[BioC] design a modelMatrix with no common references

[Giusy Della Gatta]

Hi Robert,
I saw the example used in chapter  11.5. The problem is that
I don't have any common reference that I can use to normalize
the data, while in the chapter they are a comparing everything against
the "Pool".


G




-----Original Message-----
From: Robert Castelo [mailto:robert.castelo at upf.edu] 
Sent: Monday, July 13, 2009 6:00 AM
To: James W. MacDonald
Cc: Giusy Della Gatta; bioconductor at stat.math.ethz.ch
Subject: Re: [BioC] design a modelMatrix with no common references

James, Guisy,

if you let me make a question about what you're discussing.

why do you say that the two-color 2x2 factorial design without a common
reference RNA source is "a lot of work" ??

(i was actually wondering how would be the 11.5 example of the Limma
user's guide without a common RNA source)

thanks!
robert.

On Fri, 2009-07-10 at 10:14 -0400, James W. MacDonald wrote:
> Hi Guisy,
> 
> You really have two different experiments here, so I don't know if limma 
> is going to want to do things automatically for you without warnings or 
> incorrect model matrices. However, I think you want to use the 
> parameters argument to modelMatrix() rather than the ref argument (since 
> you have two different reference samples).
> 
>  > targets <- matrix(paste(rep(c("Myc","Rag"), each=4), 
> rep(c("CD3","PBS"), each=2, times=3)[2:9], sep = "_"), byrow=T, ncol=2)
>  > targets
>       [,1]      [,2]
> [1,] "Myc_CD3" "Myc_PBS"
> [2,] "Myc_PBS" "Myc_CD3"
> [3,] "Rag_CD3" "Rag_PBS"
> [4,] "Rag_PBS" "Rag_CD3"
>  > colnames(targets) <- c("Cy3","Cy5")
>  > rownames(targets) <- paste("Array", 1:4)
>  > targets
>          Cy3       Cy5
> Array 1 "Myc_CD3" "Myc_PBS"
> Array 2 "Myc_PBS" "Myc_CD3"
> Array 3 "Rag_CD3" "Rag_PBS"
> Array 4 "Rag_PBS" "Rag_CD3"
>  > parameters <- cbind(First=c(-1,1,0,0), Second=c(0,0,-1,1))
>  > rownames(parameters) <- c("Myc_PBS","Myc_CD3","Rag_PBS","Rag_CD3")
>  > parameters
>          First Second
> Myc_PBS    -1      0
> Myc_CD3     1      0
> Rag_PBS     0     -1
> Rag_CD3     0      1
>  > modelMatrix(targets, parameters)
> Found unique target names:
>   Myc_CD3 Myc_PBS Rag_CD3 Rag_PBS
>          First Second
> Array 1    -1      0
> Array 2     1      0
> Array 3     0     -1
> Array 4     0      1
> Warning message:
> In modelMatrix(targets, parameters) :
>    number of parameters should be one less than number of targets
> 
> But that seems like a lot of work, as the parameters matrix is exactly 
> the model matrix you want.
> 
> Best
> 
> Giusy Della Gatta wrote:
> > Hi everybody,
> > 
> > I have Agilent two colors expression arrays in which have been analyzed 
> > two KO mice samples (myc-/- and Rag -/-) treated with CD3 and with PBS.
> > I have a total of 4 arrays composed as follows:
> >      Sample   Cy3         Cy5
> > 1. Myc24CD3	Myc_CD3	Myc_PBS (Swap)	
> > 2. Myc24PBS	Myc_PBS	Myc_CD3	
> > 3. Rag24CD3	Rag_CD3	Rag_PBS (Swap)		
> > 4. Rag24PBS	Rag_PBS	Rag_CD3	
> > 
> > After the normalization I don't know
> > how to proceed for the  construction of the model matrix.
> > 
> > By using the suggestions of the  "Direct Two Color Designs" example (chapter 7.4 LIMMA guide)
> > I did:
> > 
> > 
> >> targets
> >                  FileName     Cy3     Cy5 Collection_time
> > 1 3_Myc24CD3gr_Myc24PBSre Myc_CD3 Myc_PBS             24h
> > 2 9_Myc24PBSgr_Myc24CD3re Myc_PBS Myc_CD3             24h
> > 3 5_Rag24CD3gr_Rag24PBSre Rag_CD3 Rag_PBS             24h
> > 4 4_Rag24PBSgr_Rag24CD3re Rag_PBS Rag_CD3             24h
> > 
> >> designmyc= modelMatrix(targets, ref="Myc_PBS")
> > Found unique target names:
> >  Myc_CD3 Myc_PBS Rag_CD3 Rag_PBS
> > 
> >> designmyc
> >      Myc_CD3 Rag_CD3 Rag_PBS
> > [1,]      -1       0       0
> > [2,]       1       0       0
> > [3,]       0      -1       1
> > [4,]       0       1      -1
> > 
> >> fit = lmFit(MA.Rq, designmyc)
> > Coefficients not estimable: Rag_PBS 
> > Warning message:
> > Partial NA coefficients for 45018 probe(s)
> > 
> > 
> > But at this point I calculated just the ratios of Myc_CD3/Myc_PBS
> > and Rag_Myc/Myc_PBS (I am not really interested in this last one!).
> > How can I specify in the model matrix design that I need two different references
> > to calculate the following logratios:  Myc_CD3/Myc_PBS, Rag_Myc/Rag_PBS?
> > 
> > 
> > Thank you in advance!
> > Giusy
> >  
> > 
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