Hello all, I'm studing how to use duplicateCorrelation() of LIMMA package in order to evaluate the between-arrays correlation. I have a simple experiment of direct comparison with dye-swap as follows: targets SlideNumber FileName Cy3 Cy5 Date 1 Ag_1.gpr wt1 1RP 20/9/2008 2 Ag_2.gpr 1RP wt1 20/9/2008 3 Ag_3.gpr wt2 2RP 8/11/2008 4 Ag_4.gpr 2RP wt2 8/11/2008 I use duplicateCorrelation() as follows: design <- c(1,-1,1,-1) > biolrep<-c(1,1,2,2) > corfit<-duplicateCorrelation(MA,design,ndups=1,block=biolrep) > corfit$consensus [1] -0.5543286 The correlation is negative because of dye-swap. Then, I evaluate linear model as explained in limma userguide: > fit<-lmFit(MA,design, block=biolrep,cor=corfit$consensus,weights=NULL) > summary(fit) Length Class Mode coefficients 10807 -none- numeric stdev.unscaled 10807 -none- numeric sigma 10807 -none- numeric df.residual 10807 -none- numeric ndups 1 -none- numeric spacing 1 -none- numeric block 4 -none- numeric correlation 1 -none- numeric cov.coefficients 1 -none- numeric pivot 1 -none- numeric genes 5 data.frame list Amean 10807 -none- numeric method 1 -none- character design 4 -none- numeric fit2<-ebayes(fit) summary(fit2) Length Class Mode df.prior 1 -none- numeric s2.prior 1 -none- numeric s2.post 10807 -none- numeric t 10807 -none- numeric p.value 10807 -none- numeric var.prior 1 -none- numeric lods 10807 -none- numeric > toptable(fit2,adjust="fdr") Error in dim(data) <- dim : attempt to set an attribute on NULL What does it means this error message and, above all, where is the mistake in my analysis procedure? I do not understand why in fit2 there are not any coefficients! I would like to evaluate the dye effect also. How can I do this? I tryed the inclusion of a dye effect coefficient in the design, design<-cbind(Dye=1,design) but when I calculate duplicateCorrelation corfit$consensus is NaN. Is it correct? Thanks very much for any kind of help in advance! Best regards Erika [[alternative HTML version deleted]]

Dear Gordon, I fixed the problem with eBayes....I used ebayes() and not eBayes() and, as reported in LIMMA help, "ebayes is the earlier and leaner function. eBayes is intended to have a more object-orientated flavor as it produces objects containing all the necessary components for downstream analysis. " Then, I fixed the problem by using eBayes() However, I did not manage to fix the problem of duplicateCorrelation() When I define the design including the dye effect and I use this design in duplicateCorrelation(), the calculated consensus correlation is NaN, where without including the dye effect is -0.55 (I have a dye swap design). I have tryed to "unswap" the design also (as you suggested in a previous message), but I have obtained the same result, that is NaN. I have used a different dataset with the same experimental design also and the result was the same: NaN I have tryed to use the simple dye swap design for duplicateCorrelation computation >design<-c(-1,1-1,1) >biolrep<-c(1,1,2,2) >corfit<-duplicateCorrelation(MA,design,ndups=1,block=biolrep) then I have defined a new design >design<-cbind(Dye=1,design) and I have used this together with the correlation previously calculated by duplicateCorrelation (-0.55) for model computing >fit<-lmFit(MA,design, block=biolrep,cor=corfit$consensus,weights=NULL) Is this a right way of mouving aroud the problem? Any suggestion will be appreciated. Erika ----- Original Message ----- From: "Gordon K Smyth" <smyth@wehi.edu.au> To: "Erika Melissari" <erika.melissari at="" bioclinica.unipi.it=""> Cc: "Bioconductor mailing list" <bioconductor at="" stat.math.ethz.ch=""> Sent: Saturday, January 24, 2009 03:40 AM Subject: [BioC] Use of duplicateCorrelation > Dear Erika, > > The output from fit() and eBayes() cannot be as you've given below, > because eBayes() does not remove the $coefficient component of the fitted > model object. Can you please rerun your code from scratch in a new R > session, re-reading the data and so on. > > Best wishes > Gordon > >> Date: Thu, 22 Jan 2009 15:51:43 +0100 >> From: "Erika Melissari" <erika.melissari at="" bioclinica.unipi.it=""> >> Subject: [BioC] Use of duplicateCorrelation >> To: <bioconductor at="" stat.math.ethz.ch=""> >> Message-ID: <00c001c97ca0$f1a27fe0$ba517283 at maanalysis> >> Content-Type: text/plain >> >> Hello all, >> >> I'm studing how to use duplicateCorrelation() of LIMMA package in order >> to evaluate the between-arrays correlation. >> I have a simple experiment of direct comparison with dye-swap as follows: >> targets >> SlideNumber FileName Cy3 Cy5 Date >> 1 Ag_1.gpr wt1 1RP >> 20/9/2008 >> 2 Ag_2.gpr 1RP wt1 >> 20/9/2008 >> 3 Ag_3.gpr wt2 2RP >> 8/11/2008 >> 4 Ag_4.gpr 2RP wt2 >> 8/11/2008 >> >> I use duplicateCorrelation() as follows: >> >> design <- c(1,-1,1,-1) >>> biolrep<-c(1,1,2,2) >>> corfit<-duplicateCorrelation(MA,design,ndups=1,block=biolrep) >>> corfit$consensus >> [1] -0.5543286 >> >> The correlation is negative because of dye-swap. >> Then, I evaluate linear model as explained in limma userguide: >> >>> fit<-lmFit(MA,design, block=biolrep,cor=corfit$consensus,weights=NULL) >>> summary(fit) >> Length Class Mode >> coefficients 10807 -none- numeric >> stdev.unscaled 10807 -none- numeric >> sigma 10807 -none- numeric >> df.residual 10807 -none- numeric >> ndups 1 -none- numeric >> spacing 1 -none- numeric >> block 4 -none- numeric >> correlation 1 -none- numeric >> cov.coefficients 1 -none- numeric >> pivot 1 -none- numeric >> genes 5 data.frame list >> Amean 10807 -none- numeric >> method 1 -none- character >> design 4 -none- numeric >> fit2<-ebayes(fit) >> summary(fit2) >> Length Class Mode >> df.prior 1 -none- numeric >> s2.prior 1 -none- numeric >> s2.post 10807 -none- numeric >> t 10807 -none- numeric >> p.value 10807 -none- numeric >> var.prior 1 -none- numeric >> lods 10807 -none- numeric >>> toptable(fit2,adjust="fdr") >> Error in dim(data) <- dim : attempt to set an attribute on NULL >> >> >> >> What does it means this error message and, above all, where is the >> mistake in my analysis procedure? >> I do not understand why in fit2 there are not any coefficients! >> I would like to evaluate the dye effect also. How can I do this? >> I tryed the inclusion of a dye effect coefficient in the design, >> >> design<-cbind(Dye=1,design) >> >> but when I calculate duplicateCorrelation corfit$consensus is NaN. Is it >> correct? >> >> Thanks very much for any kind of help in advance! >> Best regards >> Erika ---------------------------------------------------------------------- ---------- Database dei virus interno non c aggiornato. Controllato da AVG - http://www.avg.com Versione: 8.0.176 / Database dei virus: 270.9.10/1809 - Data di rilascio: 24/11/2008 09:03 AM