Dear Maintainer, Last week, I submitted a post to bioconductor. However, I have been working on my HTqPCR scripts to form a qPCRset- ExpressionSet. I am almost there, but I'm getting hung up with some structural errors, perhaps, when creating new(eSet): > frame<-read.delim("dreamy.csv", skip=2, sep="\t", colClasses="character") > n=30 > head(frame) Actin_022.Triton.X.control.hskg.20.59.1 1 Actin_022\tTriton-X\tcontrol\thskg\t20.00\t2 2 Actin_022\tTriton-X\tcontrol\thskg\t22.96\t3 3 Actin_122\tTriton-X\tcontrol\thskg\t20.95\t1 4 Actin_122\tTriton-X\tcontrol\thskg\t20.86\t2 5 Actin_122\tTriton-X\tcontrol\thskg\t20.96\t3 6 Actin_322\tTriton-X\tcontrol\thskg\t20.97\t1 ## new("eSet", class=matrix) of Ct values > mat=matrix(as.numeric(frame$Ct), ncol=1, nrow=30, byrow=FALSE) > summary(mat) V1 V2 V3 V4 V5 Min. : NA Min. : NA Min. : NA Min. : NA Min. : NA 1st Qu.: NA 1st Qu.: NA 1st Qu.: NA 1st Qu.: NA 1st Qu.: NA Median : NA Median : NA Median : NA Median : NA Median : NA Mean :NaN Mean :NaN Mean :NaN Mean :NaN Mean :NaN 3rd Qu.: NA 3rd Qu.: NA 3rd Qu.: NA 3rd Qu.: NA 3rd Qu.: NA Max. : NA Max. : NA Max. : NA Max. : NA Max. : NA NA's :30 NA's :30 NA's :30 NA's :30 NA's :30 ## use eSet formatting new("ExpressionSet", phenoData = new("AnnotatedDataFrame"), featureData = new("AnnotatedDataFrame"), experimentData = new("MIAME"), annotation = character(0), exprs = new("matrix")) ... > rawdog<-new("qPCRset", exprs=mat, phenoData=as(data.frame(frame$featureType, rep(c("control", "target"), each=15), frame$featureClass, rep(c("hkgs, "jasmonic pathway", each=15), featureData=as(data.frame(frame$featureNames, rep(c("Gene", 1:n, sep="\t"))))) ##But get undetermined error. Error: unexpected symbol in "rawdog<-new("qPCRset", exprs=mat, phenoData=as(data.frame(frame$featureType, rep(c("control", "target"), each=15), as(data.frame(frame$featureClass, rep(c("hkgs, "jasmonic" ##Also tried to use methods: "AnnotatedDataFrameFrom", phenoData=(phenodata, "AnnotatedDataFrame"), rbind() <-get error message that data.frame must be of AssayData type ##tried adding paretheses to ("frame$columnName") > rawdog<-new("qPCRset", exprs=mat,phenoData=as(data.frame(frame$featureType), rep(c("control", "target"), each=15), (frame$featureClass, rep(c("hkgs, "jasmonic pathway", each=15), featureData=as(data.frame(frame$featureNames, rep(c("Gene", 1:n, sep="\t")))) Error: unexpected ',' in "rawdog<-new("qPCRset", exprs=mat,phenoData=as(data.frame(frame$featureType), rep(c("control", "target"), each=15), (frame$featureClass," ##Mixed from HTqPCR vinette and R scripts > raw=new("qPCRset", exprs=mat, phenoData=AnnotatedDataFrame(array(frame$featureNames, frame$SampleName),featureData=AnnotatedDataFrame(frame$featureType, frame$featureCategory))) Error in function (classes, fdef, mtable) : unable to find an inherited method for function "AnnotatedDataFrame", for signature "NULL", "missing" >Dreamy.csv Sample Detector featureNames sampleNames featureType featureClass Ct replicateType Actin_022 Triton-X control hskg 20.59 1 Actin_022 Triton-X control hskg 20.00 2 Actin_022 Triton-X control hskg 22.96 3 Actin_122 Triton-X control hskg 20.95 1 Actin_122 Triton-X control hskg 20.86 2 Actin_122 Triton-X control hskg 20.96 3 Actin_322 Triton-X control hskg 20.97 1 Actin_322 Triton-X control hskg 21.18 2 Actin_322 Triton-X control hskg 21.81 3 Actin_722 Triton-X control hskg 21.14 1 Actin_722 Triton-X control hskg 20.85 2 Actin_722 Triton-X control hskg 21.78 3 Actin_1422 Triton-X control hskg 21.54 1 Actin_1422 Triton-X control hskg 21.11 2 Actin_1422 Triton-X control hskg 22.45 3 Lox22_022 Triton-X target jasmonic pathway 23.93 1 Lox22_022 Triton-X target jasmonic pathway 23.36 2 Lox22_022 Triton-X target jasmonic pathway 23.52 3 Lox22_122 Triton-X target jasmonic pathway 24.82 1 Lox22_122 Triton-X target jasmonic pathway 24.71 2 Lox22_122 Triton-X target jasmonic pathway 25.61 3 Once I figure out the eSet formatting, I Would also like to add a column to phenoData: [[replicateType]] to tell R that I have replicates. I have simulated the data above, however, I have N=3 samples (untreated control, negative control, and treated) each having 2 technical replicates Actin_022 is paired with Lox22_022, each having 3 biological replicates, respectively. If possible, could you offer help on rawdog<-new(eSet, "AnnotatedDataFrameS") Best Regards, Franklin -- output of sessionInfo(): > sessionInfo() R version 2.15.1 (2012-06-22) Platform: x86_64-pc-mingw32/x64 (64-bit) locale: [1] LC_COLLATE=English_United States.1252 LC_CTYPE=English_United States.1252 LC_MONETARY=English_United States.1252 LC_NUMERIC=C [5] LC_TIME=English_United States.1252 attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] HTqPCR_1.12.0 limma_3.12.1 RColorBrewer_1.0-5 Biobase_2.18.0 BiocGenerics_0.4.0 loaded via a namespace (and not attached): [1] affy_1.36.1 affyio_1.26.0 BiocInstaller_1.8.3 gdata_2.11.0 gplots_2.11.0 gtools_2.7.0 preprocessCore_1.20.0 [8] stats4_2.15.1 tools_2.15.1 zlibbioc_1.3.0 > -- Sent via the guest posting facility at bioconductor.org.