Hello, I had arrays with 4 replicate spots per gene. I used limma package for data analysis. > targets SlideNumber FileName Cy3 Cy5 Name Field1 1 13617731 WBM WC Field1 Field2 2 13617730 WBM WC Field2 Field3 3 13617724 WC WBM Field3 Field4 4 13617627 WC WBM Field4 Field5 5 13617626 WBM WC Field5 After read in data, normalization, I used the following codes for within-array replicate spots. design <- modelMatrix(targets, ref="WC") corfit <- duplicateCorrelation(g2, design, ndups=4) # A slow computation! fit <- lmFit(g2, design, ndups=4, correlation=corfit$consensus, method="ls") fit2 <- lmFit(g2, design, ndups=4, correlation=corfit$consensus, method="robust") # eBayes fit5 <- eBayes(fit) fit6 <- eBayes(fit2) Then, topTable(fit5, number=30, adjust="BH") gives me a list of differentially expressed genes. However, some of genes show up mutiple times, such as 37A-C02.g in the list. According to limma guide, 11.6, gene names should appear only once. Block Row Column ID Name 597 4 15 15 37B-B10.g P25782 519 4 2 17 37A-C02.g P00765 531 4 4 17 37A-C02.g P00765 314 2 24 18 37A-B08.g AC186398 308 2 23 19 35B-F12.g AC122261 513 4 1 17 37A-C02.g P00765 Another question, is there any difference between methods of "ls" and "robust" in lmFit? > sessionInfo() R version 2.6.0 (2007-10-03) i486-pc-linux-gnu locale: LC_CTYPE=en_US.UTF-8;LC_NUMERIC=C;LC_TIME=en_US.UTF-8;LC_COLLATE=en_US .UTF-8;LC_MONETARY=en_US.UTF-8;LC_MESSAGES=en_US.UTF-8;LC_PAPER=en_US. UTF-8;LC_NAME=C;LC_ADDRESS=C;LC_TELEPHONE=C;LC_MEASUREMENT=en_US.UTF-8 ;LC_IDENTIFICATION=C attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] MASS_7.2-37 statmod_1.3.1 limma_2.12.0 loaded via a namespace (and not attached): [1] rcompgen_0.1-15 Best wishes, Tiandao

Hi Tiandao, You seem to only be following the vignette without checking to see if the code it uses is appropriate for your data. In addition to the vignette, you should read through the help page for each main function for more information on the options/arguments available and what the different choices mean. For example, checking ?duplicateCorrelation shows that there is an argument called "spacing" with a default value of 1. The description of spacing: the spacing between the rows of object corresponding to duplicate spots, spacing=1 for consecutive spots. Are all 4 of your duplicate spots right after each other? Probably not, else you would not be getting duplicate gene names in your output. If the spots do not have a consistent spacing between them, you can sort your g2 object (after all pre-processing and normalization) so that they are in consecutive order. You'll have to be careful about blanks & buffers, or other spots that may show up >4 times. >Another question, is there any difference between methods of "ls" and >"robust" in lmFit? Of course there is a difference! Read through the entire help page of lmFit, and the links from there for more information. Cheers, Jenny > > sessionInfo() >R version 2.6.0 (2007-10-03) >i486-pc-linux-gnu > >locale: >LC_CTYPE=en_US.UTF-8;LC_NUMERIC=C;LC_TIME=en_US.UTF-8;LC_COLLATE=en_U S.UTF-8;LC_MONETARY=en_US.UTF-8;LC_MESSAGES=en_US.UTF-8;LC_PAPER=en_US .UTF-8;LC_NAME=C;LC_ADDRESS=C;LC_TELEPHONE=C;LC_MEASUREMENT=en_US.UTF- 8;LC_IDENTIFICATION=C > >attached base packages: >[1] stats graphics grDevices utils datasets methods base > >other attached packages: >[1] MASS_7.2-37 statmod_1.3.1 limma_2.12.0 > >loaded via a namespace (and not attached): >[1] rcompgen_0.1-15 > >Best wishes, > >Tiandao > >_______________________________________________ >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 uiuc.edu