[BioC] Limma time course analysis - defining comparisons

[Helen Zhou]

Dear Jim

Thanks you for your answer. As I understand you are
recommending me to do direct comparisons between for
example 12-4h and 24-12h. However, could there not in
theory be a gene that was up a little bit in 12vs4h
and 24vs12h, so that the difference for neither of
these would be large enough to be significant, but for
24vs4h the combined change might be significant? In
that case I guess I would need all 3 comparisons?

Thanks you
Helen

--- "James W. MacDonald" <jmacdon at med.umich.edu>
wrote:

> Hi Helen,
> 
> Helen Zhou wrote:
> > Dear Sir/Madam
> > 
> > I am trying to analyse a short time series,
> roughly
> > following section 8.8 in the limma user guide. I
> am
> > interested in differences between all time points.
> I
> > am not sure whether I have to make all the
> pariwise
> > comparisons directly, or whether they can be done
> > indirectly as well.
> > 
> > For example, if I want to compare to time points,
> what
> > is the differences between the two methods listed
> > below.
> > 
> > library(bronchialIL13)
> > # Just for the IL13 samples
> > data <- HAHrma[,7:15]
> > # Design
> > targets	<-
> >
>
c("h12","h12","h12","h24","h24","h24","h4","h4","h4")
> > lev	<- c("h12","h24","h4")
> > f	<- factor(targets, levels=lev)
> > design	<- model.matrix(~0+f)
> > colnames(design)	<- lev
> > fit <- lmFit(data, design)
> > # 2-step contrasts, used to indirectly get 24 to 4
> > hours as well as the other two comparisons
> > contrasts <- makeContrasts("h24-h12", "h12-h4",
> > levels=design)
> > fit2 <- contrasts.fit(fit, contrasts)
> > fit2 <- eBayes(fit2)
> > # Direct contrast of 24 to 4 hours
> > contrasts2 <- makeContrasts("h24-h4",
> levels=design)
> > fit3 <- contrasts.fit(fit, contrasts2)
> > fit3 <- eBayes(fit3)
> > # Comparison
> > topTable(fit2, coef=1:2)
> > topTable(fit3, coef=1)
> 
> In the first case you are asking the question 'which
> reporters are 
> different in either h24 vs h4 _or_ h12 vs h4',
> whereas in the second 
> case you are asking 'which reporters are different
> between H24 and h4'.
> 
> It is entirely possible that you could have a gene
> that isn't different 
> between h24 and h4, but is different at h12. This
> would show up in the 
> first comparison but not the second, so if you want
> to compare time 
> points you are better off making direct contrasts
> rather than using the 
> F-statistic for multiple contrasts (which will then
> require the 
> additional step of figuring out which contrast(s)
> caused the statistic 
> to be significant).
> 
> Best,
> 
> Jim
> 
> 
> > 
> > More or less the same probe sets are present, but
> in
> > different order and with different p values. Is
> the
> > difference because using coef=1:2 will go via an
> > F-test? And if I want the change from 24h-0h as
> well
> > as 42-12h and 12-4h, is it most correct for me to
> > specify that contrast directly? In my actual
> > experiment I have 4 time points, so will it be
> enough
> > for me with 3 possible comparisons, or will I have
> to
> > write all the 6 possible combinations?
> > 
> > Thank you in advance for all your help.
> > 
> > Yours truly
> > Mrs Helen Zhou
> > 
> > P.S. I think this might have been mentioned on the
> > list before, but I could not find the email. In
> that
> > case, please excuse me for repeating this.
> > 
> > _______________________________________________
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>
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> 
> -- 
> James W. MacDonald, M.S.
> Biostatistician
> Affymetrix and cDNA Microarray Core
> University of Michigan Cancer Center
> 1500 E. Medical Center Drive
> 7410 CCGC
> Ann Arbor MI 48109
> 734-647-5623
>