HI Vishal, I only briefly glanced through your code, but here's one problem (quoted from my response to a similar question on the list just last week): >Did you read through the help file for duplicateCorrelation? You >can't do both block and ndups: > >"At this time it is not possible to estimate correlations between >duplicate spots and between technical replicates simultaneously. If >block is not null, then the function will set ndups=1." HTH, Jenny At 09:11 AM 4/30/2009, Vishal Thapar wrote: >Dear List, > >Hi! I am new to this list so here is a brief introduction: My name is Vishal >and I am a post doc at Cold Spring Harbor Lab working on Chip-chip / seq >data analysis. I have my background in computer algorithms so pardon me if I >make some errors with my Biological and Statistical terminology. > >Here is the problem that I am facing: > >1) I have data from Nimblegen tiling arrays. I have 3 Bioreps each having 1 >technical rep. There are no dye swaps. In each rep, there are duplicate >spots on the array. In this experiment, as I reconstructed the images from >the data, I see some "quite" bad spots in the red channel specially for >biorep2. I am sure most of you have faced this so do you usually include >this rep in your analysis, or not? How do you handle the statistical >confidence with your results if you do or dont? > >2) I want to use the duplicate spots on each rep for my analysis. As of now, >I do the normalization, I average the duplicate spots and use that as my >input to the lmfit() function. I notice that after the average, the >correlation between the reps is better. I guess that is expected but I am >not satisfied with the averaging of the Spots. I believe that there is a >better way to do this than just take the average but I am just not aware of >that. I have used the duplicateCorrelation() function in Limma which gives >me a -0.04 correlation and its probably because the probes are position >randomized (even the duplicates are). So can anyone help me and tell me how >should I proceed and use these duplicate spots in a better way than just >simply averaging them? I appreciate any pointers that I can get. > > >Source code for this: > >ma.loess<-normalizeWithinArrays(rg,method="loess", bc.method="none") > >ma.quantile <-normalizeBetweenArrays(ma.loess, method="quantile") > >ma.spot1.quantile<-ma.quantile[grep("SPOT1",ma.quantile$genes$GENE_EX PR_OPTION),] > > >ma.spot2.quantile<-ma.quantile[grep("SPOT2",ma.quantile$genes$GENE_EX PR_OPTION),] > > >ma.spot1.quantile<-ma.spot1.quantile[order(ma.spot1.quantile$genes$GE NE_EXPR_OPTION,ma.spot1.quantile$genes$POSITION),] > > >ma.quantile <- ma.quantile[order(ma.quantile$genes$GENE_EXPR_OPTION, >ma.quantile$genes$POSITION),] > >ma.spot2.quantile<-ma.spot2.quantile[order(ma.spot2.quantile$genes$GE NE_EXPR_OPTION,ma.spot2.quantile$genes$POSITION),] > > >ma.avr.quantile<-ma.spot1.quantile >ma.avr.quantile$M<-(ma.spot1.quantile$M + ma.spot2.quantile$M)/2 > >fit.avg <- lmFit(ma.avr.quantile, design) >fit <- lmFit(ma.quantile, design) > >-------------------------------- >function: duplicateCorrelation() in limma as follows: > >biolrep=c(1,1,2,2) >corfit.avr=duplicateCorrelation(ma.avr.quantile, ndups=2, block=biolrep) >-------------------------------- > >This did not work. I got a negative corelation of -0.04 > >I appreciate your time and help . > >Sincerely, > >Vishal > >ps: Thank you Gordan Smith for writing Limma. I think its really a great >tool to have and I am very appreciative of it. > > [[alternative HTML version deleted]] > >_______________________________________________ >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 Jenny Drnevich, Ph.D. Functional Genomics Bioinformatics Specialist W.M. Keck Center for Comparative and Functional Genomics Roy J. Carver Biotechnology Center University of Illinois, Urbana-Champaign 330 ERML 1201 W. Gregory Dr. Urbana, IL 61801 USA ph: 217-244-7355 fax: 217-265-5066 e-mail: drnevich at illinois.edu