Dear sir firstly sorry that iam again writing mail to you directly, two days before i worte mail to bioconductor at stat.math.ethz.ch but i did not got any reply from that Almost all analysis are working well. Thank you for all your helps throughout the process. HTqPCR package was very help full because i was able generate heat maps and performed clustering analysis. Now i am having few dought which i didt find in the vintage also. What is iqr.value and for what this is done. For me when i have done t test it showed some error but mann whitney test worked. After mann whitney test i got fold change mean value of all sample in a tumor group compared to normal group. Can we get fold change value of individual samples in tumor group compared to mean of normal samples. Could we get result in excel or as separate sheet as we get plots. Once again thank you for your help during the analysis. > iqr.values <- apply(exprs(d.norm), 1, IQR) Error in quantile.default(as.numeric(x), c(0.25, 0.75), na.rm = na.rm, : missing values and NaN's not allowed if 'na.rm' is FALSE > traceback() 6: stop("missing values and NaN's not allowed if 'na.rm' is FALSE") 5: quantile.default(as.numeric(x), c(0.25, 0.75), na.rm = na.rm, names = FALSE) 4: quantile(as.numeric(x), c(0.25, 0.75), na.rm = na.rm, names = FALSE) 3: diff(quantile(as.numeric(x), c(0.25, 0.75), na.rm = na.rm, names = FALSE)) 2: FUN(newX[, i], ...) 1: apply(exprs(d.norm), 1, IQR) > qDE.ttest <- ttestCtData(sr.norm[,1:4], groups=files$Treatment[1:4], + calibrator="Normal") Error in ttestCtData(sr.norm[, 1:4], groups = files$Treatment[1:4], calibrator = "Normal") : Two factor levels required for 'groups > sessionInfo() R version 2.11.0 (2010-04-22) x86_64-pc-mingw32 locale: [1] LC_COLLATE=English_India.1252 LC_CTYPE=English_India.1252 LC_MONETARY=English_India.1252 LC_NUMERIC=C LC_TIME=English_India.1252 attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] HTqPCR_1.2.0 limma_3.4.1 RColorBrewer_1.0-2 Biobase_2.8.0 loaded via a namespace (and not attached): [1] affy_1.26.1 affyio_1.16.0 gdata_2.8.0 gplots_2.7.4 gtools_2.6.2 preprocessCore_1.10.0 tools_2.11.0 > -- Deepak Roshan V G Laboratory Of Cell Cycle Regulation & Molecular Oncology Division of Cancer Research Regional Cancer Centre Thiruvananthapuram Kerala, India 695 011

Hello Deepak, > Dear sir > > > Almost all analysis are working well. Thank you for all your helps > throughout the process. HTqPCR package was very help full because i > was able generate heat maps and performed clustering analysis. Now i > am having few dought which i didt find in the vintage also. What is > iqr.value and for what this is done. > The IQR value is the interquartile range. It measures how much variation you see across your data for each gene, so it's a way for you to remove genes that show only little or almost no variation across your samples, since these are unlikely to be differentially expressed anyway. For high-content microarrays removing these genes is an advantage, since removing "uninteresting" genes before the downstream analysis means that when you correct your p-values for multiple testing, this correction will be less strict. For qPCR cards the number of genes is typically quite low, so this is less of an issue. Removing the low-IQR genes prior to the analysis of differential expression probably doesn't make much difference for your p-values. > For me when i have done t test > it showed some error but mann whitney test worked. After mann whitney > test i got fold change mean value of all sample in a tumor group > compared to normal group. Can we get fold change value of individual > samples in tumor group compared to mean of normal samples. > I'm afraid that this is something you'd have to calculate "manually", i.e. using non-HTqPCR functions. It shouldn't be too difficult though. If you e.g. have 20 samples and a qPCRset names "q", and the first 10 are test and the the last 10 are controls, you can say e.g. data <- getCt(q) data.test <- data[,1:10] data.control <- data[,11:20] mean.control <- rowMeans(data.control) fc.test <- data.test - mean.control > Could we > get result in excel or as separate sheet as we get plots. > In R you can always write results to a file using for example write.table. To get a tab-separated excel-like file, say: # Load example preprocessed data data(qPCRpros) # Test between two groups, collapsing replicated features diff.exp <- mannwhitneyCtData(qPCRpros[,1:4], groups=factor(c("A", "B", "B", "A")), calibrator="B") # Write to file write.table(diff.exp, file="test.xls", sep="\t", quote=FALSE, row.names=FALSE) > Once again thank you for your help during the analysis. > > You're welcome. > > iqr.values <- apply(exprs(d.norm), 1, IQR) > > Error in quantile.default(as.numeric(x), c(0.25, 0.75), na.rm = > na.rm, : > > missing values and NaN's not allowed if 'na.rm' is FALSE > As the error says, this doesn't work if you have NAs in your data. If you look at the help file for IQR, ?IQR, you'll see that there's an option na.rm which you can set to TRUE. When using the apply function, this would be: qr.values <- apply(exprs(d.norm), 1, IQR, na.rm=TRUE) > > qDE.ttest <- ttestCtData(sr.norm[,1:4], groups=files$Treatment[1:4], > > + calibrator="Normal") > > Error in ttestCtData(sr.norm[, 1:4], groups = files$Treatment[1:4], > calibrator = "Normal") : > > Two factor levels required for 'groups > Do the first 4 samples actually belong to different groups?? I.e. are there two different groups listed in files$Treatment[1:4]? Otherwise you're trying to perform a t-test of all samples against nothing, which isn't possible. HTH \Heidi > > sessionInfo() > > R version 2.11.0 (2010-04-22) > > x86_64-pc-mingw32 > > > locale: > > [1] LC_COLLATE=English_India.1252 LC_CTYPE=English_India.1252 > LC_MONETARY=English_India.1252 LC_NUMERIC=C > LC_TIME=English_India.1252 > > > attached base packages: > > [1] stats graphics grDevices utils datasets methods base > > > other attached packages: > > [1] HTqPCR_1.2.0 limma_3.4.1 RColorBrewer_1.0-2 > Biobase_2.8.0 > > > loaded via a namespace (and not attached): > > [1] affy_1.26.1 affyio_1.16.0 gdata_2.8.0 > gplots_2.7.4 gtools_2.6.2 preprocessCore_1.10.0 > tools_2.11.0 > > > > > -- > Deepak Roshan V G > Laboratory Of Cell Cycle Regulation & Molecular Oncology > Division of Cancer Research > Regional Cancer Centre > Thiruvananthapuram > Kerala, India 695 011