[BioC] design in mixed ref and dye-swap experiment

[Silvano Piazza]

Hello to everyones,
the experiments that I have to consider is very simple:

I want  to find significant genes between 2 conditions A and B, but I 
have only few experiment so I have to collect both ref versus 
conditions (A or B) either dye swap experiment (A versus B and B versus 
A)

so targets is
SlideNumber	Cy3	Cy5
array1		ref	A
array2		ref	B
array3		ref	B
array4		ref	B
array5		A	B
array6		B	A

of course array5 and array6 are the dye-swap.

So to design the procedure, I follow the LIMMA user guide (by Gordon 
Smith), Chapter 14.5 Weaver Mutant Data.

so
 >design <- modelMatrix(targets, ref = "ref")
	Found unique target names:
  	B A ref
 >design
	A	B
	array1    0    1
	array2    1    0
	array3    1    0
	array4    1    0
	array5   -1    1
	array6    1   -1
 >fit <- lmFit(MA,design)
 >cont.matrix <- makeContrasts(A.B=A-B,levels=design,weight=MA$weights)
 >fit2 <- contrasts.fit(fit, cont.matrix)
 > fit2 <- eBayes(fit2)
 >topTable(fit2,adjust.method="fdr")
	....omissis...
	       		M        	A         		t  		P.Value         B
	209		3.801460 	6.538782  8.315672 1.0000000 -4.209468
	2328	1.184194 	7.343676  6.717978 1.0000000 -4.228492
	7877	1.904360 	6.504330  6.114349 1.0000000 -4.239110
	27187	-4.0759493.771499 -5.783558 1.0000000 -4.246099
	3709	3.434542 3.467492  5.639159 1.0000000 -4.249459
	7561	2.002753 5.159913  5.616194 1.0000000 -4.250013
	7130	2.580527 3.863867  5.600047 1.0000000 -4.250405
	19983 	-2.1176246.836539 -5.567882 1.0000000 -4.251194
So all genes have P.Value equal to 1!!!!!!
in previous posts I read that this happen when you have to consider 
multivariate test, which i don't known how to manage..., but anyway

1) Am I doing something wrong in the design?
2) Am I doing something wrong in the subsequent evaluation steps?
Any ideas



Thank you to all

Silvano



	







Dr.Silvano Piazza
LNCIB,
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Trieste, ITALY
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