Limma script - Opinions requested
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Orfe, Lisa ▴ 20
@orfe-lisa-4235
Last seen 7.1 years ago
Aha! Thanks for the tip - am getting gene names now! And thanks for the look-see at my script. Cheers, Lisa -A couple of corrections. Background correct is yet doing everything it -should with EList objects, so the background subtraction has to be done -manually. And I now see why you're not getting gene names in your -toptable. You can use - - x <- - read.maimages(targets,columns=list(G="Median",Gb="MedBackground"),gr een.only=TRUE) - x$genes <- read.delim("Annotation file.txt",stringsAsFactors=FALSE) - x$E <- x$E-x$Eb - x <- backgroundCorrect(x, method="normexp", offset=50) - y <- normalizeBetweenArrays(x, method="quantile") - -Then lmFit(y,design,) etc and you'll have annotation in your toptable. -Gordon -On Mon, 30 Aug 2010, Gordon K Smyth wrote: > Dear Lisa, > > limma now has a representation for single colour arrays (ELists), so you can > simplify your pre-processing code somewhat: > > E <- > read.maimages(targets,columns=list(G="Median",Gb="MedBackground"),gr een.only=TRUE) > E$genes <- read.delim("Annotation file.txt",stringsAsFactors=FALSE) > E <- backgroundCorrect(E, method="subtract") > E <- backgroundCorrect(E, method="normexp", offset=50) > NormE <- normalizeBetweenArrays(E, method="quantile") > > I don't understand you question about getting gene names in the toptable, as > this should happen automatically. > > Best wishes > Gordon > > >> Date: Fri, 27 Aug 2010 10:59:10 -0700 >> From: "Orfe, Lisa" <lorfe at="" vetmed.wsu.edu=""> >> To: "'bioconductor at stat.math.ethz.ch'" >> <bioconductor at="" stat.math.ethz.ch=""> >> Subject: [BioC] Limma script - Opinions requested >> Message-ID: >> <44F1D6D7EB8CC84F92859EE5C4E6ECB4011374445539 at CVMMBX.vetmed.wsu.edu> >> Content-Type: text/plain; charset="us-ascii" >> >> I am VERY new to R/Bioconductor and over the past two weeks have managed, I >> hope, to put together a limma script to process my single color generic >> arrays. I was hoping that some of the experts that read these posts could >> comment on it - specifically, is it valid? Is there something I could be >> doing that is easier/more appropriate? I know it works as I have been >> getting the genelists back out at the end, so, my question is very >> general...is this how you would process a home-made single color array? If >> not, I would LOVE some pointers. >> >> >>> library(limma) >>> targets <- readTargets("targets.txt") >>> RG <- read.maimages(targets,source="generic" >>> +columns=list(R="Median",G="Median",Rb="MedBackground",Gb="MedBack ground")) >>> RG$genes <- read.delim("Annotation file.txt") >>> BsubRG <- backgroundCorrect(RG, method="normexp", offset=50) >>> NormRG <- normalizeBetweenArrays(BSubRG$G, method="quantile") >>> MA <- log2(NormRG) >> >> ---Now - if I have a single factor I would set up my design as such: >>> design <- model.matrix(~0+factor(c(1,2,3,4,1,2,3,4,1,2,3,4))) >>> colnames(design) <- c("A", "B", "C", "D") >>> corfit <- duplicateCorrelation(MA, design, ndups=4) >>> fit <- lmFit(MA, design, ndups=4, correlation=corfit$consensus) >>> fit <- eBayes(fit) >>> cont.matrix <- makeContrasts(AvsB=A-B, levels=design) >>> fit2 <- contrasts.fit(fit, cont.matrix) >>> fit2 <- eBayes(fit2) >>> GeneList <- topTable(fit2, "AvsB", n=20, adjust="BH", lfc=1) >>> write.table(GeneList, file = "file name.txt", quote = FALSE, sep = "\t") >> >> >> ---however, if I have multiple factors, then I set up the design as... >>> TS <- paste(targets$columnname, targets$columnname, sep=".") >>> TS <- factor(TS, levels=c("xx.xx", "xx.xx", "xx.xx")) >>> design <- model.matrix(~0+TS) >>> colnames(design) <- levels(TS) >> >> Everything after is the same as above...in pasting this in I do have a >> couple of questions. >> >> 1. Is it recommended/necessary to apply a Bayesian smoothing to both fits? >> 2. How do I get back out my gene names *in* the topTable? >> >> Thanks for your time and I apologize if the answers seem remarkably >> obvious. >> >> Lisa >> Lisa Orfe >> Bustad 405 >> 509-335-6320 >> "Science is a wonderful thing when one does not have to earn one's living >> at it." Albert Einstein > ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:6}}
Annotation Bayesian limma PROcess Annotation Bayesian limma PROcess • 649 views
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