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Question: limma topTable doesn't work without replicates [was: Help with limma]
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gravatar for Gordon Smyth
5.1 years ago by
Gordon Smyth32k
Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
Gordon Smyth32k 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 intend...{{dropped:4}}
ADD COMMENTlink modified 5.1 years ago by Venu Pullabhatla40 • written 5.1 years ago by Gordon Smyth32k
0
gravatar for Venu Pullabhatla
5.1 years ago by
Venu Pullabhatla40 wrote:
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}}
ADD COMMENTlink written 5.1 years ago by Venu Pullabhatla40

when fit2$coefficients is used, log2FC is displayed for each one of the samples. In this case, what is the reference sample which we can compare the logFC against?

 

Thanks

ADD REPLYlink written 20 days ago by Nithisha10
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