Dear colleagues: I am analyzing the result of some cancer samples miRNA NGS experiments with edgeR, with the following setup: R version 2.15.3 (2013-03-01) Platform: x86_64-w64-mingw32/x64 (64-bit) locale: [1] LC_COLLATE=Italian_Italy.1252 LC_CTYPE=Italian_Italy.1252 [3] LC_MONETARY=Italian_Italy.1252 LC_NUMERIC=C [5] LC_TIME=Italian_Italy.1252 attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] edgeR_3.0.8 limma_3.14.4 I am following a rather standard workflow - please note that we have 8 samples and only 1 control > targets <- read.delim("targets.txt", stringsAsFactors = FALSE) > d <- readDGE(targets, header=FALSE) > colnames(d) <- c("ARMS1","ARMS2","ARMS3","ARMS4","ERMS1","ERMS2","ERMS3","ERMS4","NMS ") > dim(d) [1] 2038 9 >keep <- rowSums(cpm(d)> 5) >= 3 > dim(d) [1] 685 9 > d$samples$lib.size <- colSums(d$counts) > d<-calcNormFactors(d) > d <- estimateCommonDisp(d, verbose=TRUE) Disp = 0.71417 , BCV = 0.8451 > d <- estimateTagwiseDisp(d, trend="none",verbose=TRUE) Using interpolation to estimate tagwise dispersion. > de.com <- exactTest(d) > sumde.com$table$PValue < 0.05) [1] 97 > topValues <- topTagsde.com,n=97) > summary(decideTestsDGEde.com,p.value=0.05)) [,1] -1 44 0 641 1 0 What we noticed is that there is a strong asimmetry in the corrected P values, in that only the downregulated miRNAs have a significant corrected P value - the upregulated miRNAs are less when examing the uncorreetd counts, basically we have alf of the CPM Questions: => is the unbalanced experimental design affecting the results ? this unbalance is coherent with the literature, in cancers the majority of miRNAs are downregulated => if yes, can I correct it or we should just take the results as they are and validate extensively if we want to explore also the upregulated miRNAs ? Thanks a lot in advance for any help, Alessandro & colleagues ----------------------------------------------------- Alessandro Guffanti - Head, Bioinformatics Genomnia srl Via Nerviano, 31/B – 20020 Lainate (MI) Tel. +39-0293305.702 / Fax +39-0293305.777 www.genomnia.com [http://www.genomnia.com/] alessandro.guffanti@genomnia.com [mailto:alessandro.guffanti@genomnia.com] Per cortesia, prima di stampare questa e-mail pensate all'ambiente. Please consider the environment before printing this mail note. ----------------------------------------------------------- Il Contenuto del presente messaggio potrebbe contenere informazioni confidenziali a favore dei soli destinatari del messaggio stesso. Qualora riceviate per errore questo messaggio siete pregati di cancellarlo dalla memoria del computer e di contattare i numeri sopra indicati. Ogni utilizzo o ritrasmissione dei contenuti del messaggio da parte di soggetti diversi dai destinatari è da considerarsi vietato ed abusivo. The information transmitted is intended only for the per...{{dropped:10}}