Dear limma experts, I have direct experiments with two biological replicates and two technical replicates. In each array sots are printted in 4 replicates. In duplicateCorrelation help it is written that "At this time it is not possible to estimate correlations between duplicate spots and between technical replicates simultaneously." The question is it possible to average on both technical and spot replicates but not simultaneously and if yes then how? If not which least-squares analysis should I drop technical sample replicates or spots replicates? Thanks in advance Ron [[alternative HTML version deleted]]

> Date: Sun, 17 Apr 2005 17:24:14 +0300 > From: "Ron Ophir" <ron.ophir@weizmann.ac.il> > Subject: [BioC] technical replicates and spots in limma > To: <bioconductor@stat.math.ethz.ch> > > Dear limma experts, > I have direct experiments with two biological replicates and two > technical replicates. In each array sots are printted in 4 replicates. > In duplicateCorrelation help it is written that "At this time it is not > possible to estimate correlations between duplicate spots and between > technical replicates simultaneously." > The question is it possible to average on both technical and spot > replicates but not simultaneously and if yes then how? > If not which least-squares analysis should I drop technical sample > replicates or spots replicates? The between spot correlation is usually in the range 0.5-0.9. Correlations between technical replicates are usually not so strong, seldom higher than around 0.2-0.3 and often less. If you're going to ignore one of these correlations, it should be the technical replication. If you're going to average over one of the replicate structures, it should be over the replicate spots. The measurement error is often larger than the biological variation, so that treating the technical replicates as biological replicates is often not as bad as it sounds. This is what I would usually do, having checked the between technical rep correlation is not large. Gordon > Thanks in advance > Ron

> > >>>> "Gordon K Smyth" <smyth@wehi.edu.au> 04/18/05 2:24 PM >>> >> Date: Sun, 17 Apr 2005 17:24:14 +0300 >> From: "Ron Ophir" <ron.ophir@weizmann.ac.il> >> Subject: [BioC] technical replicates and spots in limma >> To: <bioconductor@stat.math.ethz.ch> >> >> Dear limma experts, >> I have direct experiments with two biological replicates and two >> technical replicates. In each array sots are printted in 4 replicates. >> In duplicateCorrelation help it is written that "At this time it is not >> possible to estimate correlations between duplicate spots and between >> technical replicates simultaneously." >> The question is it possible to average on both technical and spot >> replicates but not simultaneously and if yes then how? >> If not which least-squares analysis should I drop technical sample >> replicates or spots replicates? > >The between spot correlation is usually in the range 0.5-0.9. Correlations between technical >replicates are usually not so strong, seldom higher than around 0.2-0.3 and often less. > >If you're going to ignore one of these correlations, it should be the technical replication. If >you're going to average over one of the replicate structures, it should be over the replicate >spots. Thanks. Averaging over spot replicates using duplicateCorrelation() assuming equal space between replicates coordinates or I can give a vector of spots location like in block for technical replicates. If the latter is not possible, does the following commands are what should be done: spotRep<-as.factor(c(1,1,2,2,3,1,1,3,3,2,2,3,...)) vvRaw$R<-unlist(by(vvRaw$R,spotRep,mean)) vvRaw$G<-unlist(by(vvRaw$G,spotRep,mean)) vvRaw$Rb<-unlist(by(vvRaw$Rb,spotRep,mean)) vvRaw$Gb<-unlist(by(vvRaw$Gb,spotRep,mean)) Ron > >The measurement error is often larger than the biological variation, so that treating the >technical replicates as biological replicates is often not as bad as it sounds. This is what I >would usually do, having checked the between technical rep correlation is not large. > >Gordon > >> Thanks in advance >> Ron