Hello,

Thank you for providing limma user guide, which is of great help. It is very clear on the guide of how to analyze time course data. But I was asked to answer different questions.

The study design of my data is quite similar to that described on the guide. There are duplicates for each of the 6 conditions. The code below is from the guide.
> lev <- c("wt.0hr","wt.6hr","wt.24hr","mu.0hr","mu.6hr","mu.24hr")
> f <- factor(targets$Target, levels=lev)
> design <- model.matrix(~0+f)
> colnames(design) <- lev
> fit <- lmFit(eset, design)
#1. Which genes respond at either the 6 hour or 24 hour times in the wild-type? Any two contrasts between the three times would give the same result.
> cont.wt <- makeContrasts("wt.6hr-wt.0hr", "wt.24hr-wt.6hr", levels=design)
> fit2 <- contrasts.fit(fit, cont.wt)
> fit2 <- eBayes(fit2)
> topTableF(fit2, adjust="BH")
#2. Which genes respond (i.e., change over time) in the mutant?
> cont.mu <- makeContrasts("mu.6hr-mu.0hr", "mu.24hr-mu.6hr", levels=design)
> fit2 <- contrasts.fit(fit, cont.mu)
……

#3. Which genes respond diﬀerently over time in the mutant relative to the wild-type?
> cont.dif <- makeContrasts(Dif6hr =(mu.6hr-mu.0hr)-(wt.6hr-wt.0hr), Dif24hr=(mu.24hr-mu.6hr)-(wt.24hr-wt.6hr), levels=design)
> fit2 <- contrasts.fit(fit, cont.dif)
……

But I was asked to find the changed genes under the following specific conditions.

1.       Comparisons of the two types at each time point:

wt.0hr vs. mu.0hr

wt.6hr vs. mu.6hr

wt.24hr vs. mu.24hr

2.       Comparisons of different time points in wt:

wt.0hr vs. wt.6hr

wt.6hr vs. wt.24hr

3.       Comparisons of different time points in mu:

mu.0hr vs. mu.6hr

mu.6hr vs. mu.24hr
In this case, I was thinking to do the following but I do not feel it right. Or should I subset the data according to each comparison to do the analysis?
makeContrasts(“mu.0hr-wt.0hr”, “mu.6hr-wt.6hr”, “mu.24hr-wt.24hr”, levels=design) for 1
makeContrasts("wt.6hr-wt.0hr", "wt.24hr-wt.6hr", "wt.24hr-wt.0hr", levels=design) for 2
makeContrasts("mu.6hr-mu.0hr", "mu.24hr-mu.6hr", "mu.24hr-mu.0hr", levels=design) for 3

Any suggestions? Thank you very much in advance!

Thanks,
Xiayu


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