Dear R-Users, I use home-made spotted arrays and every probe is printed in triplicate. I normalized my data using limma package and I got mean from these three replicates by using duplicateCorrelation function: design<-modelMatrix(targets, ref="HL60") duplicateCorrelation(MAl.pAq, design, ndups=3,spacing=1) corfit <- duplicateCorrelation(MAl.pAq, ndups=3, spacing=1, design) all.correlations <- tanh(corfit$atanh.correlations) fit <- lmFit(MAl.pAq, design, ndups=3, spacing=1, correlation=corfit$consensus) contrast.matrix<-makeContrasts(AML.M2-K,levels=design) fit2<-contrasts.fit(fit,contrast.matrix) fit2<-eBayes(fit2) topTable(fit2,adjust="BH") However, this function does not change object dimmensions (plot shows all spots, not only mean from all replicates), so I try to use avedups function (a<-avedups(MA.normAq, ndups=3, spacing=1, weights=NULL)), but I have problem with creating fit2, because of differences in dimmentions. I would like to get a topTable object. What shall I do?? Regards, Barbara Cegielska

When I use duplicateCorrelation, I get one contrast per gene, not per spot. I am not sure what the problem is here. The spots are aggregated in the fit, but of course any function such as plots that use the original data will not have this aggregation. --Naomi At 04:40 PM 7/3/2009, michael watson \(IAH-C\) wrote: >If I were doing this, I would use the aggregate function to >calculate the mean for each of my genes, and then pass this averaged >data as a matrix to lmFit. > >There shouldn't be too many problems. > >Why don't you post your full code and any errors? > >________________________________ > >From: bioconductor-bounces at stat.math.ethz.ch on behalf of Barbara Cegielska >Sent: Fri 03/07/2009 8:13 AM >To: bioconductor at stat.math.ethz.ch; r-help-bounces at r-project.org >Subject: [BioC] how to merge replicate spots? > > > >Dear R-Users, > >I use home-made spotted arrays and every probe is printed in >triplicate. I normalized my data using limma package and I got mean >from these three replicates by using duplicateCorrelation function: > > >design<-modelMatrix(targets, ref="HL60") >duplicateCorrelation(MAl.pAq, design, ndups=3,spacing=1) >corfit <- duplicateCorrelation(MAl.pAq, ndups=3, spacing=1, design) >all.correlations <- tanh(corfit$atanh.correlations) >fit <- lmFit(MAl.pAq, design, ndups=3, spacing=1, >correlation=corfit$consensus) >contrast.matrix<-makeContrasts(AML.M2-K,levels=design) >fit2<-contrasts.fit(fit,contrast.matrix) >fit2<-eBayes(fit2) >topTable(fit2,adjust="BH") > >However, this function does not change object dimmensions (plot >shows all spots, not only mean from all replicates), so I try to use >avedups function (a<-avedups(MA.normAq, ndups=3, spacing=1, >weights=NULL)), but I have problem with creating fit2, because of >differences in dimmentions. I would like to get a topTable object. >What shall I do?? > >Regards, > >Barbara Cegielska > >_______________________________________________ >Bioconductor mailing list >Bioconductor at stat.math.ethz.ch >https://stat.ethz.ch/mailman/listinfo/bioconductor >Search the archives: >http://news.gmane.org/gmane.science.biology.informatics.conductor > >_______________________________________________ >Bioconductor mailing list >Bioconductor at stat.math.ethz.ch >https://stat.ethz.ch/mailman/listinfo/bioconductor >Search the archives: >http://news.gmane.org/gmane.science.biology.informatics.conductor Naomi S. Altman 814-865-3791 (voice) Associate Professor Dept. of Statistics 814-863-7114 (fax) Penn State University 814-865-1348 (Statistics) University Park, PA 16802-2111

If I were doing this, I would use the aggregate function to calculate the mean for each of my genes, and then pass this averaged data as a matrix to lmFit. There shouldn't be too many problems. Why don't you post your full code and any errors? ________________________________ From: bioconductor-bounces@stat.math.ethz.ch on behalf of Barbara Cegielska Sent: Fri 03/07/2009 8:13 AM To: bioconductor at stat.math.ethz.ch; r-help-bounces at r-project.org Subject: [BioC] how to merge replicate spots? Dear R-Users, I use home-made spotted arrays and every probe is printed in triplicate. I normalized my data using limma package and I got mean from these three replicates by using duplicateCorrelation function: design<-modelMatrix(targets, ref="HL60") duplicateCorrelation(MAl.pAq, design, ndups=3,spacing=1) corfit <- duplicateCorrelation(MAl.pAq, ndups=3, spacing=1, design) all.correlations <- tanh(corfit$atanh.correlations) fit <- lmFit(MAl.pAq, design, ndups=3, spacing=1, correlation=corfit$consensus) contrast.matrix<-makeContrasts(AML.M2-K,levels=design) fit2<-contrasts.fit(fit,contrast.matrix) fit2<-eBayes(fit2) topTable(fit2,adjust="BH") However, this function does not change object dimmensions (plot shows all spots, not only mean from all replicates), so I try to use avedups function (a<-avedups(MA.normAq, ndups=3, spacing=1, weights=NULL)), but I have problem with creating fit2, because of differences in dimmentions. I would like to get a topTable object. What shall I do?? Regards, Barbara Cegielska _______________________________________________ Bioconductor mailing list Bioconductor at stat.math.ethz.ch https://stat.ethz.ch/mailman/listinfo/bioconductor Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor

Michael, No, it wasn't deliberate. I've change it and it seems to work. Is it correct? ################################################################ > fit <- lmFit(a, design) > contrast.matrix<-makeContrasts(AML.M2-K,levels=design) > fit2<-contrasts.fit(fit,contrast.matrix) > fit2<-eBayes(fit2) > topTable(fit2,adjust="BH") Block Row Column ID Name Status logFC AveExpr t P.Value adj.P.Val B 850 35 6 1 6767 ST13 Oligos 3.075192 7.234434 9.388662 7.710111e-05 0.02516929 2.306532 284 11 5 4 6280 S100A9 Oligos -3.005805 8.759735 -9.303701 8.122192e-05 0.02516929 2.257757 859 35 9 1 23166 STAB1 Oligos 2.929309 7.131428 9.017997 9.706814e-05 0.02516929 2.089691 841 35 3 1 6726 SRP9 Oligos 2.847275 7.485457 8.970909 1.000094e-04 0.02516929 2.061377 557 21 6 4 3717 JAK2 Oligos 2.315902 8.071873 8.413855 1.439105e-04 0.02516929 1.712576 854 35 7 4 4772 NFATC1 Oligos 2.453980 7.217102 8.247170 1.611193e-04 0.02516929 1.603018 785 33 2 4 6093 ROCK1 Oligos 2.256970 7.425680 8.150150 1.722239e-04 0.02516929 1.538097 436 17 2 1 3563 IL3RA Oligos 2.554791 7.891780 7.911541 2.034941e-04 0.02530803 1.374718 937 37 10 10 291 SLC25A4 Oligos 2.507008 8.085160 7.737719 2.304150e-04 0.02530803 1.252276 972 37 17 10 369 ARAF Oligos 2.282989 6.985434 7.484587 2.772916e-04 0.02530803 1.068606 > dim(fit2) [1] 1023 1 #################################################################### P.s Sorry for not keeping discussions "on the list". Regards, B.