Dear all,
I am analizyng Agilent G4121A mouse chips with LIMMA.
My first three problems:
1) Layout: from the GAL file provided with the chip (and from the flat
output file), I can only obtain the rows (105), and the columns (215).
No
information about the layout... is this due to the different
technology
(without printing tips)? If this is the case, I guess I don't need the
printtiploess normalization. Is that correct? But what about the
imageplot?
How should I set the layout parameters in this function?
2) Is there a way (I am sure there is) to use the spot type
information
(stored in RG$genes$Status) also after the lmFit analysis (i.e. in the
qqt
plot and in the plot(A,M,pch=16,cex=0.1))? I would like to have the
same
colors for control spots also in these plots.
3) about the internal controls and duplicate spots on the Agilent
arrays,
their meaning is not clearly exposed in the accompanying
documentation. I
emailed the technical support, and they said that this information was
developed with Rosetta (their analysis software), so it is not
available to
their customers... Don't you think this is a limitation of the
analysis? Do
you have more information about this?
Thank you for your help
** hoping I did not post too much RTFM questions :-] **
Giovanni
At 08:39 AM 27/08/2004, Giovanni Coppola wrote:
>Dear all,
>I am analizyng Agilent G4121A mouse chips with LIMMA.
>My first three problems:
>1) Layout: from the GAL file provided with the chip (and from the
flat
>output file), I can only obtain the rows (105), and the columns
(215). No
>information about the layout... is this due to the different
technology
>(without printing tips)?
Yes it is. Agilent doesn't have print tips.
> If this is the case, I guess I don't need the
>printtiploess normalization. Is that correct?
I recommend method="loess".
> But what about the imageplot?
>How should I set the layout parameters in this function?
Set ngrid.c=1, ngrid.r=1. BTW this will cause method="printtiploess"
and
method="loess" to be the same at the normalization step.
>2) Is there a way (I am sure there is) to use the spot type
information
>(stored in RG$genes$Status) also after the lmFit analysis (i.e. in
the qqt
>plot and in the plot(A,M,pch=16,cex=0.1))? I would like to have the
same
>colors for control spots also in these plots.
You can pass an argument col to qqt() or to plot(). This is a vector
of
integers or colour names ("red", "green" etc) of the same length as
the
number of points. E.g.,
plotcol <- RG$genes$Status
plotcol[RG$genes$Status=="Control"] <- "red"
plotcol[RG$genes$Status=="Gene"] <- "black"
qqt(fit$t, col=plotcol)
>3) about the internal controls and duplicate spots on the Agilent
arrays,
>their meaning is not clearly exposed in the accompanying
documentation. I
>emailed the technical support, and they said that this information
was
>developed with Rosetta (their analysis software), so it is not
available to
>their customers... Don't you think this is a limitation of the
analysis? Do
>you have more information about this?
We haven't been able to get further information on the controls from
Agilent either. It would certainly be nice to know they are but it
doesn't
limit the analysis. Many other arrays don't have these controls in the
first place.
Gordon
>Thank you for your help
>** hoping I did not post too much RTFM questions :-] **
>
>Giovanni
>
>_______________________________________________
>Bioconductor mailing list
>Bioconductor@stat.math.ethz.ch
>https://stat.ethz.ch/mailman/listinfo/bioconductor
At 08:39 AM 27/08/2004, Giovanni Coppola wrote:
>Dear all,
>I am analizyng Agilent G4121A mouse chips with LIMMA.
>My first three problems:
>1) Layout: from the GAL file provided with the chip (and from the
flat
>output file), I can only obtain the rows (105), and the columns
(215). No
>information about the layout... is this due to the different
technology
>(without printing tips)? If this is the case, I guess I don't need
the
>printtiploess normalization. Is that correct? But what about the
imageplot?
>How should I set the layout parameters in this function?
You can't use imageplot() with Agilent arrays processed using
AgilentFE
software, because AgilentFE does not output rows for blank/empty spots
while limma expects to get full arrays. You can use imageplot() with
Agilent arrays processed by Genepix or similar.
It is possible to get an image plot of Agilent/AgilentFE arrays using
a
combination of the limma and marray packages. The following code works
for me:
library(limma)
RG <- read.maimages(fnames, source="agilent", path="AgilentFE")
library(convert)
y <- as(RG,"marrayRaw")
nr <- max(RG$genes$Row)
nc <- max(RG$genes$Col)
maNgr(y) <- 1
maNgc(y) <- 1
maNsr(y) <- nr
maNsc(y) <- nc
included <- rep(FALSE,nr*nc)
pos <- nc*(RG$genes$Row-1)+RG$genes$Col
included[pos] <- TRUE
maSub(y) <- included
maImage(y,x="maRb")
Gordon