[BioC] limma: design for paired data with multiple time points

[Jonathan Ellis]

Hi Jim,

On Tue, May 20, 2014 at 10:17:34AM -0400, James W. MacDonald wrote:
> Hi Jonathan,
> 
> On 5/19/2014 9:28 PM, Jonathan Ellis wrote:
> >Dear list,
> >
> >I am analysing some microarrays with the limma package, and so far have
> >discovered no significant DEGs.  I'm unsure if this is because there
> >really are no DEGs or I've misunderstood the limma package.  I'm hoping
> >someone with more experience can tell me if my analysis is correct, or,
> >if not, where I've gone wrong.
> >
> >I have arrays collected at three time points (t0, t14 and t56) from the
> >same patients, so I have a data frame that resembles:
> >
> >Patient Time
> >1       t0
> >1       t14
> >1       t56
> >2       t0
> >2       t14
> >2       t56
> >etc.
> >
> >and I'm interested in comparing t14 to t0, t56 to t0 and t56 to t14,
> >whilst accounting for fact that array are from the same patient.  My
> >analysis has been:
> >
> >design <- model.matrix(~ 0 + Time + Patient)
> >colnames(design) <- c('t0', 't14', 't56', 'p1', 'p2', 'p3', 'p4', 'p5', 'p6')
> >array.weights <- arrayWeights(x.filtered, design)
> >fitw <- lmFit(x.filtered, design, weights = array.weights)
> >contrast.matrix <- makeContrasts(t14-t0, t56-t14, t56-t0, levels = design)
> >fit2 <- contrasts.fit(fitw, contrast.matrix)
> >fit2 <- eBayes(fit2)
> >
> >I would be very grateful if someone can tell me if this analysis is
> >correct or not.
> 
> Looks OK to me. When you say 'no significant DEGs', what exactly do
> you mean? You can certainly use a relatively large FDR, if you are
> willing to accept failures when you validate.
> 
> Best,
> Jim

Thanks for your reply.  By no significant DEGs I mean that all FDR
values are around 0.9.  I'm aware that this is a perfectly legitimate
result if the arrays really don't provide evidence for differential
expression.

Jonathan

> 
> >
> >Cheers,
> >Jonathan
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> 
> -- 
> James W. MacDonald, M.S.
> Biostatistician
> University of Washington
> Environmental and Occupational Health Sciences
> 4225 Roosevelt Way NE, # 100
> Seattle WA 98105-6099
>