Mohamed Lajnef a ?crit : > Dear R-Users, > > I can not uderstand a result I have ( see Toptable). I used LIMMA to > find differentially expressed genes by 3 treatments, my database ( > illumina files) includes (48803 probes (rows) and 120 columns ( 40 by > level)), my program as follows > library(beadarray) > BSData<-readBeadSummaryData(fichier,skip=0,columns = list(exprs = > "AVG_Signal", se.exprs="BEAD_STDERR",NoBeads = "Avg_NBEADS", > Detection="Detection.Pval")) > BSData.quantile=normaliseIllumina(BSData, > method="quantile",transform="log2") # > detection<-Detection(BSData) # Matrix contain detection P value which > estimates the probability of a probe being detected above the > background level > > # Filtring after normalization > library(genefilter) > filtre<-function (p = 0.05, A = 100, na.rm = TRUE) > > { > function(x) { > if (na.rm) > x <- x[!is.na(x)] > sum(x <= A)/length(x) >= p > } > } > ff<-filtre(p=0.80, A=0.01) # i keep rows if pvalues<=0.01, the probe > has to be over expressed in at least 80% per level ( i have 3 levels) > > i<-genefilter(detection[,1:40],ff) > j<-genefilter(detection[,41:80],ff) # I will now keep 10156 probes > (after filtring tools) > k<-genefilter(detection[,81:120],ff) > > # Differential expression using Limma after normalization & filtering > tools > library(limma) > donne<-exprs(BSData.quantile) > OBSnormfilter<-donne[j,] # keep 10156 probes after normalization > groups<-as.factor(c(rep("Tem",40),rep("EarlyO",40),rep("LateO",40))) > design<-model.matrix(~0+groups) > colnames(design)=levels(groups) > fit<-lmFit(OBSnormfilter,design) > cont.matrix<-makeContrasts(Tem-EarlyO,Tem-LateO,EarlyO-LateO, > levels=design) > fit2<-contrasts.fit(fit, cont.matrix) > ebfit<-eBayes(fit2) > gene1<-topTable(ebfit, coef=1) > gene2<-topTable(ebfit, coef=2) > gene3<-topTable(ebfit, coef=3) > > gene1 ( result of Toptable between the control and first treatment > groups) > > ID logFC AveExpr t > P.Value adj.P.Val B > 9300 520255 -0.3209704 6.429487 -3.643323 0.0003963748 0.9998062 > -0.6345996 > 6192 7650097 -0.2677064 6.243968 -3.581163 0.0004921590 0.9998062 > -0.7817435 > 5528 10161 0.2022500 8.002581 3.434507 0.0008116961 0.9998062 > -1.1212432 > 6077 4180725 0.1380486 5.922805 3.423087 0.0008434258 0.9998062 > -1.1472217 > 3569 5080487 -0.1621675 7.717032 -3.308604 0.0012326133 0.9998062 > -1.4039040 > 2265 270332 -0.1996710 6.599011 -3.257771 0.0014545247 0.9998062 > -1.5156669 > 4643 5360301 0.5115730 6.616680 3.188442 0.0018176145 0.9998062 > -1.6658702 > 3885 110523 -0.1489957 6.165416 -3.130799 0.0021819409 0.9998062 > -1.7887772 > 8220 6280053 -0.1379738 6.603755 -3.057891 0.0027397895 0.9998062 > -1.9416230 > 4355 1430626 -0.1867890 6.624203 -3.054026 0.0027727561 0.9998062 > -1.9496424 > > looking at the results, Toptable show no any signficant genes, how > do you explain this?? ( because I have a lot of replication ( 40 by > level) ???) > > Any help would be appreciated > > Regards > ML > > > > > > > -- Mohamed Lajnef INSERM Unit? 955. 40 rue de Mesly. 94000 Cr?teil. Courriel : Mohamed.lajnef at inserm.fr tel. : 01 49 81 31 31 (poste 18470) Sec : 01 49 81 32 90 fax : 01 49 81 30 99