[BioC] duplicate correlation on Agilent 4x44 arrays

[Mitch Levesque]

Gordon,

Thanks for the reply. I am not using any particular instruction set, just
what I have put together from the User Guide. 

You were right about the file dimensions, they are different:

> dim(RG)
[1] 44407     4
> gal <- readGAL()
> dim(gal)
[1] 180880     10

Is it possible to read the duplicate positions directly off of the gal file?
I tried:

layout <- getLayout(gal, guessdups=TRUE)

and I get the following:

$ngrid.r
[1] 1

$ngrid.c
[1] 4

$nspot.r
[1] 170

$nspot.c
[1] 266

$ndups
[1] 8

$spacing
[1] NA

attr(,"class")
[1] "PrintLayout"


I haven't tried without the normexp, but I will test it. Thanks again.

Mitch



-----Original Message-----
From: Gordon Smyth [mailto:smyth at wehi.EDU.AU] 
Sent: Tuesday, April 10, 2007 1:03 PM
To: Mitch Levesque
Cc: bioconductor at stat.math.ethz.ch
Subject: [BioC] duplicate correlation on Agilent 4x44 arrays

Dear Mitch,

You don't say what instructions you are trying to follow here. I 
think you may be trying to use code which was intended for other data 
sets. I suspect that there may be more than one problem.

Firstly, why do you need to use readGAL()? This is only needed with 
SPOT data. Your RG object from read.maimages() will already contain 
annotation information from the Agilent output files. Look at

    names(RG$genes)

to see what you have.

Secondly, does your GAL file match your data files? Type

    dim(RG)

and

    gal <- readGAL()
    dim(gal)

Do the row numbers agree? I am guessing they may have different 
numbers of rows.

BTW, do you need to use "normexp"? I've found the AgilentFE 
background estimator is already pretty good, and doesn't produce 
negative intensities anyway.

Best wishes
Gordon

>Date: Mon, 9 Apr 2007 12:21:57 +0200
>From: "Mitch Levesque" <Mitch.Levesque at tuebingen.mpg.de>
>Subject: [BioC] duplicate correlation on Agilent 4x44 arrays
>To: <bioconductor at stat.math.ethz.ch>
>
>Hi Bioconductors,
>
>I am using R 2.4.1 and limma to analyze the new Agilent 4x44 array design
>and am having trouble with the duplicate correlation function using the
>following script:
>
>
>library(limma)
>targets <- readTargets("Targets.txt")
>RG <- read.maimages(targets$FileName, source="agilent")
>RG$genes<-readGAL()
>RG$printer<-getLayout(RG$genes)
>RG <- backgroundCorrect(RG, method="normexp", offset=50)
>MA <- normalizeWithinArrays(RG, method="loess")
>MA <- MA[order(RG$genes[,"ID"]),]
>
>I get the following error:
>
>Error in `[.MAList`(MA, order(RG$genes[, "ID"]), ) :
>        subscript out of bounds
>
>I would like to treat the duplicate probes on each array as a technical
>replicate, but since the spacing is not consistent for each gene, I must
>first order the list by reference number. Are there any suggestions about
>how I may do this?
>
>Mitch