[BioC] Technical replicates in limma

[Steve Taylor]

Hi,

Apologies if this is a naive limma question but the experimental design in this analysis is quite confusing, which has made me want to confirm the approach with the experts!

I have the following set of Affy CEL files that are from 19 treatment samples and 5 control samples.

My targets file is as follows:

file		expt	
---------	-----	
Batch1_1.CEL	s_1		
Batch1_2.CEL	s_2		
Batch1_3.CEL	s_3		
Batch1_4.CEL	s_4		
Batch1_5.CEL	s_5		
Batch1_6.CEL	s_6		
Batch1_7.CEL	control_T	
Batch1_9.CEL	control_B	
Batch1_11.CEL	control_P	
Batch2_2.CEL	s_7		
Batch2_3.CEL	s_8		
Batch2_5.CEL	s_9		
Batch2_6.CEL	s_10		
Batch2_7.CEL	s_11		
Batch2_9.CEL	s_12		
Batch2_10.CEL	s_13		
Batch2_11.CEL	s_14		
Batch2_12.CEL	s_15		
Batch2_13.CEL	s_6		
Batch2_14.CEL	control_P	
Batch2_15.CEL	control_B	
Batch2_16.CEL	control_T	
Batch3_1.CEL	control_P	
Batch3_2.CEL	control_T	
Batch3_4.CEL	control_B	
Batch3_6.CEL	control_O	
Batch3_8.CEL	control_O_LP	
Batch3_9.CEL	s_16		
Batch3_10.CEL	s_17		
Batch3_11.CEL	s_18		
Batch3_12.CEL	s_13		
Batch3_13.CEL	s_13		
Batch3_14.CEL	s_19		

I want to make several different comparisons of treatment vs control combinations so I am using the makeContrast function. The main issue is how to handle the mixture of technical and biological 
replicates.

For example, I wish to compare samples s_1,s_3,s_5,s_6 and s_7 vs control samples and get the top 100 significant probe sets. So after reading in the targets file and normalising the data...

design<-model.matrix(~-1+expt)
contr.mat<-makeContrasts(TvsN = (s_1+s_3+s_5+s_6+s_7)/5-(control_T+control_P+control_B+control_O+control_O_LP)/5,levels=design)
fit  <- lmFit(normeset, design ) # normeset is the normalised data
fit2 <- contrasts.fit( fit, contr.mat )
fit3 <- eBayes( fit2 )
topTable(fit3, n=100, adjust="BH", sort.by="B")

does the above correctly take into account the technical replicates? If not what is the best way to handle this?

Thanks for any help,

Steve

-------------------------
Medical Sciences Division
Oxford University