Separate channel analysis of two-color microarray
Entering edit mode
7.8 years ago

Hello everyone,

I'm sort of new to the world of bioinformatics and microarrays data analysis, and I've been having some trouble with limma's 'separate channel analysis of two-color data'. I've tried to implement the pipeline as suggested in the user's guide, but I got an error message saying dimmensions of the objects in the argument of intraspotCorrelation function:

> corfit <- intraspotCorrelation(MA, design)
Error in intraspotCorrelation(MA, design) : The number of rows of the design matrix should match the number of channel intensities, i.e., twice the number of arrays

I checked the structure of the input objects and they seem to be fine:

> nrow(design)
[1] 8
> M<- as.matrix(MA$M)
> dim(M)
[1] 45018 8

Any thoughts on what could be going on here? Of note, the data went through normalization by normalizeWithinArrays and nomalizeBetweenArrays, and then avereps before trying the correlation fit.

I hope you can help me. Thanks in advance!

R software error limma microarray single-channel • 2.7k views
Entering edit mode
7.8 years ago
Vinay Singh ▴ 50

Try the following one this work fine for me.

> library(limma)
> dat<-read.maimages(files=dir(), source="agilent")
> dat2<-normalizeWithinArrays(dat, method="loess","normexp", offset=50)
> dat3<-normalizeBetweenArrays(dat2, method="scale")

# extracting M values (normalized expression values)

> dat.m<-dat3$M
> rownames(dat.m)<-dat2$genes$ProbeName
> write.table(dat.m, "datm.txt", sep="\t",row.names=T, col.names=T, quote=F)

# making density plots

> plotMA3by2(dat3, device="pdf")
> plotDensities(dat3)

#finding differentially expressed genes

> library(limma)
> biolrep <- c(24, 24, 24, 48, 48, 48, 24, 24, 24, 48, 48, 48, 24, 24, 24, 48, 48, 48)
> corfit <- duplicateCorrelation(dat3, ndups = 1, block = biolrep)
> fit <- lmFit(dat3, block = biolrep, cor = corfit$consensus)
> fit2 <- eBayes(fit)
> topTable(fit2)

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