[BioC] [limma] Factorial refernce design

Ron Ophir ron.ophir at weizmann.ac.il
Thu Oct 6 11:40:32 CEST 2005

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 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,

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