[BioC] Help on Loading AgilentData into LIMMA

[Jarno Tuimala]

Hi Wendy!

> No my files were not created with a genePix program. My raw data were
> obtained with agilent's Feature extraction. That was why I was using
> source="agilent"

Sorry, my mistake!

> I thought F635 and F532 were codes to imply foreground and background but I
> guess I was wrong. If my datafiles were not created with a GenePix program
> does that mean F635 and F532 does not apply? What should I use then?

Yes, F still stands for foreground, and B for background.

It seems that you have tried to read in your data with the following command:

> RG<-read.maimages("targets.txt", source="agilent",
columns=list(gBGMeanSignal="F635", gBGMeanSignal="F532",
bBGMedianSignal="B635 Median", bBGMedianSignal="B532 Median"),
annotation=c("Block", "Column", "Row", "Name", "ID"))

For limma, you need the specify the columns argument as:

columns=list(R="", G="", Rb"", Gb="")

There have to be an entry for each of R, G, Rb and Gb. Maybe this is the reason 
for the error message.

> but I cannot put the only channel I have as both red and green channels as I
> only columns in my data for the green channel. i.e. I do not have a
> rBGMeanSignal or rBGMedianSignal.

And I meant is that you have to specify the same columns for both R and G and Rb 
and Gb in the columns argument.

So, if your datafile is Agilent, something like this should work (you should 
have columns gMeanSignal, gBGMedianSignal and ProbeName in your datafile):

RG<-read.maimages(files="targets.txt", columns=list(R="gMeanSignal", 
G="gMeanSignal", Rb="gBGMedianSignal", Gb="gBGMedianSignal"), 
annotation=c("ProbeName"))

This reads the green channel intensities from the file to an limma object, where 
both red and green channels have exactly the same values. This is the way to 
read in one-color Agilent data using read.maimages(), since limma expects that 
there are two channels, and both have to be filled in.

Best Regards,
Jarno

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Jarno Tuimala, PhD, bioinformatics, CSC, P.O.Box 405, FI-02101 Espoo, Finland
tel.: +358 9 457 2226, fax: +358 9 457 2302, e-mail: jarno.tuimala at csc.fi
CSC is the Finnish IT Center for Science, http://www.csc.fi/molbio