Dear All, I'am using limma package to extract the genes diffrentially expressed by 3 treatment,my database includes 48803 genes (rows) and 120 colones (40 replicates per treatment),my program is as follows: donne<-exprs(BSData.quantile) groups<-as.factor(c(rep("Tem",40),rep("EarlyO",40),rep("LateO",40))) design<-model.matrix(~0+groups) colnames(design)=levels(groups) fit<-lmFit(donne,design) cont.matrix<-makeContrasts(TemvsEarlyO=Tem-EarlyO,TemvsLateO=Tem- LateO,EarlyOvsLateO=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) results<-decideTests(ebfit) # I have a matrix that contains only 0 because in the ligne 26 results <- new("TestResults", sign(tstat) * (p < p.value)) of decideTests program the condition (p<p.value) is="" always="" false="" in="" my="" case!!="" something="" is="" wrong="" in="" my="" program,="" but="" i="" dont="" know="" where??="" any="" help="" would="" be="" appreciated.="" regards="" [[alternative="" html="" version="" deleted]]="" <="" div="">

On Sat, Mar 28, 2009 at 3:58 AM, Mohamed lajnef <lajnef@gmail.com> wrote: > Dear All, > > I'am using limma package to extract the genes diffrentially expressed by 3 > treatment,my database includes > > 48803 genes (rows) and 120 colones (40 replicates per treatment),my program > is as follows: > > donne<-exprs(BSData.quantile) > groups<-as.factor(c(rep("Tem",40),rep("EarlyO",40),rep("LateO",40))) > design<-model.matrix(~0+groups) > colnames(design)=levels(groups) > fit<-lmFit(donne,design) > > cont.matrix<-makeContrasts(TemvsEarlyO=Tem-EarlyO,TemvsLateO=Tem- LateO,EarlyOvsLateO=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) Hi, Mohamed. Do any of these topTable results show any significant genes? If not, then that would explain your decideTest() results. Sean > > > results<-decideTests(ebfit) # I have a matrix that contains only 0 because > > in the ligne 26 results <- new("TestResults", sign(tstat) * (p < p.value)) > of decideTests program the condition (p<p.value) is="" always="" false="" in="" my=""> case!! > something is wrong in my program, but I dont know where?? > > Any help would be appreciated. > > Regards > > [[alternative HTML version deleted]] > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > [[alternative HTML version deleted]]

Hi Mohamed: I think you can try two things: (1) Try alternative normalization methods; (2) Remove probes which are not expressing in all your arrays. Hope this helps. Cheers, Wei Mohamed lajnef wrote: > Dear All, > > I'am using limma package to extract the genes diffrentially expressed by 3 > treatment,my database includes > > 48803 genes (rows) and 120 colones (40 replicates per treatment),my program > is as follows: > > donne<-exprs(BSData.quantile) > groups<-as.factor(c(rep("Tem",40),rep("EarlyO",40),rep("LateO",40))) > design<-model.matrix(~0+groups) > colnames(design)=levels(groups) > fit<-lmFit(donne,design) > cont.matrix<-makeContrasts(TemvsEarlyO=Tem-EarlyO,TemvsLateO=Tem- LateO,EarlyOvsLateO=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) > > results<-decideTests(ebfit) # I have a matrix that contains only 0 because > > in the ligne 26 results <- new("TestResults", sign(tstat) * (p < p.value)) > of decideTests program the condition (p<p.value) is="" always="" false="" in="" my=""> case!! > something is wrong in my program, but I dont know where?? > > Any help would be appreciated. > > Regards > > [[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 >

Hi Mohamed: Nonexpressed probes are not those probes which have missing values. We do not know probes with missing values are expressing or not. You can use "Detection" scores to try to find those probes which do not express in all of your arrays and then filter them out before carrying out differential expression analysis to see if you can get some DE genes. You should be able to find "Detection" columns in your expression profiles. Cheers, Wei Mohamed lajnef wrote: > Dear Wei & Sean, > > Sean: result of topTbale > > gene1 > ID logFC AveExpr t P.Value > adj.P.Val B > 9623 7570474 -0.11659435 4.991279 -3.826220 0.0002074251 0.9999788 > 0.4950223 > 43599 5550736 -0.18627341 8.316864 -3.800538 0.0002275799 0.9999788 > 0.4162686 > 931 4570630 -0.10900890 4.834441 -3.776569 0.0002480571 0.9999788 > 0.3431365 > 20081 5560544 -0.22781231 8.704546 -3.770575 0.0002534446 0.9999788 > 0.3249047 > 33970 6280711 -0.08123219 5.013244 -3.521244 0.0006066949 0.9999788 > -0.4135212 > 19419 4010192 -0.09110624 4.903243 -3.516622 0.0006163498 0.9999788 > -0.4268336 > 37533 3290356 0.08945260 4.876439 3.512031 0.0006260841 0.9999788 > -0.4400442 > 10727 1260035 -0.14783999 5.784915 -3.506596 0.0006377940 0.9999788 > -0.4556641 > 9921 2260719 -0.09260158 4.927686 -3.474897 0.0007103085 0.9999788 > -0.5463862 > 17444 6180360 0.08237090 4.932764 3.474807 0.0007105246 0.9999788 > -0.5466424 > > > > Wei: 1) i used beadarray methods( quantile) to normalise the log2 > transoformed data > 2) i dont have a missing values in my data! > > Thank you again for help > > > > > > > 2009/3/30 Wei Shi <shi@wehi.edu.au <mailto:shi@wehi.edu.au="">> > > Hi Mohamed: > > I think you can try two things: > > (1) Try alternative normalization methods; > (2) Remove probes which are not expressing in all your arrays. > > Hope this helps. > > Cheers, > Wei > > Mohamed lajnef wrote: > > Dear All, > > I'am using limma package to extract the genes diffrentially > expressed by 3 > treatment,my database includes > > 48803 genes (rows) and 120 colones (40 replicates per > treatment),my program > is as follows: > > donne<-exprs(BSData.quantile) > groups<-as.factor(c(rep("Tem",40),rep("EarlyO",40),rep("LateO",40))) > design<-model.matrix(~0+groups) > colnames(design)=levels(groups) > fit<-lmFit(donne,design) > cont.matrix<-makeContrasts(TemvsEarlyO=Tem-EarlyO,TemvsLateO =Tem-LateO,EarlyOvsLateO=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) > > results<-decideTests(ebfit) # I have a matrix that contains > only 0 because > > in the ligne 26 results <- new("TestResults", sign(tstat) * > (p < p.value)) > of decideTests program the condition (p<p.value) is="" always=""> false in my > case!! > something is wrong in my program, but I dont know where?? > > Any help would be appreciated. > > Regards > > [[alternative HTML version deleted]] > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > <mailto:bioconductor@stat.math.ethz.ch> > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > > > [[alternative HTML version deleted]]