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 [[alternative HTML version deleted]]

Hi Xiayu, On 7/21/2014 3:20 PM, Rao,Xiayu wrote: > 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 di???erently 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 You don't have to do three different calls to makeContrast(). Simply put all the contrasts you care about in one contrasts matrix: contrast <- makeContrasts("mu.0hr-wt.0hr", "mu.6hr-wt.6hr", "mu.24hr-wt.24hr","wt.6hr-wt.0hr", "wt.24hr-wt.6hr", "wt.24hr-wt.0hr","mu.6hr-mu.0hr", "mu.24hr-mu.6hr", "mu.24hr-mu.0hr", levels=design) Best, Jim > > Any suggestions? Thank you very much in advance! > > Thanks, > Xiayu > > > [[alternative HTML version deleted]] > > > > _______________________________________________ > Bioconductor mailing list > Bioconductor at r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor > -- James W. MacDonald, M.S. Biostatistician University of Washington Environmental and Occupational Health Sciences 4225 Roosevelt Way NE, # 100 Seattle WA 98105-6099