[BioC] [limma] Factorial refernce design
ron.ophir at weizmann.ac.il
Thu Oct 6 13:05:12 CEST 2005
>>> "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>
> I work with dual channel technology and I would like to use limma
> 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
> 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
Not exactly, in the user guide the contrasts.matrix as I described here
is run by lmFit() whereas contrasts.fit() gets
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().
>> I allowed myself to use the example in chapter 14 since reference
>> design equivalent to affymetrix experiment after parametrizing it
>> mentioned above with my design matrix and fit it.
>> So is it right the way I did it although in the example for
>> 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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