Entering edit mode
Dear Dario,
In truth, limma shouldn't need to know the layout for "control"
normalization. I'll change the code to remove the check in the
future.
In the meantime, you can set
RG$printer <- list(ngrid.r=1, ngrid.c=1,
nspot.r=nrow(RG), nspot.c=ncol(RG))
then the code will work fine.
However, I suspect that the Nimblegen probes marked "random" may
actually
be negative control probes, whereas control normalization requires
positive control probes covering a wide range of intensities.
Nimblegen
arrays usually have an annotation column called ControlType, which is
equal to 0 for negative controls. You can also view where the
"random"
probes fall using plotMA(). If the control probes are all at the
lowest
intensities, then they are not suitable for use with control
normalization. On the other hand, you could still make use of the
fact
that these probes shouldn't change between samples, by using "loess"
normalization and upweighting these probes.
BTW, the reason why the layout isn't set automatically is that limma
doesn't have a read method for Nimblegen arrays, so you probably read
the
data using source="generic", so limma doesn't know whether it's a new
commercial array or an old spotted array.
Best wishes
Gordon
> Date: Tue, 25 Jan 2011 13:00:29 +1100 (EST)
> From: Dario Strbenac <d.strbenac at="" garvan.org.au="">
> To: bioconductor at r-project.org
> Subject: [BioC] limma Within Array Normalisation By Controls
> Message-ID: <20110125130029.BKP14739 at gimr.garvan.unsw.edu.au>
> Content-Type: text/plain; charset=us-ascii
>
> Hello,
>
> I have an experiment where there are some Nimblegen 2.1 million
probe
> Mouse promoter arrays with IPs always in the red channel and input
DNA
> always in the green channel. I notice that the arrays have some
probes
> of type Random, which I assume should not be changing between
channels.
> So, I'd like to use normalizeWithinArrays with method = "control". I
> call the method as :
>
> normalizeWithinArrays(RG, method = "control", controlspots =
RG$genes$Status == "Random")
>
> and get the error :
>
> Error in normalizeWithinArrays(RG, method = "control", controlspots
= RG$genes$Status == :
> Layout argument not specified
>
> I'm confused as to why it requires the layout ? I didn't think newer
array designs had print tip groups or replicate spots ?
>
> --------------------------------------
> Dario Strbenac
> Research Assistant
> Cancer Epigenetics
> Garvan Institute of Medical Research
> Darlinghurst NSW 2010
> Australia
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