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
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@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
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
----------------------------------------------------------------------
-------
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
