limma missing green channel (Agilent two color microarray)

Question

[moroch]

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 (i.e. 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.

[moroch]

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)

Answer 1

[Gordon Smyth]

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.

[moroch]

Thank you very much!

Answer 2

[Madelaine Gogol]

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"))

[moroch]

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!

[moroch]

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 uninstalling/installing and still nothing...

[wd]

Hi

Today I exactly encountered the same problem (explained here)

Have you already found a solution for your problem?

Regards
Wannes