Question: Single-color Agilent array analyzing in R
2
gravatar for Leite
21 months ago by
Leite410
Leite410 wrote:

Hello everyone,

I'm working with data that was performed using Agilent-014850 Whole Human Genome Microarray 4x44K G4112F arrays and the One Color Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, USA).

I'm trying to set up a code in R:

#Load limma
library(limma)

#Set-up
targetinfo <- readTargets("Targets.txt",row.names="FileName",sep="")

#Read files
project <- read.maimages(targetinfo,source="agilent", green.only=TRUE)

#Background correction
project.bgc <- backgroundCorrect(project, method="normexp", offset=16)

#Normalize the data with the 'quantile' method for 1-color
project.NormData <-normalizeBetweenArrays(project.bgc,method="quantile")

#Create the study design and comparison model
design <- paste(targetinfo$Target, sep="")
design <- factor(design)
comparisonmodel <- model.matrix(~0+design)
colnames(comparisonmodel) <- levels(design)
#Checking the experimental design
design
comparisonmodel

project.fit <- lmFit(project.NormData, comparisonmodel)
#When I used > project.fit <- lmFit(project.NormData$M, comparisonmodel)
Error in array(x, c(length(x), 1L), if (!is.null(names(x))) list(names(x),  : 
'data' must be of a vector type, was 'NULL'


#So, I used without a $M 
project.fit <- lmFit(project.NormData,comparisonmodel)
  
#Applying the empirical Bayes method
project.fit.eBayes <- eBayes(project.fit)
names(project.fit.eBayes)

#Make individual contrasts and fit the new model
CaseControl <- makeContrasts(CaseControl="D0A-Control", levels=comparisonmodel)
CaseControl.fitmodel <- contrasts.fit(project.fit.eBayes, CaseControl)
CaseControl.fitmodel.eBayes <- eBayes(CaseControl.fitmodel)

#Filtering Results
 nrow(topTable(CaseControl.fitmodel.eBayes, coef="CaseControl", number=99999, lfc=2))
 probeset.list <- topTable(CaseControl.fitmodel.eBayes, "CaseControl", number=99999, adjust.method="BH", sort.by="P", lfc=2)

#To save results
write.table(probeset.list, "results.txt", sep="\t", quote=FALSE)

Well, now it seems correct, and you guys what do you think?

Best regards,

Leite

ADD COMMENTlink modified 21 months ago • written 21 months ago by Leite410

Hi Leite,

When I read the targets.txt file, will the individual files be accessible through R?

Thanks

ADD REPLYlink written 3 months ago by vinayjrao140

Please elaborate on what you want to do.

ADD REPLYlink written 3 months ago by Kevin Blighe48k

I want to be able to see the contents of each file imported using readTargets. Is that possible?

ADD REPLYlink written 3 months ago by vinayjrao140
1

I suppose that you can simply read them into your R session separately. I have not processed Agilent data for a long time, so, perhaps there is another way.

If you execute the str() function on, for example, the project object, you may see how each sample is arranged inside this object.

ADD REPLYlink modified 3 months ago • written 3 months ago by Kevin Blighe48k
3
gravatar for Kevin Blighe
21 months ago by
Kevin Blighe48k
Kevin Blighe48k wrote:

You have to pass a comparison model to lmFit(). Also, for Agilent processing, the expression values are eventually stored in the 'M' or 'E' variables of your object, and not accessed via the exprs() function. The choice of M or E depends on whether it is 2- or 1-channel / colour array.

comparisonmodel <- model.matrix(~0+design)
colnames(comparisonmodel) <- levels(design)
comparisonmodel

fit <- lmFit(project.NormData$M, comparisonmodel)
ADD COMMENTlink modified 11 weeks ago • written 21 months ago by Kevin Blighe48k
1

Hi Kevin,

You are always willing to help!

I'll try it and post de full code later.

ADD REPLYlink written 21 months ago by Leite410

I edited the code in the question, now I think it's ok, what you think about?

ADD REPLYlink written 21 months ago by Leite410

Looks okay!

ADD REPLYlink written 21 months ago by Kevin Blighe48k
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