[BioC] Fwd: limma questions (normalization) - correction

[Gordon Smyth]

Dear Brooke,

Oops, I wrote something too hastily yesterday which was incorrect.

>To: Brooke LaFlamme <bal44 at cornell.edu>
>From: Gordon Smyth <smyth at wehi.edu.au>
>Subject: [BioC] limma questions (normalization)
>Cc: bioconductor at stat.math.ethz.ch
>Bcc:
>X-Eudora-Signature: <<No Default>>
>Date: Thu, 20 Sep 2007 10:57:05 +1000
>
>Dear Brooke,
>
>>Date: Tue, 18 Sep 2007 17:28:23 -0400 (EDT)
>>From: Brooke LaFlamme <bal44 at cornell.edu>
>>Subject: [BioC] limma questions (normalization)
>>To: bioconductor at stat.math.ethz.ch
>>
>>I would like to flag out all spots with fluorescence intensity 
>>lower than that of the mean background intensity of the array.
>>The weight function I am using to do this is (using Genepix output 
>>file (*.gpr) column names):
>>
>>myfun<-function(x){
>>                 okred<-mean(x[,"B635"])-x[,"F635 Mean"]<=0
>>                 okgreen<-mean(x[,"B532"])-x[,"F532 Mean"]<=0
>>                 as.numeric(okgreen & okred)
>>                 }
>>
>>Does this actually do what I want? I?m not certain of how to 
>>calculate ?mean background intensity? for the whole array.
>
>I assume you're trying to use the wt.fun argument of 
>read.maimages(). You cannot use this approach to compute the mean 
>background intensity of the array. You need to read in the whole 
>array before you can do that.

Oops, my last comment is wrong. wt.fun acts on the whole gpr 
data.frame, so you actually can compute the mean background intensity 
as part of the spot quality weight calculation.

Your code here could do what you want. However three warnings:

1. You are assuming that the column "B635" exists, and this will only 
be true if you are using a custom GenePix background measure. If you 
are, you didn't mention it. What background measure are you using?

2. It would be easier to do this calculation after reading in the 
data using read.maimages() rather than using the wt.fun argument.

3. As I said yesterday, this spot weight calculation seems a bad 
idea. It would especially be a bad idea if you're using a custom 
background measure in GenePix 6.0 or later such as morphological 
opening, which makes this sort of intensity-based weighting unnecessary.

Best wishes
Gordon