[BioC] Help on Loading AgilentData into LIMMA

[Wendy Chen]

Hi Jarno,

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"

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?

With 
RG<-read.maimages("targets.txt", columns=list(gBGMeanSignal="xxxxx",
gBGMedianSignal="yyyyy"), annotation=c("Block", "Column", "Row", "Name",
"ID"))

I'll still get the error:
columns must specify foreground G and R

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.


-----Original Message-----
From: Jarno Tuimala [mailto:jtuimala at csc.fi] 
Sent: Tuesday, 22 April 2008 4:51 PM
To: Wendy Chen
Cc: bioconductor at stat.math.ethz.ch
Subject: Re: [BioC] Help on Loading AgilentData into LIMMA

Hello!

> 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"))

You seem to have a datafile that has been created with GenePix program, so 
maybe setting source="genepix" in read.maimages() would help? In limma the 
source does not refer to the manufacturer of the chip, but to the program
that 
was used to scan the image and produce the data file.

> My data is read as an agilent single colour and therefore I've only a Cy3
> output. How then do I define the foreground for R?

If you have a single color slide, then you probably need to change the
default 
columns that read.maimages() uses:

columns=list(R = "F635 Median", G = "F532 Median", Rb = "B635 Median", Gb = 
"B532 Median")

into

columns=list(R = "F635 Median", G = "F635 Median", Rb = "B635 Median", Gb = 
"B632 Median")

So you put the only channel you have in as both red and green channels.

> also what does F635 and B532 means?

Chips are scanned using two wavelenghts, 635 and 532. These are the green
and 
red colors/channels. Labels F and B in front of the wavelenghts refer to the

Foreground and Background for that particular wavelenght. So, F635 is the 
foreground (spot) intensity for the green color, and B532 is the background
for 
the red color.

Best Regards,
Jarno Tuimala
Finland