[BioC] Contrast matrices in limma

[Dejian Zhao]

Dear all,

I wrote this email to know whether my contrast matrices are
conrrect. Many thanks in advance for any advice.

I have a difficulty in constructing the contrast matrix of factorial
design in limma. In section 8.7 of limma user's guide, a factorial
design is discussed. My experimental design resembles the example
very much except that my arrays are direct two-color arrays.
Therefore the hybridization diagram is a square as follows:

WS-MS
|..|
WU-MU

W: wild type
M: mutant
U: unstimulated
S: stimulated
(The horizontal and vertical lines stand for direct hybridizations,
and the dots in the square have no meanings but to fill the space)

What I'm interested in are exactly the same as those in the example.
1. Which genes respond to stimulation in wild type,
2. Which genes respond to stimulation in mutants,and
3. Which genes respond differently in mutants compared to wild type.

I constructed the contrast matrices (see below in the code and
results) without knowing their validity.
Are they right?



Part of my code and results are as follows:
> sessionInfo()
R version 2.6.2 (2008-02-08)
i386-pc-mingw32

locale:
LC_COLLATE=Chinese_People's Republic of
China.936;LC_CTYPE=Chinese_People's Republic of
China.936;LC_MONETARY=Chinese_People's Republic of
China.936;LC_NUMERIC=C;LC_TIME=Chinese_People's Republic of
China.936

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base

other attached packages:
[1] statmod_1.3.5 limma_2.12.0
> targets
  Cy5 Cy3 FileName
1  WS  WU    1.gpr
2  WU  WS    2.gpr
3  MS  MU    3.gpr
4  MU  MS    4.gpr
5  WS  MS    5.gpr
6  MS  WS    6.gpr
7  WU  MU    7.gpr
8  MU  WU    8.gpr
> design<-modelMatrix(targets,ref="WU")
Found unique target names:
 MS MU WS WU
> design
     MS MU WS
[1,]  0  0  1
[2,]  0  0 -1
[3,]  1 -1  0
[4,] -1  1  0
[5,] -1  0  1
[6,]  1  0 -1
[7,]  0 -1  0
[8,]  0  1  0
> contrast.matrix<-makeContrasts("WS-WU"=WS,"MS-MU"=MS-MU,"(MS-MU)-(WS-WU)"=(MS-MU)-WS,levels=design)
> contrast.matrix
      Contrasts
Levels WS-WU MS-MU (MS-MU)-(WS-WU)
    MS     0     1               1
    MU     0    -1              -1
    WS     1     0              -1