[BioC] [limma] Factorial refernce design

[Ron Ophir]

>>> "Gordon K Smyth" <smyth at wehi.EDU.AU> 10/06/05 12:45 PM >>>

> Date: Thu, 06 Oct 2005 12:40:32 +0300
> From: "Ron Ophir" <ron.ophir at weizmann.ac.il>
> Subject: [BioC] [limma] Factorial refernce design
> To: "<bioconcutor" <bioconductor at stat.math.ethz.ch>
>
> Hi,
> I work with dual channel technology and I would like to use limma
for
> two way analysis. My experiment is as follow:
> FileName	                               Cy3	Cy5
> GL_251322310100_S02_.gpr	WT3h	NI3h
> GL_251322310101_S02_.gpr	WT3h	NI3h
> GL_251322310103_S02_.gpr	Mut3h	NI3h
> GL_251322310104_S02_.gpr	Mut3h	NI3h
> GL_251322310107_S02_.gpr	WT18h	NI3h
> GL_251322310136_S02_.gpr	WT18h	NI3h
> GL_251322310108_S02_.gpr	Mut18h	NI3h
> GL_251322310368_S02_.gpr	Mut18h	NI3h
> where I have two factors genotype: WT and Mut and Time: early and
late
> and NI3h is the reference.
> Therefore it seems to be natural that my design matrix would be as
> 	WT3h	Mut3h	WT18h	Mut18h
> GL_251322310100_S01	-1	0	0	0
> GL_251322310101_S01	-1	0	0	0
> GL_251322310103_S01	0	-1	0	0
> GL_251322310104_S01	0	-1	0	0
> GL_251322310107_S01	0	0	-1	0
> GL_251322310136_S01	0	0	-1	0
> GL_251322310108_S01	0	0	0	-1
> GL_251322310368_S01	0	0	0	-1
>
> then run fit<-lmFit(Marray,design)
>
> I want to answer the following questions:
> What is the effect of genotype on genes' expression?
> What is the effect of time on genes' expression?
> What is the synergistic effect of time and genotype?
>
> Since the most intuitive for me is to use the sum to zero
> parameterization method I created such contrast matrix
>
> 	Genotype.WTvsMut	Time.EarlyvsLate	Int
> WT3h	1	-1	1
> Mut3h	-1	-1	-1
> WT18h	1	1	-1
> Mut18h	-1	1	1
>
> then run contrasts<-contrasts.fit(fit,cotrasts.matrix)
>
> However from the example in chapter 14 in Limma users guide it seems
> that I had to run the my contrats matrix with lmFit function.

>I can't guess what you mean, or what difficulty you are imagining. 
The section on factorial
>designs in the users guide is exactly equivalent to what you have
given above.

>Gordon

Not exactly, in the user guide the contrasts.matrix as I described here
is run by lmFit() whereas contrasts.fit() gets
such matrix
     WT.SvsU Mu.SvsU Diff
[1,]       0       0    0
[2,]       0       0    0
[3,]      -2      -2    0
[4,]      -2       2    4

to implement other questions.

I guess that dealing with single channel experiment let run contrasts
matrix directly in lmFit().

Ron


>
>
>
>> I allowed myself to use the example in chapter 14 since reference
>> design equivalent to affymetrix experiment after parametrizing it
as
>> mentioned above with my design matrix and fit it.
>> So is it right the way I did it although in the example for
factorial
>> design my design of  contrasts matrix is run with lmFit function?
>> Is OK to run it without the intercept WT=c(1,1,1,1) if I'm not
>> interesting in it?
>>
>> Thanks in advance,
>> Ron
>