Dear Venu, Please keep the discussion on the list. log2-fold-changes are contained in fit2$coefficients. Best wishes Gordon On Mon, 15 Oct 2012, Venu Pullabhatla wrote: > Dear Gordon > > Many thanks for your reply. Apologies I should have been a little more > descriptive in my problem. I am aware that Limma cannot calculate any > statistics without replicates. So I wanted to get the fold change only using > topTable, but I am not aware that topTable does not work without replicates. > I assumed I can get the toptable output, but without the statistics due to > lack of replicates. Can you please guide me how to find out fold changes > without replicates using Limma. I am using agilent single channel. Once again > thanks a lot for your continued help and support with Limma. > > Best wishes > > Venu > > On 14/10/12 23:47, Gordon K Smyth wrote: >> Dear Venu, >> >> limma requires replicates. Pretty much any statistical testing method >> requires replicates. >> >> You can run lmFit() without replicates, which gives you fold changes, but >> you cannot go on to compute eBayes statistics or to use topTable(). >> >> The same answer has been given to many posts asking this question over the >> years. >> >> I am a bit puzzled what you expect limma to do for you in the absence of >> replication. >> >> Best wishes >> Gordon >> >>> Date: Fri, 12 Oct 2012 10:06:40 +0100 >>> From: Venu Pullabhatla <venu.pullabhatla at="" kcl.ac.uk=""> >>> To: <bioconductor at="" stat.math.ethz.ch=""> >>> Subject: [BioC] Help with Limma >>> >>> Dear Limma users >>> >>> After finishing the analysis successfully with replicates, I am now >>> trying to analyze the same without replicates. After following a >>> suggestion that I can use Limma without applying the fit2 <- >>> eBayes(fit2) step (for without replicates), I tried to do the same for >>> just one of the patients as a test, but it failed because of the >>> following error. >>> >>> Error in array(x, c(length(x), 1L), if (!is.null(names(x))) >>> list(names(x), : >>> attempt to set an attribute on NULL >>> Calls: topTable ... toptable -> as.matrix -> as.matrix.default -> array >>> Execution halted >>> >>> I get this error when applying topTable function to get the list of DE >>> genes. I have searched a lot for getting possible answers, but only >>> ended in vain. I came across this post in many forums, but with no >>> working solution. Can anyone please rectify my mistake in the script. >>> >>> I used the same script successfully for analyzing with replicates except >>> applying the fit2 <- eBayes(fit2) step because of lack of replicates. >>> >>> Here is the script I am using >>> >>> >>> library(limma) >>> library(annotate) >>> library(hgug4845a.db) >>> >>> targets <- readTargets("targets_individual_test", sep="\t") >>> data <- read.maimages(targets, path=".", source="agilent.median", >>> green.only=TRUE) >>> bgdata <- backgroundCorrect(data, method="normexp", offset=16) >>> normdata <- normalizeBetweenArrays(bgdata, method="quantile") >>> normdata_avg <- avereps(normdata, ID=normdata$genes$ProbeName) >>> >>> condition <- factor(targets$Condition, levels = unique(targets$Condition)) >>> design <- model.matrix(~0 + condition) >>> colnames(design) <- levels(condition) >>> >>> fit <- lmFit(normdata_avg, design) >>> fit$genes$Symbol <- getSYMBOL(fit$genes$ProbeName,"hgug4845a.db") >>> >>> contrast.matrix <- makeContrasts("Healthy-State1", "Healthy- State2", >>> "Healthy-State3", levels=design) >>> fit2 <- contrasts.fit(fit, contrast.matrix) >>> >>> ######## FINE UNTIL THIS STEP ########################### >>> >>> output <- topTable(fit2, coef="Healthy-State1", genelist=fit$genes, >>> number=Inf) >>> >>> ###### I GET THE ERROR AFTER RUNNING THE ABOVE COMMAND ####### >>> >>> Error in array(x, c(length(x), 1L), if (!is.null(names(x))) >>> list(names(x), : >>> attempt to set an attribute on NULL >>> Calls: topTable ... toptable -> as.matrix -> as.matrix.default -> array >>> Execution halted >>> >>> ################################################# >>> >>> write.table(output, file="output.txt", sep="\t", quote=FALSE) >>> >>> Many million thanks for the continued help and support. >>> >>> Best wishes >>> >>> Venu >>> > > > -- > Venu Pullabhatla, Ph.D. > Bioinformatician > NIHR GSTFT/KCL Comprehensive Biomedical Research Centre > Guy's & St. Thomas' NHS Foundation Trust > 8th Floor, Tower Wing, Guy's Hospital > Great Maze Pond, London SE1 9RT > > > ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:4}}

Dear Gordon Thank you very much for your help. I got it working now and also sorry for not keeping the discussion on the list. Best wishes Venu On 15/10/12 23:02, Gordon K Smyth wrote: > Dear Venu, > > Please keep the discussion on the list. > > log2-fold-changes are contained in fit2$coefficients. > > Best wishes > Gordon > > > > On Mon, 15 Oct 2012, Venu Pullabhatla wrote: > >> Dear Gordon >> >> Many thanks for your reply. Apologies I should have been a little >> more descriptive in my problem. I am aware that Limma cannot >> calculate any statistics without replicates. So I wanted to get the >> fold change only using topTable, but I am not aware that topTable >> does not work without replicates. I assumed I can get the toptable >> output, but without the statistics due to lack of replicates. Can you >> please guide me how to find out fold changes without replicates using >> Limma. I am using agilent single channel. Once again thanks a lot for >> your continued help and support with Limma. >> >> Best wishes >> >> Venu >> >> On 14/10/12 23:47, Gordon K Smyth wrote: >>> Dear Venu, >>> >>> limma requires replicates. Pretty much any statistical testing >>> method requires replicates. >>> >>> You can run lmFit() without replicates, which gives you fold >>> changes, but you cannot go on to compute eBayes statistics or to use >>> topTable(). >>> >>> The same answer has been given to many posts asking this question >>> over the years. >>> >>> I am a bit puzzled what you expect limma to do for you in the >>> absence of replication. >>> >>> Best wishes >>> Gordon >>> >>>> Date: Fri, 12 Oct 2012 10:06:40 +0100 >>>> From: Venu Pullabhatla <venu.pullabhatla at="" kcl.ac.uk=""> >>>> To: <bioconductor at="" stat.math.ethz.ch=""> >>>> Subject: [BioC] Help with Limma >>>> >>>> Dear Limma users >>>> >>>> After finishing the analysis successfully with replicates, I am now >>>> trying to analyze the same without replicates. After following a >>>> suggestion that I can use Limma without applying the fit2 <- >>>> eBayes(fit2) step (for without replicates), I tried to do the same for >>>> just one of the patients as a test, but it failed because of the >>>> following error. >>>> >>>> Error in array(x, c(length(x), 1L), if (!is.null(names(x))) >>>> list(names(x), : >>>> attempt to set an attribute on NULL >>>> Calls: topTable ... toptable -> as.matrix -> as.matrix.default -> >>>> array >>>> Execution halted >>>> >>>> I get this error when applying topTable function to get the list of DE >>>> genes. I have searched a lot for getting possible answers, but only >>>> ended in vain. I came across this post in many forums, but with no >>>> working solution. Can anyone please rectify my mistake in the script. >>>> >>>> I used the same script successfully for analyzing with replicates >>>> except >>>> applying the fit2 <- eBayes(fit2) step because of lack of replicates. >>>> >>>> Here is the script I am using >>>> >>>> >>>> library(limma) >>>> library(annotate) >>>> library(hgug4845a.db) >>>> >>>> targets <- readTargets("targets_individual_test", sep="\t") >>>> data <- read.maimages(targets, path=".", source="agilent.median", >>>> green.only=TRUE) >>>> bgdata <- backgroundCorrect(data, method="normexp", offset=16) >>>> normdata <- normalizeBetweenArrays(bgdata, method="quantile") >>>> normdata_avg <- avereps(normdata, ID=normdata$genes$ProbeName) >>>> >>>> condition <- factor(targets$Condition, levels = >>>> unique(targets$Condition)) >>>> design <- model.matrix(~0 + condition) >>>> colnames(design) <- levels(condition) >>>> >>>> fit <- lmFit(normdata_avg, design) >>>> fit$genes$Symbol <- getSYMBOL(fit$genes$ProbeName,"hgug4845a.db") >>>> >>>> contrast.matrix <- makeContrasts("Healthy-State1", "Healthy- State2", >>>> "Healthy-State3", levels=design) >>>> fit2 <- contrasts.fit(fit, contrast.matrix) >>>> >>>> ######## FINE UNTIL THIS STEP ########################### >>>> >>>> output <- topTable(fit2, coef="Healthy-State1", genelist=fit$genes, >>>> number=Inf) >>>> >>>> ###### I GET THE ERROR AFTER RUNNING THE ABOVE COMMAND ####### >>>> >>>> Error in array(x, c(length(x), 1L), if (!is.null(names(x))) >>>> list(names(x), : >>>> attempt to set an attribute on NULL >>>> Calls: topTable ... toptable -> as.matrix -> as.matrix.default -> >>>> array >>>> Execution halted >>>> >>>> ################################################# >>>> >>>> write.table(output, file="output.txt", sep="\t", quote=FALSE) >>>> >>>> Many million thanks for the continued help and support. >>>> >>>> Best wishes >>>> >>>> Venu >>>> >> >> >> -- >> Venu Pullabhatla, Ph.D. >> Bioinformatician >> NIHR GSTFT/KCL Comprehensive Biomedical Research Centre >> Guy's & St. Thomas' NHS Foundation Trust >> 8th Floor, Tower Wing, Guy's Hospital >> Great Maze Pond, London SE1 9RT >> >> >> > > ______________________________________________________________________ > The information in this email is confidential and inte...{{dropped:17}}