Question: limma missing green channel (Agilent two color microarray)
gravatar for moroch
5.5 years ago by
moroch0 wrote:

Hi everyone, 

I am trying to analyse microarray data using the limma package. When I do it at my work computer I get no problems but when I try the same exact pipeline in my computer I see that the green channel is "seen" as red (ie when I use plotDensities(MA) or plotDensities(RG) there are two red lines and no green). In the end, I get a bunch of NAs in the final topTable() filtered by adjPval. 

Both computers are Mac, Mavericks OS, and both R and limma are updated to the last available versions. I have no idea of what's going on but I would really appreciate your help to solve it! 

Thanks so much for your time. 




limma microarray R • 2.1k views
ADD COMMENTlink modified 5.4 years ago by Gordon Smyth1.8k • written 5.5 years ago by moroch0

just in case: my analysis is based in the one published in the Mattick lab wiki, as follows:

targets <- readTargets("targets.txt")
RG <- read.maimages(targets, path="somedirectory", source="agilent.median")
RG <- backgroundCorrect(RG, method="normexp", offset=16)
MA <- normalizeWithinArrays(RG, method="loess")
MA.avg <- avereps(MA, ID=MA$genes$ProbeName)
design <- modelMatrix(targets, ref="Pool")
fit <- lmFit(MA.avg, design)
fit2 <- eBayes(fit)
output <- topTable(fit2, adjust="BH", coef="Time1", number=100000)
write.table(output, file="Time1_vs_Pool.txt", sep="\t", quote=FALSE)

ADD REPLYlink written 5.5 years ago by moroch0
gravatar for Gordon Smyth
5.4 years ago by
Gordon Smyth1.8k
Gordon Smyth1.8k wrote:

Sorry, I introduced this bug when I tried to make the methods dispatching of plotDensities() more "elegant".

Now fixed in limma 3.22.6.

Note that the bug only affected plotDensities(). The green channel was read and was included correctly in any differential expression analysis.


ADD COMMENTlink written 5.4 years ago by Gordon Smyth1.8k

thank you very much! 

ADD REPLYlink written 5.2 years ago by moroch0
gravatar for Madelaine Gogol
5.5 years ago by
Madelaine Gogol5.2k
Kansas City
Madelaine Gogol5.2k wrote:

I don't know what exactly is going on, but you can specify exactly which columns from the agilent file should be read in as R and G... Maybe that would help narrow down the problem?

read.maimages(targets$FileName, source="agilent", path=datapath, names=targets$Description, columns= list( R = "rMedianSignal", G = "gMedianSignal", Rb = "rBGMedianSignal", Gb = "gBGMedianSignal"), annotation = c("FeatureNum","Row","Col","ProbeName","ControlType","GeneName", "Description","SystematicName"))

ADD COMMENTlink written 5.5 years ago by Madelaine Gogol5.2k

thanks! I will try it and see what happens... I really don't know what could be causing this except OS/R/limma versions. 

thanks again! 

ADD REPLYlink written 5.5 years ago by moroch0

well, still no luck. I can see that the green channel is read but then it somehow disappears and is turned into red. I get no differentially expressed genes of course. I tried unistalling/installing and still nothing... 

ADD REPLYlink written 5.5 years ago by moroch0


Today I exactly encountered the same problem (explained here: )

Have you already found a solution for your problem?




ADD REPLYlink written 5.4 years ago by wd0
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