[BioC] Strange signal Log-Ratios with MA.RG
Gordon Smyth
smyth at wehi.edu.au
Sat Apr 2 12:27:32 CEST 2005
>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
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