[BioC] two color arrays normalization

[Giusy Della Gatta]

Hi Naomi,
I don't know if I understood correct. I switched the signs of the design  and the 
contrast matrices, but I still have the same results: controls going at the opposite way.

> library(limma)
> # Read in data files
> targets=readTargets("target_frame_ltbarc_pgedit.giusy")
> RG<-read.maimages(targets$FileName, source="agilent", ext="txt")
Read Control _1b.txt 
Re
Read Control_3b.txt 
Read dexa_dbz_lt_1.txt 
Read dexa_dbz_lt_2.txt 
Read dexa_dbz_lt_3.txt 
> # create MA list
> MA<-MA.RG(RG, bc.method="none")
> # perform background correction
> RG<-backgroundCorrect (RG,method="none")
> # perform within array normalization
> MA<-normalizeWithinArrays(RG, method="loess")
> # Create design matrix 
> design <- modelMatrix(targets, ref="ref")
Found unique target names:
 ctrl ref treat 
> design<- cbind(ctrl= c(1,1,1,0,0,0), treat= c(0,0,0,1,1,1))
> design
     ctrl treat
[1,]    1     0
[2,]    1     0
[3,]    1     0
[4,]    0     1
[5,]    0     1
[6,]    0     1
> fit<-lmFit(MA,design)
> cont.matrix<-cbind("ctrl-ref"=c(-1,0), "treat-ref"=c(0,-1))
> cont.matrix
     ctrl-ref treat-ref
[1,]       -1         0
[2,]        0        -1
> fit2<-contrasts.fit(fit, cont.matrix)
> d1 <- ebayes(fit2)




Thank you
Giusy


-----Original Message-----
From: Naomi Altman [mailto:naomi at stat.psu.edu]
Sent: Tue 2/17/2009 7:50 PM
To: Giusy Della Gatta; Naomi Altman; bioconductor at stat.math.ethz.ch
Subject: RE: [BioC] two color arrays normalization
 
Hi Giusy,
Move the minus signs from the first design matrix to the 2nd and I 
think it will work fine.

--Naomi

At 06:07 PM 2/17/2009, Giusy Della Gatta wrote:
>Hi Naomi,
>
>I performed the analysis of my micorarrays,but still I don't manage 
>to revert the channels!
>My experiment consisted into infect cells with an adenovirus: an emty one
>and an adenovirus expressing for a specific protein.  Then I
>treated the same cells with a specific drug or simply with the vehicle (DMSO).
>I have 6 microarrays: 3 controls DMSO0-treated and 3 samples drug-treated.
>In each microarray the green channel is expressing the levels of 
>infected and treated
>cells while the red channel are not infected cells. For all the experiments
>I have the same RED CHANNEL reference.
>I composed the target file as follows:
>
>Filename        Cy3     Cy5
>Control_1b.txt  ctrl    ref
>Control_2b.txt  ctrl    ref
>Control_3b.txt  ctrl    ref
>dexa_dbz_lt_1.txt       treat   ref
>dexa_dbz_lt_2.txt       treat   ref
>dexa_dbz_lt_3.txt       treat   ref
>
>and the I used the following script:
>
> >targets=readTargets("target_frame_ltbarc_pgedit.giusy")
> >RG<-read.maimages(targets$FileName, source="agilent", ext="txt")
> >MA<-MA.RG(RG, bc.method="normexp")
> >MA<-normalizeWithinArrays(RG, method="loess")
> >design <- modelMatrix(targets, ref="ref")
> > design
>  ctrl treat
>[1,]   -1     0
>[2,]   -1     0
>[3,]   -1     0
>[4,]    0    -1
>[5,]    0    -1
>[6,]    0    -1
> >fit<-lmFit(MA,design)
> >cont.matrix<-cbind("ctrl-ref"=c(1,0), "treat-ref"=c(0,1))
> >cont.matrix
>      ctrl-ref treat-ref
>[1,]        1         0
>[2,]        0         1
>
> >fit2<-contrasts.fit(fit, cont.matrix)
> >d1 <- ebayes(fit2)
> >toptable(fit2,adjust="fdr")
>
>I don't know if I am still omitting
>something, because I have the positive
>controls of this experiment  that
>are going exactly in the opposite way!!
>
>May you can help me?
>
>Thank you in advance!
>Giusy
>
>
>-----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"?
>
>--Naomi
>
>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)
Associate Professor
Dept. of Statistics                              814-863-7114 (fax)
Penn State University                         814-865-1348 (Statistics)
University Park, PA 16802-2111