagent
9 Animal 3 5 Pre control
10 Animal 3 5 Pre control
11 Animal 3 6 Post control
12 Animal 3 6 Post control
13 Animal 4 7 Pre reagent
14 Animal 4 7 Pre reagent
15 Animal 4 8 Post reagent
16 Animal 4 8 Post reagent
17 Animal 5 9 Pre control
18 Animal 5 9 Pre control
19 Animal 5 10 Post control
20 Animal 5 10 Post control
21 Animal 6 11 Pre reagent
22 Animal 6 11 Pre reagent
23 Animal 6 12 Post reagent
24 Animal 6 12 Post reagent
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