[BioC] Paired two-color design
january.weiner at gmail.com
Wed Aug 29 10:12:51 CEST 2012
Dear Gordon, thank you for your answer.
Gordon K Smyth wrote:
> I would analyse it like this:
That makes totally sense; however, I have one more question (sorry!
and thank you for your patience).
In the meanwhile, I have taken an alternative approach, as described
in the second part of the chapter on technical replicates in the limma
page 42): fit for each biological replicate separately, then create a
contrast corresponding to the average of these and subtract the
design <- cbind( ctrl= c( 1, -1, rep( 0, 6 ) ), e1= c( 0, 0, 1, -1,
rep( 0, 4 ) ), e2= c( rep( 0, 4 ), 1, -1, 0, 0 ), e3= c( rep( 0, 6 ),
1, -1 ) )
cmtx <- makeContrasts( "(e1+e2+e3)/3 - ctrl", levels= design )
fit <- lmFit( MA, design ) ; fit <- contrast.fit( fit, cmtx ) ; fit <-
eBayes( fit )
If I understand the text of the limma guide, these are alternative
approaches and should give at least similar results.
And yes, the estimated logFC are exactly the same. However, the
p-values are much different. Using duplicateCorrelation causes a bunch
of genes to become statistically non-significant (not the other way
round). Either duplicateCorrelation is less sensitive or more
specific, and I wonder which is the case. Unfortunately, this bunch of
genes changes the results of the functional analysis.
Also, maybe I'm lost, but after reading and thinking I don't see why I
can't use intraspotCorrelation here. I'm not saying I can, in fact
this gives me vastly different results I don't really trust (given the
later results of the functional analysis), but I just don't see the
problem, since the correlations are calculated within the arrays. I
got quite used to apply that, since often I'm confronted with the
where the job is to compare A1 with A0 and B1 with B0. (the dye swaps
are technical replicates). I think that this is an unconnected design,
and there is no way of doing that with a normal model, so that the
channels should be analysed separately.
-------- Dr. January Weiner 3 --------------------------------------
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