[BioC] Limma: technical replicates in multi-group design

[Gordon K Smyth]

Dear Mike, Wei, Adam, James,

I have to agree with Mike.  If I understand it correctly, there is no 
biological replication at all in this experiment.  In that situation, 
there is no way to assess statistical significance relative to biological 
variation.

limma has ways of making use of technical replicates, but there needs to 
be biological replication as well.

Best wishes
Gordon

> Date: Fri, 26 Mar 2010 09:49:13 +0100 (CET)
> From: Mike Walter <michael_walter at email.de>
> To: bioconductor at stat.math.ethz.ch, Adam Kiezun <akiezun at gmail.com>,
> 	Wei Shi <shi at wehi.edu.au>
> Subject: Re: [BioC] Limma: technical replicates in multi-group design
>
> Dear Wei,
>
> In my opinion the way you construct the design matrix the technical 
> replicates are treated as independent samples. The technical replicates 
> are handled with the block argument in the duplicateCorrelation() 
> function. As I understand the experimental design, however, there are 
> only technical but no biological replicates. I have data from the same 
> type of experiment and told the guys that there is no way of using any 
> statistical method to analyse their data. Am I right or is it possible 
> to use limma in such a case?
>
> Kind regards,
>
> Mike
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> -----Urspr?ngliche Nachricht-----
> Von: Wei Shi
> Gesendet: 25.03.2010 23:37:26
> An: Adam Kiezun [
> Betreff: Re: [BioC] Limma: technical replicates in multi-group design
>
>> Dear Adam:
>>
>>    You can use the following commands to create a design matrix for
>> your experiment:
>>
>> s <- factor(c("A","A","B","B","C","C"))
>> design <- model.matrix(~0+s)
>> colnames(design) <- levels(s)
>> fit <- lmFit(x,design)
>>
>>    To do a pairwise comparison, you can use the makeConstrasts function:
>>
>> contr <- makeContrasts(A-B,A-C,B-C,levels=design)
>> fit.contr <- eBayes(contrasts.fit(fit,contr))
>>
>> Cheers,
>> Wei
>>
>> Adam Kiezun wrote:
>>> Hi,
>>> How do I write the design matrix to specify technical replicates in
>>> multi-group experiment on a single-channel array (data coming from
>>> lumi)?
>>>
>>> I have 3 groups of samples (A, B, C), each group has 2 technical
>>> replicates (so I have 6 expression vectors: A1, A2, B1, B2, B3). How
>>> do I write the design matrix to tell limma that A1 and A2 etc are
>>> technical replicates and that I want to do all pairwise comparisons
>>> between groups (ie. AvsB, AvsC, BvsC)?
>>>
>>> The limma manual covers technical replicates for two-channel arrays,
>>> and the multi-group experiments with biological replicates. I'd like
>>> to know how to deal with a one-channel arrays, multi-group experiment
>>> with technical replicates.
>>>
>>> regards
>>> Adam

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