[BioC] help with paired-test in limma
Ariel Chernomoretz
ariel.chernomoretz at crchul.ulaval.ca
Tue Jul 5 18:09:46 CEST 2005
Dear list,
This is my first time with limma (and also with linear models!).
After reading the vignettes, papers and previously posted messages I came out
with something, but I am not sure it is the correct procedure....in particular
I have doubts related to how to handle with limma the paired nature of my
design. I would greatly appreciate some remarks, comments, help, etc.
I want to study the differences between two treatments: allocationA and
allocationB, using single color affymetrix arrays, in a paired design.
Two samples were taken from 13 patients, one before, and the other after
treatment. There were 6 and 7 patients treated with allocationA, and
allocationB, respectively. Each sample was technically replicated, so at the
end we have 4 chip per patient.
The experimental setup looks like this:
Sample BeforeAfter Patient allocation
1 0 1 A
2 0 1 A
3 1 1 A
4 1 1 A
5 0 2 A
6 0 2 A
7 1 2 A
8 1 2 A
9 0 3 B
10 0 3 B
11 1 3 B
12 1 3 B
. . . .
. . . .
52 1 13 B
After reading some posts I decided to take the average of technical
replicates. Then I used a block in order to get the lmFit, and I calculated
the contrasts of interest:
> design<-model.matrix(~allocation*AntesDespues,data=pData(eset))
> bblock<-rep(1:13,each=2)
> fit2<-lmFit(eset,design,block=bblock)
>
> cont.matrix<-cbind(A.AfterVSBefore=c(0,0,1,0),
> B.AfterVSBefore=c(0,0,1,1),
> Interac =c(0,0,0,1))
> fit2<-contrasts.fit(fit2,cont.matrix)
> fit2eb<-eBayes(fit2)
I think that in this way I am implicitly assuming an intrablock correlation
level of 0.75. Is this somehow appropriate in order to consider this
a paired t-test like analysis?
With duplicateCorrelation, I get a value of 0.21 (!)
Is this value the one I should consider for lmFit ?
Using this rather low value isn't like disregarding
the before-after pairing for each patient? Shouldn't I tweak the correlation
level to an artificially higher value (for instance 0.9) instead?
Any comments would be highly appreciated
Thanks
Ariel./
--
Ariel Chernomoretz, Ph.D.
Centre de recherche du CHUL
2705 Blv Laurier, bloc T-367
Sainte-Foy, Qc
G1V 4G2
(418)-525-4444 ext 46339
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