[BioC] Paired two-color design

[Gordon K Smyth]

Dear January,

I would analyse it like this:

   DyeSwapPair <- c(1,1,2,2,3,3,4,4)
   ctrlBvsA <- c(1,-1,0,0,0,0,0,0)
   BvsA=c(0,0,1,-1,1,-1,1,-1)
   design <- cbind(Dye=1,ctrlBvsA,BvsA)

This produces a design matrix with a treatment BvsA effect, and a 
control BvsA effect, plus a probe-specific dye effect just for good 
measure.

Then estimate the technical correlation.  I'll write MA for your 
normalized MAList object.  The value of corfit$consensus should be 
negative:

   corfit <- duplicateCorrelation(MA,design,block=DyeSwapPair)
   fit <- lmFit(MA,design,block=DyeSwapPair,correlation=corfit$consensus)

Then test for the interaction effect:

   fit2 <- contrasts.fit(fit, c(0,-1,1))
   fit2 <- eBayes(fit2)
   topTable(fit2)

Best wishes
Gordon

On Tue, 28 Aug 2012, Gordon K Smyth wrote:

>> The second problem is this: we have the same setup as above, except
>> that there are controls for A and B (before treatment). I call them
>> ctrlA and ctrlB, respectively. There is only one biological replicate
>> with a dye swap for each of these controls. The setup looks now like
>> that:
>> 
>> targets:
>> Cy3 Cy5
>> ctrlA ctrlB
>> ctrlB ctrlA
>> A1  B1
>> B1  A1
>> A2  B2
>> B2  A2
>> A3  B3
>> B3  A3
>> 
>> I want to look for interaction between the treatment effect and the
>> group, in other words I want the difference between (A vs controlA) -
>> (B vs controlB).
>> 
>> I use more or less the same approach as above (using intraspotCorrelation):
>> 
>> t2 <- targetsA2C( t )
>> design <- model.matrix( ~ 0 + t$group )
>> colnames( design ) <- levels( t$group )
>> # colnames( design ) are: ctrlA, ctrlB, A1, A2, A3, B1, B2, B3
>> 
>> corfit <- intraSpotCorrelation( rg, design )
>> fit <- lmsciFit( rg, design, correlation= corfit$consensus.correlation )
>> cmtx <- makeContrasts( "(( A1 + A2 + A3 )/3 - ctrlA) - ( B1 + B2 + B3
>> )/3 - ctrlB))", levels= design )
>> fit <- contrasts.fit( fit, cmtx )
>> fit <- eBayes( fit )
>> 
>> Is that correct?
>> 
>> Kind regards,
>> 
>> J.
>> 
>> -- 
>> -------- Dr. January Weiner 3 --------------------------------------
>> 
>

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