[BioC] Limma time course analysis - defining comparisons

[Helen Zhou]

Dear Sir/Madam

I am trying to analyse a short time series, roughly
following section 8.8 in the limma user guide. I am
interested in differences between all time points. I
am not sure whether I have to make all the pariwise
comparisons directly, or whether they can be done
indirectly as well.

For example, if I want to compare to time points, what
is the differences between the two methods listed
below.

library(bronchialIL13)
# Just for the IL13 samples
data <- HAHrma[,7:15]
# Design
targets	<-
c("h12","h12","h12","h24","h24","h24","h4","h4","h4")
lev	<- c("h12","h24","h4")
f	<- factor(targets, levels=lev)
design	<- model.matrix(~0+f)
colnames(design)	<- lev
fit <- lmFit(data, design)
# 2-step contrasts, used to indirectly get 24 to 4
hours as well as the other two comparisons
contrasts <- makeContrasts("h24-h12", "h12-h4",
levels=design)
fit2 <- contrasts.fit(fit, contrasts)
fit2 <- eBayes(fit2)
# Direct contrast of 24 to 4 hours
contrasts2 <- makeContrasts("h24-h4", levels=design)
fit3 <- contrasts.fit(fit, contrasts2)
fit3 <- eBayes(fit3)
# Comparison
topTable(fit2, coef=1:2)
topTable(fit3, coef=1)

More or less the same probe sets are present, but in
different order and with different p values. Is the
difference because using coef=1:2 will go via an
F-test? And if I want the change from 24h-0h as well
as 42-12h and 12-4h, is it most correct for me to
specify that contrast directly? In my actual
experiment I have 4 time points, so will it be enough
for me with 3 possible comparisons, or will I have to
write all the 6 possible combinations?

Thank you in advance for all your help.

Yours truly
Mrs Helen Zhou

P.S. I think this might have been mentioned on the
list before, but I could not find the email. In that
case, please excuse me for repeating this.