[BioC] Strange signal Log-Ratios with MA.RG

[Giulio Di Giovanni]

Thank you to all and forgive me,

Simply, lost in the data, I didn't noticed the that bg values where larger 
than fg, I already had raw ratios in a column and I made the log...But now 
it's perfectly clear.

Btw, I never had the intention to calculate log-ratios by hand, and so I 
don't "suggest" to use that formula, simply was a little test I made because 
a was losing some data I needed:
In fact, I'm not interested in gene analysis, now.  I'm working on 
background signal reduction given by some added solutes, and so high 
backgrounds are important data...

Thanks again

Giulio





>From: Gordon Smyth <smyth at wehi.edu.au>
>To: "Giulio Di Giovanni" <perimessaggini at hotmail.com>
>CC: bioconductor at stat.math.ethz.ch, Sean Davis <sdavis2 at mail.nih.gov>
>Subject: Re: [BioC] Strange signal  Log-Ratios with MA.RG
>Date: Sat, 02 Apr 2005 20:27:32 +1000
>
>
>>Date: Fri, 1 Apr 2005 13:19:30 -0500
>>From: Sean Davis <sdavis2 at mail.nih.gov>
>>Subject: Re: [BioC] Strange signal  Log-Ratios with MA.RG
>>To: "Giulio Di Giovanni" <perimessaggini at hotmail.com>
>>Cc: bioconductor at stat.math.ethz.ch
>>
>>
>>On Apr 1, 2005, at 11:06 AM, Giulio Di Giovanni wrote:
>>
>> > Hi to all,
>> >
>> > I have a problem that really I cannot solve.
>> >
>> > Some signal log-ratios given to me converting a RGlist with MA.RG(RG)
>> > are different from the ones calculated directly, that's the point:
>> >
>> > Looking some .gpr files, I build a RGList with
>> >
>> > RG <- read.maimages(source="genepix", ext="gpr)
>> >
>> > and I obtain for the first 3 genes and the first sample the following
>> > Red and Green Foreground and Background intensities
>> >
>> > RG[1:3,1]
>> > An object of class "RGList"
>> > $R
>> >     63MG
>> > [1,]  407
>> > [2,] 4304
>> > [3,]  531
>> >
>> > $G
>> >     63MG
>> > [1,]  291
>> > [2,] 3571
>> > [3,]  394
>> >
>> > $Rb
>> >     63MG
>> > [1,]  518
>> > [2,]  518
>> > [3,]  493
>> >
>> > $Gb
>> >     63MG
>> > [1,]  295
>> > [2,]  295
>> > [3,]  302
>> >
>> > That's to say that log ratios are:
>> >
>> >> za <- log2((RG$R[1:3]-RG$Rb[1:3])/(RG$G[1:3]-RG$Gb[1:3]))
>> >> za
>>
>>Note here that RG$R[1:3] is not necessarily the same as RG$R[1:3,1].
>>Same goes for other RG stuff in your example.
>>
>> > [1]  4.7944159  0.2087391 -1.2756344
>> >
>> > But when I made the conversion with MA.RG(RG) (I need that for
>> > following analysis)
>> > I obtain:
>> >
>> >> RGMA <- MA.RG(RG)
>> >> RGMA$M[1:3,1]
>> > [1]         NA  0.2087391 -1.2756344
>> >
>> > And this happens for several other genes and samples. Most values are
>> > exactly equal, others have NAs ...
>> >
>>
>>Note that for the first gene, you will have a negative values in red
>>and green channels.  If you type MA.RG in your window, you will see
>>that it sets RG$R values <=0 after background substraction to NA; same
>>for RG$G.  I think MA.RG is probably doing the right thing here. Do you
>>agree?
>>
>>Sean
>
>Dear Giulo,
>
>As Sean is gently pointing out here, MA.RG() is giving different results to 
>you because the simplistic "direct calculation" that you give is wrong. 
>Taking the ratio of two negative intensities to get a positive ratio, as 
>for your first value, is nonsense. Note that the log-ratio of 4.79 that you 
>have computed isn't even of the right sign -- you are suggesting that the 
>red channel corrected intensity is much larger than the green channel for 
>this spot, but in fact it is the other way around. Any analysis based on 
>these values will be misleading.
>
>I actually recommend using a background correction method which avoids 
>negative intensities -- this is what I do myself.
>
>Gordon
>