[BioC] basic limma questions

[Endre Sebestyen]

Again :

Hi!

I'm a beginner in limma and bioconductor, and I'd like to ask a few
basic questions.

I wrote the following script :

library(limma)
targets <- readTargets("Targets1.txt")
RG <- read.maimages(targets$FileName, columns=list(R="CtrlVol",
G="DataVol", Rb="CtrlBg", Gb="DataBg"),
annotation=c("ID","Name","Rep"))
RG$genes <- readGAL("maize.gal")
RG$printer <- getLayout(RG$genes)
spottypes <- readSpotTypes()
RG$genes$Status <- controlStatus(spottypes, RG)
bgCorr <- backgroundCorrect(RG, method="movingmin")
nWithin <-normalizeWithinArrays(bgCorr, method="loess")
nBetween <- normalizeBetweenArrays(nWithin, method="Aquantile")
design <- c(1,1,1)
isGene <- nBetween$genes$Status == "cDNA"
fit <- lmFit(nBetween[isGene, ], design)
fit <- eBayes(fit)
res <- topTable(fit, number=1000)
write.table(res, file="results24top1000.txt", sep="+++")

First, limma didn't recognize the ArrayVision format, and I had to
parse the raw data and define the columns myself. Is it correct to
pass the CtrlVol to R, DataVol to G, etc? The other question is that
I'm not sure about the design. Cy3 was the treated, Cy5 the control,
but after I used the read.maimages function and defined the values
myself, this design should be OK. Am I right?

Last question : how can I combine the replicates on a chip? I have
some genes with 2,3,etc replicates, but not all. This is the 46k maize
array from www.maizearray.org

Thanks for any comment and help.


Endre Sebestyen

-- 
Agricultural Research Institute of the Hungarian Academy of Sciences
Department of Applied Genomics
H-2462 Martonvásár, Brunszvik street 2.