[BioC] two color arrays normalization
Giusy Della Gatta
Gatta at icg.cpmc.columbia.edu
Wed Feb 11 16:31:53 CET 2009
I studied the model matrix command and it seems
that is the one that I need to construct a new matrix
and specify which will be my reference.
>From LIMMA user's guide is not clear when I have to use
In the guide I found this example: It is a two-color microarray experiments in which
a common reference has been used on all the arrays (I have exactly
this kind of experiment):
> targets <- readTargets("runxtargets.txt")
SlideNumber Cy3 Cy5
1 2144 EGFP AML1
2 2145 EGFP AML1
3 2146 AML1 EGFP
4 2147 EGFP AML1.CBFb
5 2148 EGFP AML1.CBFb
6 2149 AML1.CBFb EGFP
7 2158 EGFP CBFb
8 2159 CBFb EGFP
9 2160 EGFP AML1.CBFb
10 2161 AML1.CBFb EGFP
11 2162 EGFP AML1.CBFb
12 2163 AML1.CBFb EGFP
13 2166 EGFP CBFb
14 2167 CBFb EGFP
> design <- designMatrix(targets,ref="EGFP")
AML1 AML1.CBFb CBFb
1 1 0 0
2 1 0 0
3 -1 0 0
4 0 1 0
5 0 1 0
6 0 -1 0
7 0 0 1
8 0 0 -1
9 0 1 0
10 0 -1 0
11 0 1 0
12 0 -1 0
13 0 0 1
14 0 0 -1
> fit <- lmFit(RG, design)
in this way the specific matrix is constructed, but why I have to use the
"fit<- lmfit" command?
And also, after this command can I go ahead with my analysis that will consist in:
- MA<-normalizeBetweenArrays(MA, method="Rquantile")
> d1 <- ebayes(lm)
Thank you very much for your help
From: Naomi Altman [mailto:naomi at stat.psu.edu]
Sent: Tue 2/10/2009 11:33 AM
To: Giusy Della Gatta; Naomi Altman
Cc: bioconductor at stat.math.ethz.ch
Subject: RE: [BioC] two color arrays normalization
Please keep the discussion on the list.
I have forgotten some of the specifics of the code, so I am going to
turn this over to someone else to respond.
At 11:19 AM 2/10/2009, Giusy Della Gatta wrote:
>Hi Naomi only another question,
>at this point I am wondering why I need the "Target file"
>(see in attachment)?
>I was sure that by composing this file was automatic the recognition
>of my reference and my treatment signals.
>From: Naomi Altman [mailto:naomi at stat.psu.edu]
>Sent: Tue 2/10/2009 8:33 AM
>To: Giusy Della Gatta; Naomi Altman
>Cc: bioconductor at stat.math.ethz.ch
>Subject: RE: [BioC] two color arrays normalization
>This will make no difference to the statistical tests and
>p-values. The simplest ways to get the M values right are:
>Either: Create the design matrix as in the limma manual - say
>designMatrix. Then switch sign.
>designMatrix= - designMatrix
>Then proceed as in the manual.
>Or: Create the design matrix and do the analysis (without switching
>sign). Call the output fit.out.
>The M-values are in fit.out$coef.
>fit.out$coef= - fit.out$coef
>At 10:32 PM 2/9/2009, Giusy Della Gatta wrote:
> >Hi Naomi,
> >my colleague told me that in the calculation of the M value
> >the green channel is used as reference by default.
> >You can see it also by considering the corrispondent formula:
> >(M = log(R/G) = log R - log G).
> >In my experiment, the red color is the reference
> >so I was thinking that in this case I have to use some
> >specific command to invert the M formula?
> >Thank you!
> >-----Original Message-----
> >From: Naomi Altman [mailto:naomi at stat.psu.edu]
> >Sent: Mon 2/9/2009 9:56 PM
> >To: Giusy Della Gatta; bioconductor at stat.math.ethz.ch
> >Subject: Re: [BioC] two color arrays normalization
> >If there is no dye-swap, then what do you mean by "swapping of the colors"?
> >At 07:56 PM 2/9/2009, Giusy Della Gatta wrote:
> > >Hi everybody,
> > >
> > >I am analyzing two color Agilent microarrays
> > >by using LIMMA package.
> > >In my specific case the red channel is representing
> > >"the reference" while the green channel is "the treatment".
> > >Is it enough to use the Target File composition to specify the name
> > >of the samples
> > >and their corrispondet channels? Or I have to use other specific commands
> > >to specify the "swapping" of the colors?
> > >
> > >Thank you in advance!
> > >Regards
> > >Giusy
> > >
> > >_______________________________________________
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> >Naomi S. Altman 814-865-3791 (voice)
> >Associate Professor
> >Dept. of Statistics 814-863-7114 (fax)
> >Penn State University 814-865-1348 (Statistics)
> >University Park, PA 16802-2111
>Naomi S. Altman 814-865-3791 (voice)
>Dept. of Statistics 814-863-7114 (fax)
>Penn State University 814-865-1348 (Statistics)
>University Park, PA 16802-2111
Naomi S. Altman 814-865-3791 (voice)
Dept. of Statistics 814-863-7114 (fax)
Penn State University 814-865-1348 (Statistics)
University Park, PA 16802-2111
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