Microarray
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Time Course
Limma package
- http://master.bioconductor.org/help/course-materials/2005/BioC2005/labs/lab01/drosEmbryo/ or http://bioinf.wehi.edu.au/marray/jsm2005/lab5/lab5.html. Note the package drosEmbryo is not available on Bioc although it can be downloaded from bioinf.wehi.edu.au. It still cannot be used.
> data(drosEmbryoRMA) Warning message: 'drosEmbryoRMA' looks like a pre-2.4.0 S4 object: please recreate it
- Limma Guide Section 9.6 (no .Rd file nor R code!).
# Step 1 - Read the design and create eset.
targets = readTargets("targets.txt")
eset = rma(ReadAffy())
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)
# Step 2 - Which genes respond at either the 6 hour or 24 hour times in the wild-type?
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")
# Step 3- 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)
fit2 <- eBayes(fit2)
topTableF(fit2, adjust="BH")
# Step 4- 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)
fit2 <- eBayes(fit2)
topTableF(fit2, adjust="BH")
A case study using Limma package
http://www.biomedcentral.com/1756-0500/3/81