Envisage/0000755003020100016370000000000011031122667012461 5ustar sr9signg00000000000000Envisage/R/0000755003020100016370000000000011031122552012653 5ustar sr9signg00000000000000Envisage/R/modelEntry.R0000755003020100016370000002526311031122552015133 0ustar sr9signg00000000000000## Copyright (C) 2007 Sam Robson ## ## This file is part of Envisage. ## ## Envisage is free software; you can redistribute it and/or modify ## it under the terms of the GNU General Public License as published by ## the Free Software Foundation; either version 2 of the License, or ## (at your option) any later version. ## ## Envisage is distributed in the hope that it will be useful, ## but WITHOUT ANY WARRANTY; without even the implied warranty of ## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the ## GNU General Public License for more details. ## ## You should have received a copy of the GNU General Public License ## along with Envisage; if not, write to the Free Software ## Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA ############################################################################### # BEGIN - FUNCTION - modelEntry # ############################################################################### ## Allows users to specify a minimum model for the model selection process modelEntry <- function(pData, Mentry, useParams, labParams, MEorINT, glm.formula) { ########################################################################### # 1 - GUI # ########################################################################### if (!missing(Mentry)) { ## Original variable names before removal of labelling, etc. orgParamNames <- names(pData) nOrgParam <- length(orgParamNames) ## Create starting model by pulling out the labels from the model ## that the user has supplied Minput <- tclvalue(tkget(Mentry)) if (nchar(Minput) == 0) { startingModel <- "x ~ 1" intPos <- NULL } else if (nchar(Minput) != 0) { ## Find the positions of the "+" characters plusPos <- NULL for (i in 1:nchar(Minput)) { if (substr(Minput, i, i) == "+") { plusPos <- c(plusPos, i) } } rm(i) ## Seperate out the terms MtermsP <- list() if (length(plusPos) != 0) { count <- 1 for (i in 1:length(plusPos)) { MtermsP[[i]] <- substr(Minput, count, (plusPos[i]-1)) count <- plusPos[i]+1 } MtermsP[[length(plusPos)+1]] <- substr(Minput, (plusPos[length(plusPos)]+1), nchar(Minput)) } else if (length(plusPos) == 0) { MtermsP[[1]] <- Minput } ## Find the interaction terms intPos <- NULL for (i in 1:length(MtermsP)) { for (j in 1:nchar(MtermsP[[i]])) { if (substr(MtermsP[[i]], j, j) == ":") { intPos <- c(intPos, i) } } } ## Pull out the values for the terms in the main model Mlabels <- list() for (i in 1:length(MtermsP)) { ## If not an interaction term if (!i %in% intPos) { for (j in 1:nchar(MtermsP[[i]])) { if ((substr(MtermsP[[i]], j, j)) %in% 0:9) { Mlabels[[i]] <- as.numeric(substr(MtermsP[[i]], j, j)) if ((substr(MtermsP[[i]], j, j+1)) %in% 0:99) { Mlabels[[i]] <- as.numeric(substr(MtermsP[[i]], j, j+1)) break } else break } } ## If the term is an interaction } else if (i %in% intPos) { colPos <- NULL for (j in 1:nchar(MtermsP[[i]])) { if (substr(MtermsP[[i]], j, j) == ":") { colPos <- j } } Mlabels[[i]] <- vector(length=2) for (j in 1:colPos) { if ((substr(MtermsP[[i]], j, j)) %in% 0:9) { Mlabels[[i]][1] <- as.numeric(substr(MtermsP[[i]], j, j)) if ((substr(MtermsP[[i]], j, j+1)) %in% 0:99) { Mlabels[[i]][1] <- as.numeric(substr(MtermsP[[i]], j, j+1)) break } else break } } for (j in colPos:nchar(MtermsP[[i]])) { if ((substr(MtermsP[[i]], j, j)) %in% 0:9) { Mlabels[[i]][2] <- as.numeric(substr(MtermsP[[i]], j, j)) if ((substr(MtermsP[[i]], j, j+1)) %in% 0:99) { Mlabels[[i]][2] <- as.numeric(substr(MtermsP[[i]], j, j+1)) break } else break } } } } ## Convert the numbers into variable names Mnames <- Mlabels for (i in 1:length(Mlabels)) { for (j in 1:length(Mlabels[[i]])) { if (Mlabels[[i]][j] > nOrgParam) { tkmessageBox(title="Model terms out of bounds", message=paste("Warning: Please ensure that the terms ", "specificed in\nthe starting model are in the range p1 ", "to p",nOrgParam, ".", sep=""), type="ok", icon="warning") ## Stop if a problem stop() } else Mnames[[i]][j] <- gsub(" ", "_", orgParamNames[Mlabels[[i]][j]]) } } ## Check that the variables to be used in the model have been selected ## by the user for (i in 1:length(Mnames)) { for (j in 1:length(Mnames[[i]])) { if (!(gsub("_", " ", Mnames[[i]][j]) %in% useParams)) { if (gsub("_", " ", Mnames[[i]][j]) %in% labParams) { tkmessageBox(title=paste("Selected labelling variables", " as model terms", sep=""), message=paste("You have", " selected variable '", gsub("_", " ", Mnames[[i]][j]) ,"' as a variable for the ", "starting\nmodel, which has been flagged as a ", "labelling variable. It is\nrecommended that you ", "do not use this variable in your model\nto ", "prevent overfitting.", sep=""), type="ok", icon="warning") ## Stop if a problem stop() } else if (!(gsub("_", " ", Mnames[[i]][j]) %in% labParams)) { tkmessageBox(title="Model terms not selected", message=paste("You have selected variable '", gsub("_", " ", Mnames[[i]][j]) ,"' as a variable ", "for\nthe starting model. Please ensure that you ", "have also ticked\nthe 'Choose Model Variable' box", " for this variable.", sep=""), type="ok", icon="warning") ## Stop if a problem stop() } } else if (gsub("_", " ", Mnames[[i]][j]) %in% useParams) { ## Create starting model from the selected terms startingModel <- "x ~ " for (i in 1:length(Mlabels)) { ## For main effect terms if (length(Mlabels[[i]]) == 1) { startingModel <- paste(startingModel, Mnames[[i]], " + ", sep="") } else if (length(Mlabels[[i]]) == 2) { startingModel <- paste(startingModel, Mnames[[i]][1], ":", Mnames[[i]][2], " + ", sep="") } } startingModel <- substr(startingModel, 1, nchar(startingModel)-3) } } } } terms <- NULL } ########################################################################### # 1 - Command line # ########################################################################### if(!missing(glm.formula)) { ## Find tilda character tilPos <- NULL for (i in 1:nchar(glm.formula)) { if (substr(glm.formula, i, i) == "~") { tilPos <- c(tilPos, i) } } ## Start of terms for (i in (tilPos+1):nchar(glm.formula)) { if (substr(glm.formula, i, i) != " ") { termStart <- i break } } ## Find plus characters plusPos <- NULL for (i in 1:nchar(glm.formula)) { if (substr(glm.formula, i, i) == "+") { plusPos <- c(plusPos, i) } } plusPos <- c(plusPos, (nchar(glm.formula)+2)) ## Find interactions intPos <- NULL for (i in 1:nchar(glm.formula)) { if (substr(glm.formula, i, i) == ":") { intPos <- c(intPos, i) } } ## Find model terms if (length(plusPos) > 0) { terms <- NULL count <- termStart for (i in 1:length(plusPos)) { terms <- c(terms, substr(glm.formula, count, (plusPos[i]-2))) count <- plusPos[i]+2 } } ## Create starting glm.formula startingModel <- "x ~" for (i in 1:length(terms)) { temp <- ifelse(substr(terms[i],1,1) != "'", terms[i], paste("'", terms[i], sep="")) temp <- ifelse(substr(temp, nchar(temp), nchar(temp)) != "'", temp, paste(temp, "'", sep="")) temp <- gsub(" ", "_", temp) startingModel <- paste(startingModel, temp, "+") } startingModel <- substr(startingModel, 1, (nchar(startingModel)-2)) startingModel <- as.formula(startingModel) } out <- list(startingModel=startingModel, intPos=intPos) return(out) } ############################################################################### # END - FUNCTION - modelEntry # ###############################################################################Envisage/R/mulTestCor.R0000755003020100016370000000556211031122552015112 0ustar sr9signg00000000000000## Copyright (C) 2007 Sam Robson ## ## This file is part of Envisage. ## ## Envisage is free software; you can redistribute it and/or modify ## it under the terms of the GNU General Public License as published by ## the Free Software Foundation; either version 2 of the License, or ## (at your option) any later version. ## ## Envisage is distributed in the hope that it will be useful, ## but WITHOUT ANY WARRANTY; without even the implied warranty of ## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the ## GNU General Public License for more details. ## ## You should have received a copy of the GNU General Public License ## along with Envisage; if not, write to the Free Software ## Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA ############################################################################### # BEGIN - FUNCTION - mulTestCor # ############################################################################### ## Use specified multiple testing correction to correct p values for multiple ## hypothesis testing mulTestCor <- function(pValues, MTC, pData, exprData, param4INT) { ## Create names of all possible main effect and first order ## interaction terms paramInt <- gsub(" ", "_", names(pData)) if (length(param4INT) != 0) { for (i in 1:length(param4INT)) { for (j in 1:length(param4INT)) { if (param4INT[j] != param4INT[i]) { paramInt <- c(paramInt, gsub(" ", "_", paste(param4INT[i], ":", param4INT[j], sep=""))) } } } rm(i, j) } nInt <- length(paramInt) ## Create matrix in which to store all p-value results for MTC allPVal <- as.data.frame(matrix(data=NA, nrow=nrow(exprData), ncol=nInt, dimnames=list(rownames(exprData), paramInt))) for (i in 1:nrow(exprData)) { if (!is.na(pValues[[i]])[1]) { for (j in 1:length(pValues[[i]])) { allPVal[[names(pValues[[i]])[j]]][i] <- pValues[[i]][j] } } } rm(i,j) ## MTC across all genes if (MTC != "None") { adjp <- as.data.frame(matrix(data=NA, nrow=nrow(exprData), ncol=nInt, dimnames=list(rownames(exprData), paramInt))) for (i in 1:nInt) { rawp <- allPVal[[i]] names(rawp) <- rownames(exprData) temp <- mt.rawp2adjp(rawp, proc=MTC) adjp[temp$index, i] <- temp$adjp[, 2] } rm(i) } else adjp <- allPVal return(adjp) } ############################################################################### # END - FUNCTION - mulTestCor # ###############################################################################Envisage/R/coef.glmNA.R0000755003020100016370000000272411031122552014717 0ustar sr9signg00000000000000## Copyright (C) 2007 Sam Robson ## ## This file is part of Envisage. ## ## Envisage is free software; you can redistribute it and/or modify ## it under the terms of the GNU General Public License as published by ## the Free Software Foundation; either version 2 of the License, or ## (at your option) any later version. ## ## Envisage is distributed in the hope that it will be useful, ## but WITHOUT ANY WARRANTY; without even the implied warranty of ## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the ## GNU General Public License for more details. ## ## You should have received a copy of the GNU General Public License ## along with Envisage; if not, write to the Free Software ## Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA ############################################################################### # BEGIN - FUNCTION - coef.glmNA # ############################################################################### ## Create function for a new class of glm objects - glmNA. This helps Anova to ## ignore the NA calls produced due to aliased variables coef.glmNA <- function(object, ...){na.omit(object$coefficients)} ############################################################################### # END - FUNCTION - coef.glmNA # ############################################################################### Envisage/R/modelCalcME.R0000755003020100016370000000747311031122552015121 0ustar sr9signg00000000000000## Copyright (C) 2007 Sam Robson ## ## This file is part of Envisage. ## ## Envisage is free software; you can redistribute it and/or modify ## it under the terms of the GNU General Public License as published by ## the Free Software Foundation; either version 2 of the License, or ## (at your option) any later version. ## ## Envisage is distributed in the hope that it will be useful, ## but WITHOUT ANY WARRANTY; without even the implied warranty of ## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the ## GNU General Public License for more details. ## ## You should have received a copy of the GNU General Public License ## along with Envisage; if not, write to the Free Software ## Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA ############################################################################### # BEGIN - FUNCTION - modelCalcME # ############################################################################### ## Function that calculates main effect linear models for each gene using a ## step-wise iterative process modelCalcME <- function(paramData, exprData, startingModel, progress) { errorGenes <- list() p <- list() failedAnova <- NULL if (!missing(progress)) tcl("update", "idletasks") for (i in 1:nrow(exprData)) { ## Model calculation - No interactions model <- NULL allData <- cbind(exprData[i,], paramData) colnames(allData) <- c("x", gsub(" ", "_", names(paramData))) genLin <- glm(x ~ ., data=allData, na.action=na.omit, family=gaussian) stepGenLin <- step(genLin, scope=list(upper=x ~ ., lower=startingModel), direction="backward", trace=0) model <- stepGenLin ## Information about aliasing error <- NULL temp <- alias(model)$Complete if (length(temp) > 0) { for (j in 1:nrow(temp)) { for (k in 1:ncol(temp)) { if (abs(temp[j,k]) > 0) { error <- c(error, paste(rownames(temp)[j], " & ", colnames(temp)[k], sep="")) } } } if (length(error) > 0) { errorGenes[[i]] <- error class(model) <- c("glmNA", "glm", "lm") } else if (length(error) == 0) { errorGenes[[i]] <- NA class(model) <- c("glmNA", "glm", "lm") } } else errorGenes[[i]] <- NA names(errorGenes)[i] <- rownames(exprData)[i] ## Uses a type II anova method to calculate p values for each variable ## and interaction for each gene anovaResults <- NULL if (length(model$coefficients) > 1) { anovaResults <- Anova(model,type="II",test="F") temp <- anovaResults$'Pr(>F)' if (!all(is.na(temp))) { p[[i]] <- temp[1:length(temp)-1] names(p[[i]]) <- rownames(anovaResults)[1:length(temp)-1] } else if (all(is.na(temp))) { p[[i]] <- NA failedAnova <- c(failedAnova, rownames(exprData)[i]) } } else p[[i]] <- NA ## Update progress report if (!missing(progress)) { tclvalue(progress) <- i tcl("update", "idletasks") } } rm(i) names(p) <- rownames(exprData) out <- list(pValues=p, failedAnova=failedAnova, errorGenes=errorGenes) return(out) } ############################################################################### # END - FUNCTION - modelCalcME # ############################################################################### Envisage/R/dataTransform.R0000755003020100016370000000401511031122552015606 0ustar sr9signg00000000000000## Copyright (C) 2007 Sam Robson ## ## This file is part of Envisage. ## ## Envisage is free software; you can redistribute it and/or modify ## it under the terms of the GNU General Public License as published by ## the Free Software Foundation; either version 2 of the License, or ## (at your option) any later version. ## ## Envisage is distributed in the hope that it will be useful, ## but WITHOUT ANY WARRANTY; without even the implied warranty of ## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the ## GNU General Public License for more details. ## ## You should have received a copy of the GNU General Public License ## along with envisage; if not, write to the Free Software ## Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA ############################################################################### # BEGIN - FUNCTION - dataTransform.R # ############################################################################### ## Extract expression values from ExpressionSet object, and ensure that data is ## logged before analysis dataTransform <- function(expData) { ## Read in gene expression values 'd', and exclude missing values for future ## calculations d <- exprs(expData) d <- na.exclude(d) ## Log d if it has not been logged previously (if d has been previously ## logged it will be normalized around 0 instead of 1 (log1 = 0), and so we ## will see negative gene expression values) nonZero <- 0 for (i in nrow(d):1) { for (j in 1:ncol(d)) { if (d[i,j] <= 0) { nonZero <- nonZero + 1 } } } if (nonZero == 0) { d <- log2(d) } return(d) } ############################################################################### # END - FUNCTION - dataTransform.R # ###############################################################################Envisage/R/GS2BC.R0000755003020100016370000000503111031122552013600 0ustar sr9signg00000000000000## Copyright (C) 2007 Sam Robson ## ## This file is part of Envisage. ## ## Envisage is free software; you can redistribute it and/or modify ## it under the terms of the GNU General Public License as published by ## the Free Software Foundation; either version 2 of the License, or ## (at your option) any later version. ## ## Envisage is distributed in the hope that it will be useful, ## but WITHOUT ANY WARRANTY; without even the implied warranty of ## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the ## GNU General Public License for more details. ## ## You should have received a copy of the GNU General Public License ## along with Envisage; if not, write to the Free Software ## Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA ############################################################################### # BEGIN - FUNCTION - GS2BC # ############################################################################### ## Slighlty altered version of function GSint2BC from package GeneSpring. ## Creates an object of class ExpressionSet, the updated microarray data class ## exprSet. GS2BC <- function(interpretation, what = "nor") { pd <- as.data.frame(t(interpretation@expparam)) row.names(pd) <- c(1:length(row.names(pd))) vl <- as.list(names(pd)) names(vl) <- names(pd) if (what == "nor") { exprs <- interpretation@a.nor se.exprs <- interpretation@se.nor } else if (what == "raw") { exprs <- interpretation@a.raw se.exprs <- interpretation@se.raw } else stop(paste("Unknown value for variable \"what\": ", what, " (Supported values are \"raw\" and \"nor\" for the Raw and Normalized", " values respectively")) row.names(exprs) <- interpretation@genenames if (length(se.exprs) != 0) { row.names(se.exprs) <- interpretation@genenames } metData <- NULL for (i in 1:length(vl)) { metData <- c(metData, vl[[i]]) } metData <- as.data.frame(metData) rownames(metData) <- names(vl) colnames(metData) <- "labelDescription" pdata <- new("AnnotatedDataFrame", data=pd, varMetadata=metData) new("ExpressionSet", phenoData=pdata, exprs=exprs) } ############################################################################### # END - FUNCTION - GS2BC # ###############################################################################Envisage/R/result.output.R0000755003020100016370000001532511031122552015664 0ustar sr9signg00000000000000## Copyright (C) 2007 Sam Robson ## ## This file is part of Envisage. ## ## Envisage is free software; you can redistribute it and/or modify ## it under the terms of the GNU General Public License as published by ## the Free Software Foundation; either version 2 of the License, or ## (at your option) any later version. ## ## Envisage is distributed in the hope that it will be useful, ## but WITHOUT ANY WARRANTY; without even the implied warranty of ## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the ## GNU General Public License for more details. ## ## You should have received a copy of the GNU General Public License ## along with Envisage; if not, write to the Free Software ## Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA ############################################################################### # BEGIN - FUNCTION - result.output # ############################################################################### ## Function that takes the corrected p-Values from the model calculations to ## discern genes that change significantly across the variables of interest, ## then outputs them to the user result.output <- function(adjp, pCutoff, pData, exprData, errorGenes, fileResults, MEorINT, param4INT, failedAnova) { if (MEorINT == "INT") { ## All possible interactions with the variables of interest outputNames <- gsub(" ", "_", names(pData)) if (length(param4INT) > 1) { for (i in 1:length(param4INT)) { for (j in 1:length(param4INT)) { if (param4INT[j] != param4INT[i]) { outputNames <- c(outputNames, gsub(" ", "_", paste(param4INT[i], ":", param4INT[j], sep=""))) } } rm(j) } rm(i) } } else outputNames <- gsub(" ", "_", names(pData)) ## Pulls out genes whose p values are within the cutoff limit results <- list() for (i in 1:length(outputNames)) { tempP <- NULL tempN <- NULL for (j in 1:nrow(exprData)) { if (!is.na(adjp[[outputNames[i]]][j])) { if (adjp[[outputNames[i]]][j] <= pCutoff) { tempP <- c(tempP, adjp[[outputNames[i]]][j]) tempN <- c(tempN, rownames(exprData)[j]) } } } rm(j) if (length(tempP) != 0) { names(tempP) <- tempN results[[i]] <- tempP } else results[[i]] <- NA names(results)[i] <- outputNames[i] } rm(i) ## Get rid of NA results list <- NULL for (i in 1:length(results)) { if (!is.na(results[i])) list <- c(list,i) } rm(i) results <- results[list] ## Create a directory in the working directory to store resulting ## gene lists try(dir.create(paste(fileResults, "/EnvisageResults", sep=""), showWarnings=F)) ## If results folder is non-empty, rename all old files dirs <- dir(paste(fileResults, "/EnvisageResults", sep="")) if (length(dirs) > 0) { for (i in 1:length(dirs)) { temp <- paste(substr(dirs[i],1,nchar(dirs[i])-4), "_OLD.txt", sep="") file.rename(paste(fileResults, "/EnvisageResults/", dirs[i], sep=""), paste(fileResults, "/EnvisageResults/", temp, sep="")) } } ## Write genelists to tab delimited text files in the new directory if (length(results) > 0) { Pnames <- gsub(" ","_",names(results)) Pnames <- gsub(":","_INT_",Pnames) for (i in 1:(length(results))) { if (file.exists(paste(fileResults, "/EnvisageResults/", Pnames[i],".txt",sep=""))) { file.remove(paste(fileResults, "/EnvisageResults/", Pnames[i],".txt",sep="")) } write.table(results[[i]],file=paste(fileResults,"/EnvisageResults/", Pnames[i],".txt",sep=""), append=F, row.names=T, col.names=FALSE, quote=FALSE, sep="\t") } rm(i) } else if (length(results) == 0) { warning <- tkmessageBox(message=paste("Warning: No genes found to ", "be significant.", sep=""), icon="warning", type="ok") } ## Output information for error genes list <- NULL for (i in 1:nrow(exprData)) { if (!is.na(errorGenes[[i]][1])) list <- c(list, i) } rm(i) if (length(list) > 0) { errorGenesShort <- errorGenes[list] ## Write gene list for errors if (file.exists(paste(fileResults, "/EnvisageResults/AliasGenes.txt", sep=""))) { file.remove(paste(fileResults, "/EnvisageResults/AliasGenes.txt", sep="")) } write.table(names(errorGenesShort),file=paste(fileResults, "/EnvisageResults/AliasGenes.txt", sep=""), append=F, row.names=F, col.names=FALSE, quote=FALSE, sep="\t") ## Write error report if (file.exists(paste(fileResults, "/EnvisageResults/AliasReport.txt", sep=""))) { file.remove(paste(fileResults, "/EnvisageResults/AliasReport.txt", sep="")) } for (i in 1:length(errorGenesShort)) { write(names(errorGenesShort)[i], file=paste(fileResults, "/EnvisageResults/AliasReport.txt", sep=""), append=T) write("\nAliasing is seen across the following conditions:", file=paste(fileResults, "/EnvisageResults/AliasReport.txt", sep=""), append=T) for (j in 1:length(errorGenesShort[[i]])) { write(errorGenesShort[[i]][j], file=paste(fileResults, "/EnvisageResults/AliasReport.txt", sep=""), append=T) } write("\n\n", file=paste(fileResults, "/EnvisageResults/AliasReport.txt", sep=""), append=T) } } ## Write gene list for genes that fail the Anova if (length(failedAnova) > 0) { try(file.remove(paste(fileResults, "/EnvisageResults/Failed.txt", sep=""))) write.table(failedAnova,file=paste(fileResults, "/EnvisageResults/Failed.txt", sep=""), append=F, row.names=F, col.names=FALSE, quote=FALSE, sep="\t") } return(results) } ############################################################################### # END - FUNCTION - result.output # ###############################################################################Envisage/R/INTlevels.R0000755003020100016370000000540611031122552014653 0ustar sr9signg00000000000000## Copyright (C) 2007 Sam Robson ## ## This file is part of Envisage. ## ## Envisage is free software; you can redistribute it and/or modify ## it under the terms of the GNU General Public License as published by ## the Free Software Foundation; either version 2 of the License, or ## (at your option) any later version. ## ## Envisage is distributed in the hope that it will be useful, ## but WITHOUT ANY WARRANTY; without even the implied warranty of ## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the ## GNU General Public License for more details. ## ## You should have received a copy of the GNU General Public License ## along with Envisage; if not, write to the Free Software ## Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA ############################################################################### # BEGIN - FUNCTION - INTlevels # ############################################################################### ## Function to calculate the number of levels for each main effect variable ## term and first order interaction term in the linear model INTlevels <- function(pData) { ## Number of levels for each main effect term MElevels <- NULL for (i in 1:ncol(pData)) { if (class(pData[[i]]) == "factor" && length(levels(pData[[i]])) > 1) { MElevels <- c(MElevels, (length(levels(pData[[i]]))-1)) } else MElevels <- c(MElevels, 1) } names(MElevels) <- names(pData) ## All possible interactions with the variables of interest allINT <- gsub(" ", "_", names(pData)) allINTlevels <- MElevels for (i in 1:length(pData)) { for (j in 1:length(pData)) { if (j > i && names(pData)[j] != names(pData)[i]) { allINT <- c(allINT, gsub(" ", "_", paste(names(pData)[i], ":", names(pData)[j], sep=""))) if (class(pData[[i]]) == "factor" && length(levels(pData[[i]])) > 1) { temp1 <- length(levels(pData[[i]])) } else temp1 <- 1 if (class(pData[[j]]) == "factor" && length(levels(pData[[j]])) > 1) { temp2 <- length(levels(pData[[j]])) } else temp2 <- 1 allINTlevels <- c(allINTlevels, ((temp1*temp2)-1)) } } rm(j) } rm(i) names(allINTlevels) <- allINT return(allINTlevels) } ############################################################################### # END - FUNCTION - INTlevels # ############################################################################### Envisage/R/corParams.R0000755003020100016370000000574711031122552014745 0ustar sr9signg00000000000000## Copyright (C) 2007 Sam Robson ## ## This file is part of Envisage. ## ## Envisage is free software; you can redistribute it and/or modify ## it under the terms of the GNU General Public License as published by ## the Free Software Foundation; either version 2 of the License, or ## (at your option) any later version. ## ## Envisage is distributed in the hope that it will be useful, ## but WITHOUT ANY WARRANTY; without even the implied warranty of ## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the ## GNU General Public License for more details. ## ## You should have received a copy of the GNU General Public License ## along with Envisage; if not, write to the Free Software ## Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA ############################################################################### # BEGIN - FUNCTION - corParams # ############################################################################### ## Flags all highly correlated variables which model the same data to reduce ## the number of variables and interactions in the model calculation corParams <- function(pData) { list <- NULL paramCov <- matrix(ncol=ncol(pData),nrow=ncol(pData)) rownames(paramCov) <- gsub(" ", "_", names(pData)) colnames(paramCov) <- gsub(" ", "_", names(pData)) switch <- 0 flag <- ("*") corParams <- list() count <- 1 corParamNames <- names(pData) ## Go through all variables to find high correlation, and flag any ## variables with correlation higher than 0.98 for (i in 1:length(corParamNames)) { if (switch == 1) { flag <- paste(flag,"*",sep="") switch <- 0 } if (count == 1 || (count > 1 && !(i %in% corParams[[count-1]]))) { for (j in 1:length(corParamNames)) { if (i= 0.98) { if (substr(corParamNames[i],nchar(corParamNames[i]), nchar(corParamNames[i])) != "*") { corParamNames[i] <- paste(corParamNames[i],flag,sep=" ") corParams[[count]] <- i } if (substr(corParamNames[j],nchar(corParamNames[j]), nchar(corParamNames[j])) != "*") { corParamNames[j] <- paste(corParamNames[j],flag,sep=" ") corParams[[count]] <- c(corParams[[count]],j) } switch <- 1 } } } if (switch == 1) count <- count + 1 } rm(i,j) return(list(corParamNames,corParams)) } ############################################################################### # END - FUNCTION - corParams # ###############################################################################Envisage/R/modelCalcINT.R0000755003020100016370000001351611031122552015245 0ustar sr9signg00000000000000## Copyright (C) 2007 Sam Robson ## ## This file is part of Envisage. ## ## Envisage is free software; you can redistribute it and/or modify ## it under the terms of the GNU General Public License as published by ## the Free Software Foundation; either version 2 of the License, or ## (at your option) any later version. ## ## Envisage is distributed in the hope that it will be useful, ## but WITHOUT ANY WARRANTY; without even the implied warranty of ## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the ## GNU General Public License for more details. ## ## You should have received a copy of the GNU General Public License ## along with Envisage; if not, write to the Free Software ## Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA ############################################################################### # BEGIN - FUNCTION - modelCalcINT # ############################################################################### ## Function that calculates main effect linear models using a step-wise ## iterative process, calculates first order interaction models if necessary, ## then uses a type II Anova calculation to test for significance modelCalcINT <- function(paramData, exprData, param4INT, startingModel, progress) { errorGenes <- list() p <- list() failedAnova <- NULL if (!missing(progress)) tcl("update", "idletasks") for (i in 1:nrow(exprData)) { ## Model calculation - First order interactions model <- NULL allData <- cbind(exprData[i,], paramData) colnames(allData) <- c("x", gsub(" ", "_", names(paramData))) ## If all variables are to be used for interactions if (length(param4INT) == length(paramData)) { genLin <- glm(x ~ .^2, data=allData, na.action=na.omit, family=gaussian) stepGenLin <- step(genLin, scope=list(lower=startingModel, upper="x ~ .^2"), direction="backward", trace=0) model <- stepGenLin } else if (length(param4INT) != length(paramData)) { ## If only some of the variables are to be used for ## interactions MEterms <- NULL INTterms <- NULL for (j in 1:length(paramData)) { if (names(paramData)[j] %in% param4INT) { INTterms <- c(INTterms, names(paramData)[j]) } else MEterms <- c(MEterms, names(paramData)[j]) } ## Build main effect terms into the formula formulaMain <- "x ~ " if (length(MEterms) > 0) { for (j in 1:length(MEterms)) { formulaMain <- paste(formulaMain, gsub(" ", "_", MEterms[j]), " + ", sep="") } rm(j) } ## Build terms into formula for interactions if (length(INTterms) > 0) { formulaMain <- paste(formulaMain, "(", sep="") for (j in 1:length(INTterms)) { formulaMain <- paste(formulaMain, gsub(" ", "_", INTterms[j]), " + ", sep="") } rm(j) } formulaMain <- substr(formulaMain, 1, (nchar(formulaMain)-3)) if (length(INTterms) > 0) formulaMain <- paste(formulaMain, ")^2", sep="") formulaMain <- as.formula(formulaMain) ## Stepwise modelling genLin <- glm(formulaMain, data=allData, na.action=na.omit, family=gaussian) stepGenLin <- step(genLin, scope=list(lower=startingModel, upper=formulaMain), direction="backward", trace=0) model <- stepGenLin } ## Information about aliasing error <- NULL temp <- alias(model)$Complete if (length(temp) > 0) { for (j in 1:nrow(temp)) { for (k in 1:ncol(temp)) { if (abs(temp[j,k]) > 0) { error <- c(error, paste(rownames(temp)[j], " & ", colnames(temp)[k], sep="")) } } } if (length(error) > 0) { errorGenes[[i]] <- error class(model) <- c("glmNA", "glm", "lm") } else if (length(error) == 0) { errorGenes[[i]] <- NA class(model) <- c("glmNA", "glm", "lm") } } else errorGenes[[i]] <- NA names(errorGenes)[i] <- rownames(exprData)[i] ## Uses a type II anova method to calculate p values for each variable ## and interaction for each gene anovaResults <- NULL if (length(model$coefficients) > 1) { anovaResults <- Anova(model,type="II",test="F") temp <- anovaResults$'Pr(>F)' if (!all(is.na(temp))) { p[[i]] <- temp[1:length(temp)-1] names(p[[i]]) <- rownames(anovaResults)[1:length(temp)-1] } else if (all(is.na(temp))) { p[[i]] <- NA failedAnova <- c(failedAnova, rownames(exprData)[i]) } } else p[[i]] <- NA ## Update progress report if (!missing(progress)) { tclvalue(progress) <- i tcl("update", "idletasks") } } rm(i) names(p) <- rownames(exprData) out <- list(pValues=p, failedAnova=failedAnova, errorGenes=errorGenes) return(out) } ############################################################################### # END - FUNCTION - modelCalcINT # ###############################################################################Envisage/R/envisage.R0000755003020100016370000023130311031122552014604 0ustar sr9signg00000000000000## Copyright (C) 2007 Sam Robson ## ## This file is part of Envisage. ## ## Envisage is free software; you can redistribute it and/or modify ## it under the terms of the GNU General Public License as published by ## the Free Software Foundation; either version 2 of the License, or ## (at your option) any later version. ## ## Envisage is distributed in the hope that it will be useful, ## but WITHOUT ANY WARRANTY; without even the implied warranty of ## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the ## GNU General Public License for more details. ## ## You should have received a copy of the GNU General Public License ## along with Envisage; if not, write to the Free Software ## Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA ############################################################################### ############################################################################### ## BEGIN - FUNCTION - envisage ## ############################################################################### ############################################################################### envisage <- function(expData, widget=TRUE, MTC=NULL, pCutoff=NULL, useParams=NULL, param4INT=NULL, paramType=NULL, fileResults=getwd(), MEorINT="INT", startModel=NULL) { ############################################################################### # 1 - GETTING THE DATA IN # ############################################################################### ## Check widget argument specified correctly if (!widget %in% c(TRUE, FALSE)) { stop("Please ensure that argument 'widget' is TRUE or FALSE") } ## Extract gene names and number of genes in the experiment gNames <- featureNames(expData) nGenes <- length(gNames) ## Extract sample names and number of samples in the experiment sNames <- sampleNames(expData) nSamples <- length(sNames) ## Extract the variable information paramData <- pData(expData) startParamNames <- colnames(paramData) paramNames <- startParamNames startNParam <- length(startParamNames) nParam <- startNParam ## Ensure that any variable levels labelled "NA" by the user are ## not considered to be missing data paramMatrix <- as.matrix(paramData) for (i in 1:nSamples) { for (j in 1:nParam) { if(is.na(paramMatrix[i,j])) paramMatrix[i,j] <- as.character("NAN") } } rm(i,j) paramData <- as.data.frame(paramMatrix) ## Extract gene expression values from data object d <- dataTransform(expData) ############################################################################### # 2 - REMOVING UNWANTED VARIABLES # ############################################################################### ## Flag any labelling variables labParams <- paramNames[labelParams(paramData)] ## Remove variables with a constant value constParam <- NULL for (i in 1:nParam) { if (length(levels(paramData[[i]])) == 1) { constParam <- c(constParam, i) } } rm(i) if (length(constParam) != 0) { paramData <- paramData[, -constParam] } nParam <- length(paramData) paramNames <- names(paramData) ## Avoid problems with R objects whose name begins with a number by prefixing ## the name with the character "X" for (i in 1:nParam) { if (substr(names(paramData)[i], 1, 1) %in% 0:9) { names(paramData)[i] <- paste("X", names(paramData)[i], sep="") } } rm(i) ## Ensure variable names do not contain "-" or "\" or "/" by removing them names(paramData) <- gsub("-", "_", names(paramData)) names(paramData) <- gsub("/", "_", names(paramData)) names(paramData) <- gsub(" ", "_", names(paramData)) ## Extract adjusted variable names paramNames <- names(paramData) ## Flag any highly correlated variables cor <- corParams(paramData) corParamNames <- cor[[1]] corParams <- cor[[2]] ## If widget selected if (widget==TRUE) { ############################################################################### # 3 - LAUNCH GUI # ############################################################################### ## Load up additional tcl/tk packages tclRequire("BWidget") tclRequire("Iwidgets") ## Specify fonts fontLabelsL <- tkfont.create(family="times", size=14, weight="bold") fontLabels <- tkfont.create(family="times", size=12, weight="bold") fontLabelsSmall <- tkfont.create(family="times", size=10, weight="bold") fontTiny <- tkfont.create(family="times", size=3, weight="bold") fontNames <- tkfont.create(family="times", size=12) fontHeader <- tkfont.create(family="times", size=26, weight="bold") ## Set up top level frame environment tt <- tktoplevel() tktitle(tt) <- "Envisage GUI" ## Main GUI frame frameOverall <- tkframe(tt) ## Frame for GUI title header frameHeader <- tkframe(frameOverall, relief="flat", borderwidth=2) tkgrid(tklabel(frameHeader, text="Envisage", font=fontHeader)) tkgrid(tklabel(frameHeader, text=paste("Enable Numerous Variables ", "In Significance Analysis of Gene Expression", sep=""), font=fontLabels)) tkgrid(frameHeader, columnspan=2) ## Frame for selecting variables and specifying their class (numeric ## or categoric) frameParam <- tkwidget(frameOverall, "iwidgets::labeledframe", labelpos="nw", labeltext="Select Variable Classes for Modelling") ## Column headings titleFrame <- tkframe(frameParam, borderwidth=2, width=400) labNone <- tklabel(titleFrame, text="Exclude", font=fontLabelsSmall, width=8) labCat <- tklabel(titleFrame, text="Numeric", font=fontLabelsSmall, width=7) labNum <- tklabel(titleFrame, text="Categoric", font=fontLabelsSmall, width=8) blankLab <- tklabel(titleFrame, text=" ", width=30) tkgrid(blankLab, labNone, labCat, labNum) tkgrid(titleFrame) ## Scrollable frame for variable selection sw <- tkwidget(frameParam, "ScrolledWindow", relief="sunken", borderwidth=2,bg="white") sf <- tkwidget(sw, "ScrollableFrame", width=400, height=200) tcl(sw, "setwidget",sf) subfID <- tclvalue(tcl(sf, "getframe")) usePValue <- list() paramLabel <- list() rbValue <- list() radSel <- list() radCat <- list() radNum <- list() frameBox <- list() pLab <- list() ## Assign check boxes and radioboxes for each variable for (i in 1:nParam) { ## Frame for selection of individual variables frameBox[[i]] <- tcl("frame", paste(subfID, ".", i, sep=""), relief="flat", borderwidth=2) ## Give each variable a label pLab[[i]] <- tcl("label", paste(subfID, ".", i, ".1", sep=""), text=paste("p", i, sep=""), font=fontLabels, width=3) ## Variable name paramLabel[[i]] <- tcl("label", paste(subfID, ".", i, ".3", sep=""), text=corParamNames[i], font=fontLabels, width=20) ## Class radio buttons rbValue[[i]] <- tclVar("None") ## To select variables radSel[[i]] <- tcl("radiobutton", paste(subfID, ".", i, ".4", sep=""), width=5) tkconfigure(radSel[[i]], variable=rbValue[[i]], value="None") ## For numeric class radNum[[i]] <- tcl("radiobutton", paste(subfID, ".", i, ".5", sep=""), width=5) tkconfigure(radNum[[i]], variable=rbValue[[i]], value="numeric") ## For categoric class radCat[[i]] <- tcl("radiobutton", paste(subfID, ".", i, ".6", sep=""), width=5) tkconfigure(radCat[[i]], variable=rbValue[[i]], value="categoric") ## Combine tkgrid(pLab[[i]], paramLabel[[i]], radSel[[i]], radNum[[i]], radCat[[i]]) tkconfigure(paramLabel[[i]], justify="left") tkgrid(frameBox[[i]]) } tkgrid(sw) tkgrid(tklabel(frameParam, text=paste("Please Note: *, **, etc. ", "indicates that model variables are highly correlated. Please ensure\n", "that only one of each set is chosen before continuing.",sep=""), justify="left"), sticky="w") ## Function to reset selection of variable classes resetAll <- function() { for (i in 1:nParam) { rbValue[[i]] <<- tclVar("None") tkconfigure(radSel[[i]], variable=rbValue[[i]]) tkconfigure(radNum[[i]], variable=rbValue[[i]]) tkconfigure(radCat[[i]], variable=rbValue[[i]]) } } ## Allow user to check or clear all boxes in one go frameSelect <- tkframe(frameParam, relief="groove", borderwidth=2) resetBut <- tkbutton(frameSelect, text="Reset", command=resetAll, bg="grey") tkgrid(resetBut) tkgrid(frameSelect) ## Main frame for selecting arguments for modelling frameArg <- tkwidget(frameOverall, "iwidgets::labeledframe", labelpos="nw", labeltext="Model Arguments") ## Frame for selecting multiple testing correction frameMTC <- tkframe(frameArg, relief="groove", borderwidth=2) tkgrid(tklabel(frameMTC, text="Multiple Testing Correction", font=fontLabelsSmall), sticky="w") mtcorrect <- c("None", "Benjamini & Hochberg (FDR)", "Bonferroni (FWER)", "Benjamini & Yekutieli (FWER)", "Holm (FWER)", "Hochberg (FWER)", "SidakSS (FWER)", "SidakSD (FWER)") mtcSelect<-tklistbox(frameMTC, height=7, width=25, selectmode="single", background="white") tkgrid(mtcSelect) for (i in (1:7)) { tkinsert(mtcSelect, "end", mtcorrect[i]) } tkselection.set(mtcSelect, 1) tkgrid(frameMTC,sticky="nsew") ## Select whether to model using main effect terms or first order ## interactions frameMEorINT <- tkframe(frameArg, relief="groove", borderwidth=2) tkgrid(tklabel(frameMEorINT, text="Model Terms", font=fontLabelsSmall), columnspan=2, sticky="w") radME <- tkradiobutton(frameMEorINT) radINT <- tkradiobutton(frameMEorINT) rbValINT <- tclVar("INT") tkconfigure(radME, variable=rbValINT, value="ME") tkconfigure(radINT, variable=rbValINT, value="INT") tkgrid(tklabel(frameMEorINT, text="Main Effect Terms "), radME) tkgrid(tklabel(frameMEorINT, text="1st Order Interactions "), radINT) tkgrid(frameMEorINT,sticky="nsew") ## Allow user to specify a model to specify which variables to force ## into the model frameM <- tkframe(frameArg, relief="groove", borderwidth=2) tkgrid(tklabel(frameM, text="Starting Model", font=fontLabelsSmall), columnspan=2, sticky="w") Minput <- tclVar("") Mentry <- tkentry(frameM, textvariable=Minput) tkgrid(tklabel(frameM, text="x ~ ", font=fontLabels), Mentry) tkgrid(frameM, sticky="nsew") ## Specify the p value cutoff level for significance framePCutoff <- tkframe(frameArg, relief="groove", borderwidth=2) labelP <- tklabel(framePCutoff, text="P Value Cutoff", font=fontLabelsSmall) p <- tclVar("0.05") pEntry <- tkentry(framePCutoff, textvariable=p, width=25) tkgrid(labelP, sticky="w") tkgrid(pEntry, sticky="w") tkgrid(framePCutoff, sticky="nsew") ## Combine variable and argument selection frames tkgrid(frameParam,frameArg,sticky="nsew") ## Specify the file in which to save the results frameFileSelect <- tkwidget(frameOverall, "iwidgets::labeledframe", labelpos="nw", labeltext="Select folder in which to save results:") ## Browse for file to save results and update the entry box in ## the GUI browse <- function() { file <- tclVar(tkchooseDirectory()) tkconfigure(fileInput, textvariable=file) } file <- tclVar(as.character(getwd())) browseLabel <- tklabel(frameFileSelect, text="Folder Name:") browseBut <- tkbutton(frameFileSelect, text="Browse", command=browse) fileInput <- tkentry(frameFileSelect, width=80, textvariable=file) tkgrid(browseLabel, fileInput, browseBut, columnspan=2) tkgrid(frameFileSelect, sticky="nsew", columnspan=3) ## Progress report for analysis frameProgress <- tkframe(frameOverall, relief="groove", borderwidth=2, width=400) progress <- tclVar("0") progressLabel <- tklabel(frameProgress, textvariable = progress) progressLabel2 <- tkwidget(frameProgress, "ProgressBar", variable=progress, width=450, height=20, maximum=nGenes) tkgrid(tklabel(frameProgress, font=fontLabels, text="Progress:\t"), progressLabel, tklabel(frameProgress, text=paste(" of ", nGenes, " Genes\t", sep="")), progressLabel2) tkgrid(frameProgress, sticky="nsew", columnspan=3) ############################################################################### ############################################################################### ## ## ## Function - OnOK ## ## ## ############################################################################### ############################################################################### ## Function that runs when the OK button on the main GUI window is pressed OnOK <- function() { ## Switch on hourglass cursor hgOn() ########################################################################### # 4 - PULL OUT ARGUMENT VALUES # ########################################################################### ## Get selected multiple testing correction from GUI. If no MTC is ## selected, show a warning and go back to the GUI window mtcShort <- c("None", "BH", "BY", "Bonferroni", "Holm", "Hochberg", "SidakSS", "SidakSD") MTC <- mtcShort[as.numeric(tkcurselection(mtcSelect))+1] if (length(MTC) == 0) { tkmessageBox(title="No MTC Selected", message=paste("Please ensure that you have ", "selected a multiple testing\ncorrection from the list, ", "or select 'None' if no multiple testing\ncorrection is ", "required.", sep=""), type="ok", icon="warning") ## Hourglass cursor off hgOff() } ## Get selected P cutoff from GUI pCutoff <- as.numeric(tclvalue(p)) ## Ensure that p value is between 0 and 1 if (pCutoff < 0 || pCutoff > 1) { warning <- tkmessageBox(title="P Value Cutoff Out of Range", message="p value cutoff must be between 0 and 1.", icon="warning", type="ok") ## Hourglass cursor off hgOff() } ## Main effects or interactions MEorINT <- tclvalue(rbValINT) ## Specify which variables to use in the modelling, and specify the ## class of those variables useParams <- NULL paramType <- NULL nNone <- 0 for (i in 1:nParam) { if (tclvalue(rbValue[[i]]) != "None") { useParams <- c(useParams, paramNames[i]) } else nNone <- nNone + 1 paramType <- c(paramType, tclvalue(rbValue[[i]])) } ## If no variables selected as numeric or categoric, stop if (nNone == nParam) { tkmessageBox(title="No variables selected", message=paste("You have", "chosen to exclude all variables. Please select variables for", "analysis as 'Categoric' or 'Numeric'."), icon="warning", type="ok") ## Hourglass cursor off hgOff() } ## File to save results to fileResults <- tclvalue(tkget(fileInput)) ## Ensure that highly correlated variables are not selected if (length(corParams) != 0) { for (i in 1:length(corParams)) { count <- 0 for (j in 1:length(corParams[[i]])) { if (startParamNames[corParams[[i]][j]] %in% useParams) { count <- count +1 } } ## Warning message if highly correlated variables are ## selected for modelling if (count > 1) { message <- c("Warning: The variables:\n\n") for (j in 1:length(corParams[[i]])) { if (startParamNames[corParams[[i]][j]] %in% useParams) { message <- paste(message, "\t", startParamNames[corParams[[i]][j]], "\n", sep="") } } rm(j) message <- paste(message, "\nshow very high ", "correlation. Please ensure that only\none of ", "these is selected before continuing.", sep="") warning <- tkmessageBox(title=paste("Highly Correlated ", "Variables Selected", sep=""), message=message, icon="warning", type="ok") ## Hourglass cursor off hgOff() } } } ## Window showing the selected variable classes selVar <- NULL for (i in 1:length(paramType)) { if (paramType[i] == "categoric") { selVar <- paste(selVar, paramNames[i], " - categoric\n", sep="") } else if (paramType[i] == "numeric") { selVar <- paste(selVar, paramNames[i], " - numeric\n", sep="") } } vars <- tkmessageBox(title="Selected variable classes", message=paste("Would you like to continue with the following selected", " variables?\n\n", selVar, sep=""), type="yesno", icon="question") if (tclvalue(vars) == "yes") { ## Switch on hourglass cursor hgOn() ########################################################################### # 5 - ASSIGN CLASSES TO PARAMETERS # ########################################################################### ## Ensure that numerical variables are not stored as factors in the design ## matrix adjParamData <- paramData for (i in 1:nParam) { if (paramType[i] == "numeric") { adjParamData[[i]] <- as.numeric(as.character(paramData[[i]])) } } ########################################################################### # 6 - ENSURE THAT LABELLING PARAMETERS ARE REMOVED # ########################################################################### ## Ensure that no labelling variables are selected if (length(labParams) != 0) { warnLabParams <- NULL for (i in 1:length(labParams)) { if (labParams[i] %in% useParams && !is.numeric(adjParamData[[labParams[i]]])) { warnLabParams <- c(warnLabParams, labParams[i]) } } rm(i) ## Warn user about labelling variables if (length(warnLabParams) != 0) { message <- c("Warning: The variables:\n\n") ## List possible labelling variables for (i in 1:length(warnLabParams)) { message <- paste(message, "\t", warnLabParams[i], "\n", sep="") } rm(i) ## Warning message message <- paste(message,"\nhave unique values for each sample.\n\n", "If the above variables are numeric, please ensure that you ", "have\nselected the 'numeric' radio button.\n\nIf they are ", "labelling variables, then remove them completely from\nthe ", "modelling procedure to avoid overfitting.", sep="") warning <- tkmessageBox(title="Labelling Variables Selected", message=message, icon="warning", type="ok") ## Hourglass cursor off hgOff() } } ## Extract updated variable information paramNames <- useParams nParam <- length(paramNames) ## If only one variable chosen, treat as only main effect if (nParam == 1) MEorINT <- "ME" ## Remove unwanted variables from adjParamData adjParamData <- as.data.frame(adjParamData[, paramNames]) names(adjParamData) <- paramNames ## Create starting model for the analysis tempModel <- modelEntry(pData=paramData, Mentry=Mentry, useParams=useParams, labParams=labParams, MEorINT=MEorINT) startingModel <- as.formula(tempModel[[1]]) intPos <- tempModel[[2]] if (length(intPos) != 0 && MEorINT == "ME") { tkmessageBox(title="No interactions to calculate", message=paste("Warning: Your starting model contains ", "interaction terms,\nbut you have selected to only model ", "main effect terms.\nPlease either remove the interaction ", "terms from the starting\nmodel, or select '1st Order ", "Interactions'.", sep=""), type="ok", icon="warning") ## Hourglass cursor off ## Function that changes the cursor back to the default hgOff() } ########################################################################### # 7 - CHECK INTERACTION LEVELS # ########################################################################### ## This section calculates the number of levels in the model terms (first ## order interaction terms and main effects) and compares this to the number ## of samples. If there are more terms than samples, we may see overfitting ## of the data, so the user is given the option to select variables for ## which only main effect terms are required. It is recommended to do this ## with any variables with a large number of levels to avoid problems ## further down the line. param4INT <- NULL ########################################################################### # A - INT ONLY MODEL CALCULATION # ########################################################################### ## Calculate total number of factor levels for first order interactions if (MEorINT == "INT") { allLevels <- INTlevels(adjParamData) ## Allows you to select which variables to use for interactions tt2 <- tktoplevel() tktitle(tt2) <- "First order interaction selection" mainFrame2 <- tkframe(tt2) fontLab2 <- tkfont.create(family="times",size=12,weight="bold") frameTitle2 <- tkframe(mainFrame2) tkgrid(tklabel(frameTitle2, text="Variable\nName", width=15, font=fontLab2), tklabel(frameTitle2, text="Degrees of\nFreedom", width=10, font=fontLab2), tklabel(frameTitle2, text="Select\nVariable", width=10, font=fontLab2)) tkgrid(frameTitle2,sticky="w") ## Scrollable frame for variable selection sw2 <- tkwidget(mainFrame2,"ScrolledWindow", relief="sunken",borderwidth=2) sf3 <- tkwidget(sw2, "ScrollableFrame", width=320) tcl(sw2,"setwidget",sf3) subfID3 <- tclvalue(tcl(sf3,"getframe")) frameBox2 <- list() paramName2 <- list() outputBox2 <- list() outBoxValue2 <- list() levelInfo2 <- list() ## Create scrolled frame with all variables and interactions for (i in 1:length(adjParamData)) { frameBox2[[i]] <- tcl("frame", paste(subfID3,".",i,sep=""), relief="flat", borderwidth=2) ## Variable name paramName2[[i]] <- tcl("label", paste(subfID3,".",i,".1",sep=""), text=names(adjParamData)[i], width=20, justify="left") ## Levels for each variable levelInfo2[[i]] <- tcl("label", paste(subfID3,".",i,".2",sep=""), text=allLevels[i], width=15) ## Select output variables outputBox2[[i]] <- tcl("checkbutton", paste(subfID3, ".",i,".4",sep=""), width=10) tkconfigure(outputBox2[[i]], cursor="hand2") outBoxValue2[[i]] <- tclVar("1") tkconfigure(outputBox2[[i]], variable=outBoxValue2[[i]]) ## Combine tkgrid(paramName2[[i]], levelInfo2[[i]], outputBox2[[i]]) tkgrid(frameBox2[[i]]) } rm(i) tkgrid(sw2) ############################################################################### ## OnOK2 ## ############################################################################### OnOK2 <- function() { hgOn() options(show.error.messages=TRUE) param4INT <- NULL for (i in 1:length(adjParamData)) { if (tclvalue(outBoxValue2[[i]]) == "1") { param4INT <- c(param4INT,names(adjParamData)[i]) } } rm(i) ## If no variables selected for interactions, treat as main ## effects if (length(param4INT) == 0) { MEorINT <- "ME" } ## All selected variable and interaction terms allINT <- gsub(" ", "_", param4INT) if (length(param4INT) != 0) { for (i in 1:length(param4INT)) { for (j in 1:length(param4INT)) { if (j > i && param4INT[j] != param4INT[i]) { allINT <- c(allINT, gsub(" ", "_", paste(param4INT[i], ":", param4INT[j], sep=""))) } } } rm(i,j) ## All levels for chosen variable interactions allINTLevels <- NULL temp <- NULL for (i in 1:length(allINT)) { allINTLevels <- c(allINTLevels, as.numeric(allLevels[allINT[i]])) temp <- c(temp, names(allLevels[allINT[i]])) } rm(i) names(allINTLevels) <- temp } else allINTLevels <- allLevels[1:nParam] ## If still too many levels, try again if (sum(allINTLevels) > nrow(adjParamData)) { options(show.error.messages=FALSE) warning2 <- tkmessageBox(title="Danger of overfitting", message=paste("\tMaximum number of model df: ", sum(allLevels), "\n\t\tTotal df available: ", (nSamples-1), "\n\nThere are not enough samples to fit all of the model ", "terms\nand interactions. You may see overfitting of the ", "data which\nmay cause problems with the results.\n\nPress ", "'OK' to continue anyway, or press 'Cancel' to remove\n", "more variables from the interactions list.", sep=""), icon="warning", type="okcancel") if (as.character(warning2) == "cancel") { tkfocus(tt2) hgOff() stop() } } tcl("update", "idletasks") tkfocus(tt) tcl("update", "idletasks") tkdestroy(tt2) tcl("update", "idletasks") ################################################################### # 9 - MODEL CALCULATION # ################################################################### ## Calculate optimum model for each gene by running a stepwise ## GLM calculation first on the main effect variables, then on the ## first order of interactions of the significant main effects. ## Uses a type II Anova calculation to test for significance p values if (MEorINT == "ME") { tempP <- modelCalcME(adjParamData, d, startingModel, progress) } else if (MEorINT == "INT") { tempP <- modelCalcINT(adjParamData, d, param4INT, startingModel, progress) } pVal <- tempP$pValues failedAnova <- tempP$failedAnova errorGenes <- tempP$errorGenes ## Use multiple testing correction to limit false positives adjp <- mulTestCor(pVal, MTC, adjParamData, d, param4INT) ################################################################### # 10 - RESULTS # ################################################################### tkdestroy(tt) ## Output gene lists with associated p values. Also attached is the ## gene list "ErrorGenes" which gives genes showing aliassing. This ## can mess up the model calculations, so the user should be wary ## about results for these genes if (MEorINT == "INT") { ## All possible interactions with the variables of interest outputNames <- gsub(" ", "_", names(adjParamData)) if (length(param4INT) > 1) { for (i in 1:length(param4INT)) { for (j in 1:length(param4INT)) { if (param4INT[j] != param4INT[i]) { outputNames <- c(outputNames, gsub(" ", "_", paste(param4INT[i], ":", param4INT[j], sep=""))) } } rm(j) } rm(i) } } else outputNames <- gsub(" ", "_", names(adjParamData)) tt3 <- tktoplevel() tktitle(tt3) <- "Envisage Output selection" mainFrame <- tkframe(tt3) fontLab <- tkfont.create(family="times",size=12,weight="bold") frameTitle <- tkframe(mainFrame) tkgrid(tklabel(frameTitle, text="Variable\nName", width=20, font=fontLab), tklabel(frameTitle, text="All\nChanging\nGenes", width=10, font=fontLab), tklabel(frameTitle, text="Significantly\nChanging\nGenes", width=10, font=fontLab)) tkgrid(frameTitle,sticky="w") ## Scrollable frame for variable selection sw3 <- tkwidget(mainFrame,"ScrolledWindow", relief="sunken",borderwidth=2) sf2 <- tkwidget(sw3, "ScrollableFrame", width=470) tcl(sw3,"setwidget",sf2) subfID2 <- tclvalue(tcl(sf2,"getframe")) frameBox <- list() paramName <- list() geneChange <- list() sigGeneChange <- list() outputBox <- list() outBoxValue <- list() percentGenes <- NULL sigGenes <- NULL ## Order variables based on number of genes showing changes for (i in 1:length(outputNames)) { ## Count total number of genes changing for each variable temp1 <- 0 for (j in 1:nrow(d)) { if (!is.na(adjp[[outputNames[i]]][j])) { temp1 <- temp1 + 1 } } percent1 <- ((temp1/nrow(d))*100) percent1 <- as.numeric(substr(percent1,1,5)) percentGenes <- c(percentGenes, percent1) ## Count number of significantly changing genes temp2 <- 0 for (j in 1:nrow(d)) { if (!is.na(adjp[[outputNames[i]]][j]) && adjp[[outputNames[i]]][j] <= pCutoff) { temp2 <- temp2 + 1 } } percent2 <- ((temp2/nrow(d))*100) percent2 <- as.numeric(substr(percent2,1,5)) sigGenes <- c(sigGenes, percent2) } rm(i) names(sigGenes) <- outputNames ordSigGenes <- sort(sigGenes, decreasing=T) names(percentGenes) <- outputNames ordParInt <- percentGenes[names(ordSigGenes)] ## Create scrolled frame with all variables and interactions for (i in 1:length(ordParInt)) { frameBox[[i]] <- tcl("frame", paste(subfID2,".",i,sep=""), relief="flat", borderwidth=2) ## Variable name paramName[[i]] <- tcl("label", paste(subfID2,".",i,".1",sep=""), text=names(ordParInt)[i], width=30, justify="left") ## Percentage of genes showing changes for each variable and ## interaction geneChange[[i]] <- tcl("label", paste(subfID2,".",i,".2",sep=""), text=paste(ordParInt[i],"%"), width=15) ## Percentage of genes showing significant changes for each ## variable and interaction sigGeneChange[[i]] <- tcl("label", paste(subfID2,".",i,".3",sep=""), text=paste(ordSigGenes[i],"%"), width=15) ## Select output variables outputBox[[i]] <- tcl("checkbutton", paste(subfID2, ".",i,".4",sep=""), width=10) tkconfigure(outputBox[[i]], cursor="hand2") outBoxValue[[i]] <- tclVar("1") tkconfigure(outputBox[[i]], variable=outBoxValue[[i]]) ## Combine tkgrid(paramName[[i]], geneChange[[i]], sigGeneChange[[i]], outputBox[[i]]) tkgrid(frameBox[[i]]) } rm(i) tkgrid(sw3) OnOK2 <- function() { options(show.error.messages=TRUE) mainParams <- NULL for (i in 1:length(ordParInt)) { if (tclvalue(outBoxValue[[i]]) == "1") { mainParams <- c(mainParams,names(ordParInt)[i]) } } rm(i) ## Ensure at least one variable is selected if (length(mainParams) == 0) { warning <- tkmessageBox(message=paste("Please ensure", "that at least one variable is selected"), icon="warning", type="ok") options(show.error.messages=FALSE) stop() } tkdestroy(tt3) ## Pulls out genes whose p values are within the cutoff limit results <<- list() for (i in 1:length(mainParams)) { tempP <- NULL tempN <- NULL for (j in 1:nrow(d)) { if (!is.na(adjp[[mainParams[i]]][j])) { if (adjp[[mainParams[i]]][j] <= pCutoff) { tempP <- c(tempP, adjp[[mainParams[i]]][j]) tempN <- c(tempN, rownames(d)[j]) } } } rm(j) if (length(tempP) != 0) { names(tempP) <- tempN results[[i]] <- tempP } else results[[i]] <- NA names(results)[i] <- mainParams[i] } rm(i) ## Get rid of NA results list <- NULL for (i in 1:length(results)) { if (!is.na(results[i])) list <- c(list,i) } rm(i) results <<- results[list] ## Create a directory in the working directory to store resulting ## gene lists try(dir.create(paste(fileResults, "/EnvisageResults", sep=""), showWarnings=F)) ## If results folder is non-empty, rename all old files dirs <- dir(paste(fileResults, "/EnvisageResults", sep="")) if (length(dirs) > 0) { for (i in 1:length(dirs)) { temp <- paste(substr(dirs[i],1,nchar(dirs[i])-4), "_OLD.txt", sep="") file.rename(paste(fileResults, "/EnvisageResults/", dirs[i], sep=""), paste(fileResults, "/EnvisageResults/", temp, sep="")) } } ## Write genelists to tab delimited text files in the new directory if (length(results) > 0) { Pnames <- gsub(" ","_",names(results)) Pnames <- gsub(":","_INT_",Pnames) for (i in 1:(length(results))) { if (file.exists(paste(fileResults, "/EnvisageResults/", Pnames[i],".txt",sep=""))) { file.remove(paste(fileResults, "/EnvisageResults/", Pnames[i],".txt",sep="")) } write.table(results[[i]],file=paste(fileResults,"/EnvisageResults/", Pnames[i],".txt",sep=""), append=F, row.names=T, col.names=FALSE, quote=FALSE, sep="\t") } rm(i) } else if (length(results) == 0) { warning <- tkmessageBox(message=paste("Warning: No genes found to ", "be significant.", sep=""), icon="warning", type="ok") } ## Output information for error genes list <- NULL for (i in 1:nrow(d)) { if (!is.na(errorGenes[[i]][1])) list <- c(list, i) } rm(i) if (length(list) > 0) { errorGenesShort <- errorGenes[list] ## Write gene list for errors if (file.exists(paste(fileResults, "/EnvisageResults/AliasGenes.txt", sep=""))) { file.remove(paste(fileResults, "/EnvisageResults/AliasGenes.txt", sep="")) } write.table(names(errorGenesShort),file=paste(fileResults, "/EnvisageResults/AliasGenes.txt", sep=""), append=F, row.names=F, col.names=FALSE, quote=FALSE, sep="\t") ## Write error report if (file.exists(paste(fileResults, "/EnvisageResults/AliasReport.txt", sep=""))) { file.remove(paste(fileResults, "/EnvisageResults/AliasReport.txt", sep="")) } for (i in 1:length(errorGenesShort)) { write(names(errorGenesShort)[i], file=paste(fileResults, "/EnvisageResults/AliasReport.txt", sep=""), append=T) write("\nAliasing is seen across the following conditions:", file=paste(fileResults, "/EnvisageResults/AliasReport.txt", sep=""), append=T) for (j in 1:length(errorGenesShort[[i]])) { write(errorGenesShort[[i]][j], file=paste(fileResults, "/EnvisageResults/AliasReport.txt", sep=""), append=T) } write("\n\n", file=paste(fileResults, "/EnvisageResults/AliasReport.txt", sep=""), append=T) } } ## Write gene list for genes that fail the Anova if (length(failedAnova) > 0) { try(file.remove(paste(fileResults, "/EnvisageResults/Failed.txt", sep=""))) write.table(failedAnova,file=paste(fileResults, "/EnvisageResults/Failed.txt", sep=""), append=F, row.names=F, col.names=FALSE, quote=FALSE, sep="\t") } ## Info on errors temp <- NULL tmpNames <- NULL for (i in 1:length(errorGenes)) { if (!is.na(errorGenes[[i]][1])) { temp <- c(temp, i) tmpNames <- c(tmpNames, names(errorGenes)[i]) } } temp2 <- list() if (length(temp) > 0) { for (i in 1:length(temp)) temp2[[i]] <- errorGenes[[temp[i]]] names(temp2) <- names(errorGenes)[temp] results[[length(results)+1]] <- temp2 names(results)[length(results)] <- "errorGenes" } if (length(failedAnova) > 0) { results[[length(results)+1]] <- failedAnova names(results)[length(results)] <- "failedAnova" } } ## Function to select all check boxes checkAll <- function() { for (i in 1:length(ordParInt)) { outBoxValue[[i]] <- tclVar("1") tkconfigure(outputBox[[i]], variable=outBoxValue[[i]]) } } ## Function to clear all check boxes clearAll <- function() { for (i in 1:length(ordParInt)) { outBoxValue[[i]] <- tclVar("0") tkconfigure(outputBox[[i]], variable=outBoxValue[[i]]) } } ## Allow user to check or clear all boxes in one go frameSelect <- tkframe(mainFrame, relief="groove", borderwidth=2) checkAllBut <- tkbutton(frameSelect, text="Select All", command=checkAll, bg="grey", cursor="hand2") clearAllBut <- tkbutton(frameSelect, text="Clear All", command=clearAll, bg="grey", cursor="hand2") tkgrid(checkAllBut,clearAllBut) tkgrid(frameSelect) ## Select OK to save the results to the specified file frameOk <- tkframe(mainFrame, relief="flat", borderwidth=2) OK.but3 <- tkbutton(frameOk, text=" OK ", command=OnOK2, height=1, width=10, bg="grey", cursor="hand2") ## Return has the same function as OK tkbind(tt3,"",OnOK2) blankLine2 <- tklabel(frameOk,text=" ") tkgrid(blankLine2) tkgrid(OK.but3) tkgrid(frameOk) tkgrid(mainFrame) ## Make sure that errors are shown if user quits by destroying the window tkbind(tt3,"",function() options(show.error.messages=TRUE)) } ############################################################################### ## End - OnOK2 ## ############################################################################### ## Function to select all check boxes checkAll2 <- function() { for (i in 1:length(adjParamData)) { outBoxValue2[[i]] <<- tclVar("1") tkconfigure(outputBox2[[i]], variable=outBoxValue2[[i]]) } } ## Function to clear all check boxes clearAll2 <- function() { for (i in 1:length(adjParamData)) { outBoxValue2[[i]] <<- tclVar("0") tkconfigure(outputBox2[[i]], variable=outBoxValue2[[i]]) } } OnCancel <- function() { ## Ensure error messages are switched back on (if recalled) options(show.error.messages=TRUE) tkfocus(tt) tkdestroy(tt2) hgOff() } ## Allow user to check or clear all boxes in one go frameSelect2 <- tkframe(mainFrame2, relief="groove", borderwidth=2) checkAllBut2 <- tkbutton(frameSelect2, text="Select All", command=checkAll2, bg="grey", cursor="hand2") clearAllBut2 <- tkbutton(frameSelect2, text="Clear All", command=clearAll2, bg="grey", cursor="hand2") tkgrid(checkAllBut2,clearAllBut2) tkgrid(frameSelect2) ## Explanation for selecting variables for interactions INTselectFrame <- tkframe(mainFrame2, borderwidth=2) INTselectLabel <- tklabel(INTselectFrame, text=paste("Please choose", " the variables that you would like to look at the\n", "interactions for. Please note that looking at interactions ", "increases\nthe number of terms in the model and may produce ", "overfitting if too\nmany are selected.", sep=""), justify="left") tkgrid(INTselectLabel) tkgrid(INTselectFrame) ## Select OK to run function frameOk <- tkframe(mainFrame2, relief="flat", borderwidth=2) OK.but2 <- tkbutton(frameOk, text=" OK ", command=OnOK2, height=1, width=10, bg="grey", cursor="hand2") Cancel.but2 <- tkbutton(frameOk, text=" Cancel ", command=OnCancel, height=1, width=10, bg="grey", cursor="hand2") ## Return has the same function as OK tkbind(tt2,"",OnOK2) tkgrid(OK.but2, Cancel.but2) tkgrid(frameOk) tkgrid(mainFrame2) destroyTT2 <- function() { options(show.error.messages=TRUE) hgOff() } ## Make sure that errors are shown if user quits by destroying the tkbind(tt2,"",destroyTT2()) tkfocus(tt2) tcl("update", "idletasks") } ########################################################################### # B - ME ONLY MODEL CALCULATION # ########################################################################### ## Calculate optimum model for each gene by running a stepwise ## GLM calculation first on the main effect variables, then on the ## first order of interactions of the significant main effects if (MEorINT == "ME") { tempP <- modelCalcME(adjParamData, d, startingModel, progress) pVal <- tempP$pValues failedAnova <- tempP$failedAnova errorGenes <- tempP$errorGenes ## Use multiple testing correction to limit false positives adjp <- mulTestCor(pVal, MTC, adjParamData, d, param4INT) ####################################################################### # 10 - RESULTS # ####################################################################### tkdestroy(tt) ## Output gene lists with associated p values. Also attached is the gene ## list "ErrorGenes" which gives genes showing aliassing. This can mess ## up the model calculations, so the user should be wary about results ## for these genes if (MEorINT == "INT") { ## All possible interactions with the variables of interest outputNames <- gsub(" ", "_", names(adjParamData)) if (length(param4INT) > 1) { for (i in 1:length(param4INT)) { for (j in 1:length(param4INT)) { if (param4INT[j] != param4INT[i]) { outputNames <- c(outputNames, gsub(" ", "_", paste(param4INT[i], ":", param4INT[j], sep=""))) } } rm(j) } rm(i) } } else outputNames <- gsub(" ", "_", names(adjParamData)) tt3 <- tktoplevel() tktitle(tt3) <- "Envisage Output selection" mainFrame <- tkframe(tt3) fontLab <- tkfont.create(family="times",size=12,weight="bold") frameTitle <- tkframe(mainFrame) tkgrid(tklabel(frameTitle, text="Variable\nName", width=20, font=fontLab), tklabel(frameTitle, text="All\nChanging\nGenes", width=10, font=fontLab), tklabel(frameTitle, text="Significantly\nChanging\nGenes", width=10, font=fontLab)) tkgrid(frameTitle,sticky="w") ## Scrollable frame for variable selection sw3 <- tkwidget(mainFrame,"ScrolledWindow", relief="sunken",borderwidth=2) sf2 <- tkwidget(sw3, "ScrollableFrame", width=470) tcl(sw3,"setwidget",sf2) subfID2 <- tclvalue(tcl(sf2,"getframe")) frameBox <- list() paramName <- list() geneChange <- list() sigGeneChange <- list() outputBox <- list() outBoxValue <- list() percentGenes <- NULL sigGenes <- NULL ## Order variables based on number of genes showing changes for (i in 1:length(outputNames)) { ## Count total number of genes changing for each variable temp1 <- 0 for (j in 1:nrow(d)) { if (!is.na(adjp[[outputNames[i]]][j])) { temp1 <- temp1 + 1 } } percent1 <- ((temp1/nrow(d))*100) percent1 <- as.numeric(substr(percent1,1,5)) percentGenes <- c(percentGenes, percent1) ## Count number of significantly changing genes temp2 <- 0 for (j in 1:nrow(d)) { if (!is.na(adjp[[outputNames[i]]][j]) && adjp[[outputNames[i]]][j] <= pCutoff) { temp2 <- temp2 + 1 } } percent2 <- ((temp2/nrow(d))*100) percent2 <- as.numeric(substr(percent2,1,5)) sigGenes <- c(sigGenes, percent2) } rm(i) names(sigGenes) <- outputNames ordSigGenes <- sort(sigGenes, decreasing=T) names(percentGenes) <- outputNames ordParInt <- percentGenes[names(ordSigGenes)] ## Create scrolled frame with all variables and interactions for (i in 1:length(ordParInt)) { frameBox[[i]] <- tcl("frame", paste(subfID2,".",i,sep=""), relief="flat", borderwidth=2) ## Variable name paramName[[i]] <- tcl("label", paste(subfID2,".",i,".1",sep=""), text=names(ordParInt)[i], width=30, justify="left") ## Percentage of genes showing changes for each variable and ## interaction geneChange[[i]] <- tcl("label", paste(subfID2,".",i,".2",sep=""), text=paste(ordParInt[i],"%"), width=15) ## Percentage of genes showing significant changes for each ## variable and interaction sigGeneChange[[i]] <- tcl("label", paste(subfID2,".",i,".3",sep=""), text=paste(ordSigGenes[i],"%"), width=15) ## Select output variables outputBox[[i]] <- tcl("checkbutton", paste(subfID2, ".",i,".4",sep=""), width=10) tkconfigure(outputBox[[i]], cursor="hand2") outBoxValue[[i]] <- tclVar("1") tkconfigure(outputBox[[i]], variable=outBoxValue[[i]]) ## Combine tkgrid(paramName[[i]], geneChange[[i]], sigGeneChange[[i]], outputBox[[i]]) tkgrid(frameBox[[i]]) } rm(i) tkgrid(sw3) OnOK2 <- function() { options(show.error.messages=TRUE) mainParams <- NULL for (i in 1:length(ordParInt)) { if (tclvalue(outBoxValue[[i]]) == "1") { mainParams <- c(mainParams,names(ordParInt)[i]) } } rm(i) ## Ensure at least one variable is selected if (length(mainParams) == 0) { warning <- tkmessageBox(message=paste("Please ensure", "that at least one variable is selected"), icon="warning", type="ok") options(show.error.messages=FALSE) stop() } tkdestroy(tt3) ## Pulls out genes whose p values are within the cutoff limit results <<- list() for (i in 1:length(mainParams)) { tempP <- NULL tempN <- NULL for (j in 1:nrow(d)) { if (!is.na(adjp[[mainParams[i]]][j])) { if (adjp[[mainParams[i]]][j] <= pCutoff) { tempP <- c(tempP, adjp[[mainParams[i]]][j]) tempN <- c(tempN, rownames(d)[j]) } } } rm(j) if (length(tempP) != 0) { names(tempP) <- tempN results[[i]] <- tempP } else results[[i]] <- NA names(results)[i] <- mainParams[i] } rm(i) ## Get rid of NA results list <- NULL for (i in 1:length(results)) { if (!is.na(results[i])) list <- c(list,i) } rm(i) results <<- results[list] ## Create a directory in the working directory to store resulting ## gene lists try(dir.create(paste(fileResults, "/EnvisageResults", sep=""), showWarnings=F)) ## If results folder is non-empty, rename all old files dirs <- dir(paste(fileResults, "/EnvisageResults", sep="")) if (length(dirs) > 0) { for (i in 1:length(dirs)) { temp <- paste(substr(dirs[i],1,nchar(dirs[i])-4), "_OLD.txt", sep="") file.rename(paste(fileResults, "/EnvisageResults/", dirs[i], sep=""), paste(fileResults, "/EnvisageResults/", temp, sep="")) } } ## Write genelists to tab delimited text files in the new directory if (length(results) > 0) { Pnames <- gsub(" ","_",names(results)) Pnames <- gsub(":","_INT_",Pnames) for (i in 1:(length(results))) { if (file.exists(paste(fileResults, "/EnvisageResults/", Pnames[i],".txt",sep=""))) { file.remove(paste(fileResults, "/EnvisageResults/", Pnames[i],".txt",sep="")) } write.table(results[[i]],file=paste(fileResults,"/EnvisageResults/", Pnames[i],".txt",sep=""), append=F, row.names=T, col.names=FALSE, quote=FALSE, sep="\t") } rm(i) } else if (length(results) == 0) { warning <- tkmessageBox(message=paste("Warning: No genes found to ", "be significant.", sep=""), icon="warning", type="ok") } ## Output information for error genes list <- NULL for (i in 1:nrow(d)) { if (!is.na(errorGenes[[i]][1])) list <- c(list, i) } rm(i) if (length(list) > 0) { errorGenesShort <- errorGenes[list] ## Write gene list for errors if (file.exists(paste(fileResults, "/EnvisageResults/AliasGenes.txt", sep=""))) { file.remove(paste(fileResults, "/EnvisageResults/AliasGenes.txt", sep="")) } write.table(names(errorGenesShort),file=paste(fileResults, "/EnvisageResults/AliasGenes.txt", sep=""), append=F, row.names=F, col.names=FALSE, quote=FALSE, sep="\t") ## Write error report if (file.exists(paste(fileResults, "/EnvisageResults/AliasReport.txt", sep=""))) { file.remove(paste(fileResults, "/EnvisageResults/AliasReport.txt", sep="")) } for (i in 1:length(errorGenesShort)) { write(names(errorGenesShort)[i], file=paste(fileResults, "/EnvisageResults/AliasReport.txt", sep=""), append=T) write("\nAliasing is seen across the following conditions:", file=paste(fileResults, "/EnvisageResults/AliasReport.txt", sep=""), append=T) for (j in 1:length(errorGenesShort[[i]])) { write(errorGenesShort[[i]][j], file=paste(fileResults, "/EnvisageResults/AliasReport.txt", sep=""), append=T) } write("\n\n", file=paste(fileResults, "/EnvisageResults/AliasReport.txt", sep=""), append=T) } } ## Write gene list for genes that fail the Anova if (length(failedAnova) > 0) { try(file.remove(paste(fileResults, "/EnvisageResults/Failed.txt", sep=""))) write.table(failedAnova,file=paste(fileResults, "/EnvisageResults/Failed.txt", sep=""), append=F, row.names=F, col.names=FALSE, quote=FALSE, sep="\t") } ## Info on errors temp <- NULL tmpNames <- NULL for (i in 1:length(errorGenes)) { if (!is.na(errorGenes[[i]][1])) { temp <- c(temp, i) tmpNames <- c(tmpNames, names(errorGenes)[i]) } } temp2 <- list() if (length(temp) > 0) { for (i in 1:length(temp)) temp2[[i]] <- errorGenes[[temp[i]]] names(temp2) <- names(errorGenes)[temp] results[[length(results)+1]] <- temp2 names(results)[length(results)] <- "errorGenes" } if (length(failedAnova) > 0) { results[[length(results)+1]] <- failedAnova names(results)[length(results)] <- "failedAnova" } } ## Function to select all check boxes checkAll <- function() { for (i in 1:length(ordParInt)) { outBoxValue[[i]] <- tclVar("1") tkconfigure(outputBox[[i]], variable=outBoxValue[[i]]) } } ## Function to clear all check boxes clearAll <- function() { for (i in 1:length(ordParInt)) { outBoxValue[[i]] <- tclVar("0") tkconfigure(outputBox[[i]], variable=outBoxValue[[i]]) } } ## Allow user to check or clear all boxes in one go frameSelect <- tkframe(mainFrame, relief="groove", borderwidth=2) checkAllBut <- tkbutton(frameSelect, text="Select All", command=checkAll, bg="grey", cursor="hand2") clearAllBut <- tkbutton(frameSelect, text="Clear All", command=clearAll, bg="grey", cursor="hand2") tkgrid(checkAllBut,clearAllBut) tkgrid(frameSelect) ## Select OK to save the results to the specified file frameOk <- tkframe(mainFrame, relief="flat", borderwidth=2) OK.but3 <- tkbutton(frameOk, text=" OK ", command=OnOK2, height=1, width=10, bg="grey", cursor="hand2") ## Return has the same function as OK tkbind(tt3,"",OnOK2) blankLine2 <- tklabel(frameOk,text=" ") tkgrid(blankLine2) tkgrid(OK.but3) tkgrid(frameOk) tkgrid(mainFrame) ## Make sure that errors are shown if user quits by destroying the window tkbind(tt3,"",function() options(show.error.messages=TRUE)) } } else hgOff() } ############################################################################### ############################################################################### ## ## ## End - OnOK ## ## ## ############################################################################### ############################################################################### ############################################################################### ############################################################################### ## ## ## Function - OnCancel ## ## ## ############################################################################### ############################################################################### OnCancel <- function() { ## Ensure error messages are switched back on (if recalled) options(show.error.messages=TRUE) ## Check that user wishes to quit quit <- tkmessageBox(title="Quit?", message=paste("This will end your ", "Envisage session.\nAre you sure you want to quit?", sep=""), icon="question",type="yesno",default="no") if (tclvalue(quit) == "yes") { tkdestroy(tt) } if (tclvalue(quit) == "no") { tkfocus(tt) } } ############################################################################### ############################################################################### ## ## ## End - OnCancel ## ## ## ############################################################################### ############################################################################### ## Turn all cursors into hourglass cursors hgOn <- function() { ## Ensure error messages are switched back on (if recalled) options(show.error.messages=TRUE) ## Hourglass cursor while model calculation occurs tkconfigure(tt, cursor="watch") tkconfigure(pEntry,cursor="watch") tkconfigure(mtcSelect,cursor="watch") tkconfigure(fileInput,cursor="watch") tkconfigure(browseBut,cursor="watch") tkconfigure(radME,cursor="watch") tkconfigure(radINT,cursor="watch") tkconfigure(Mentry,cursor="watch") tkconfigure(resetBut,cursor="watch") for (i in 1:nParam) { tkconfigure(radSel[[i]],cursor="watch") tkconfigure(radNum[[i]],cursor="watch") tkconfigure(radCat[[i]],cursor="watch") } tkconfigure(OK.but, cursor="watch") tkconfigure(Cancel.but, cursor="watch") } ## Turn all cursors back to their default hgOff <- function() { tkconfigure(tt,cursor="arrow") tkconfigure(pEntry, cursor="ibeam") tkconfigure(mtcSelect,cursor="hand2") tkconfigure(fileInput,cursor="ibeam") tkconfigure(browseBut,cursor="hand2") tkconfigure(radME,cursor="hand2") tkconfigure(radINT,cursor="hand2") tkconfigure(Mentry,cursor="ibeam") tkconfigure(resetBut,cursor="hand2") for (i in 1:nParam) { tkconfigure(radSel[[i]],cursor="hand2") tkconfigure(radNum[[i]],cursor="hand2") tkconfigure(radCat[[i]],cursor="hand2") } tkconfigure(OK.but, cursor="hand2") tkconfigure(Cancel.but, cursor="hand2") options(show.error.messages=FALSE) stop() } frameOkCancel <- tkframe(frameOverall,relief="flat",borderwidth=2) OK.but <- tkbutton(frameOkCancel, text=" OK ", command=OnOK, height=1, width=10, bg="grey",cursor="hand2") Cancel.but <- tkbutton(frameOkCancel, text=" Cancel ", command=OnCancel, height=1, width=10, bg="grey",cursor="hand2") ## Enter and Escape have the same function as OK and Cancel tkbind(tt,"",OnOK) tkbind(tt,"",OnCancel) blankLine <- tklabel(frameOkCancel,text=" ") tkgrid(blankLine) tkgrid(OK.but,Cancel.but) tkgrid(blankLine,sticky="ns") tkgrid(frameOkCancel) tkgrid.configure(frameOkCancel,columnspan=2) tkgrid(frameOverall) ## Make sure that errors are shown if user quits by destroying the tkbind(tt,"",function() options(show.error.messages=TRUE)) ## Ensure window is correctly loaded when the window is moved tkbind(tt,"",function() tcl("update", "idletasks")) ## Change cursors for all widgets in GUI hgOff() ## Focus on the main window tkfocus(tt) } else if (widget==FALSE) { ########################################################################### # 4 - CHECK ARGUMENT VALUES # ########################################################################### ## Check MTC mtcShort <- c("None", "BH", "BY", "Bonferroni", "Holm", "Hochberg", "SidakSS", "SidakSD") if (!MTC %in% mtcShort) stop(paste("Selected multiple testing correction", "not recognised.\nPlease choose from 'None', 'BH',", "'BY', 'Bonferroni', 'Holm', 'Hochberg',\n'SidakSS'", "or 'SidakSD'")) ## Check pCutoff if (pCutoff < 0 || pCutoff > 1) stop("P value must be between 0 and 1") ## Check MEorINT if (!MEorINT %in% c("ME", "INT")) stop("MEorINT must be either 'ME' or 'INT'") ## Check useParams and make sure the names match for (i in 1:length(useParams)) { if (substr(useParams[i], 1, 1) %in% 0:9) { useParams[i] <- paste("X", useParams[i], sep="") } } rm(i) useParams <- gsub("-", "_", useParams) useParams <- gsub("/", "_", useParams) useParams <- gsub(" ", "_", useParams) if (length(useParams) == 0) { stop(paste("Please ensure that at least one variable is selected", "\nin useParams")) } for (i in 1:length(useParams)) { if (!useParams[i] %in% paramNames) stop(paste("Please ensure that ", "the variable name '", useParams[i], "'\nin useParams matches the ", "variable names in your ExpressionSet", sep="")) } ## Check paramType if (length(paramType) != length(useParams)) { stop("Check that length of 'paramType' matches 'useParams'") } for (i in 1:length(paramType)) { if (!paramType[i] %in% c("numeric", "categoric")) { stop(paste("paramType '", paramType[i], "' not recognised. ", "Please choose from\n'numeric' and 'categoric'", sep="")) } } ## Check fileResults if (file.access(fileResults, mode=0) != 0) stop("Specified file not found") ## Check param4INT if (length(param4INT) > length(useParams)) { stop("More variables selected for interactions than main effects") } ## Check param4INT for (i in 1:length(param4INT)) { if (substr(param4INT[i], 1, 1) %in% 0:9) { param4INT[i] <- paste("X", param4INT[i], sep="") } } rm(i) param4INT <- gsub("-", "_", param4INT) param4INT <- gsub("/", "_", param4INT) param4INT <- gsub(" ", "_", param4INT) for (i in 1:length(param4INT)) { if (!param4INT[i] %in% useParams) stop(paste("Interaction variable", param4INT[i], "not included\nin useParams")) } ## Check startModel if (length(startModel) > 0) { temp <- modelEntry(glm.formula=startModel) startingModel <- temp[[1]] modelTerms <- temp[[2]] intPos <- temp[[3]] if (length(intPos) > 0 && MEorINT == "ME") { stop(paste("Specified model contains interactions, but you have", "chosen to only model main effect terms.")) } for (i in 1:length(modelTerms)) { if (!gsub("_", " ", modelTerms[i]) %in% useParams) { stop(paste("Please ensure that all terms in the selected model", "are present in useParams")) } } } else startingModel <- NULL ## Ensure that highly correlated variables are not selected if (length(corParams) != 0) { for (i in 1:length(corParams)) { count <- 0 for (j in 1:length(corParams[[i]])) { if (startParamNames[corParams[[i]][j]] %in% useParams) { count <- count +1 } } if (count > 1) { message <- c("Warning: The variables:\n\n") for (j in 1:length(corParams[[i]])) { if (startParamNames[corParams[[i]][j]] %in% useParams) { message <- paste(message, "\t", startParamNames[corParams[[i]][j]], "\n", sep="") } } rm(j) message <- paste(message, "\nshow very high ", "correlation. Please ensure that only\none of ", "these is selected", sep="") stop(message) } } } ########################################################################### # 5 - ASSIGN CLASSES TO VARIABLES # ########################################################################### ## Ensure that numerical variables are not stored as factors in the design ## matrix adjParamData <- paramData[,useParams] for (i in 1:length(useParams)) { if (paramType[i] == "numeric") { adjParamData[[i]] <- as.numeric(as.character(paramData[[useParams[i]]])) } } ########################################################################### # 6 - ENSURE THAT LABELLING PARAMETERS ARE REMOVED # ########################################################################### ## Ensure that no labelling variables are selected if (length(labParams) != 0) { warnLabParams <- NULL for (i in 1:length(labParams)) { if (labParams[i] %in% useParams) { if (class(adjParamData[[labParams[i]]]) == "factor") { warnLabParams <- c(warnLabParams, labParams[i]) } } } rm(i) ## Warn user about labelling variables if (length(warnLabParams) != 0) { message <- "The variables " ## List possible labelling variables for (i in 1:length(warnLabParams)) { message <- paste(message, "'", warnLabParams[i], "', ", sep="") } rm(i) ## Warning message message <- paste(message,"\nhave unique values for each sample.", " Either ensure they are selected as\nnumeric in paramType, ", "or do not use (to avoid overfitting)", sep="") stop(message) } } ## Extract updated variable information paramNames <- useParams nParam <- length(paramNames) ## If only one variable chosen, treat as only main effect if (nParam == 1) MEorINT <- "ME" if (length(param4INT) == 0) MEorINT <- "ME" if (MEorINT == "INT") { ################################################################### # 9 - MODEL CALCULATION # ################################################################### ## Calculate optimum model for each gene by running a stepwise ## GLM calculation first on the main effect variables, then on the ## first order of interactions of the significant main effects. ## Uses a type II Anova calculation to test for significance p values if (MEorINT == "ME") { tempP <- modelCalcME(adjParamData, d, startingModel) } else if (MEorINT == "INT") { tempP <- modelCalcINT(adjParamData, d, param4INT, startingModel) } pVal <- tempP$pValues failedAnova <- tempP$failedAnova errorGenes <- tempP$errorGenes ## Use multiple testing correction to limit false positives adjp <- mulTestCor(pVal, MTC, adjParamData, d, param4INT) ################################################################### # 10 - RESULTS # ################################################################### ## Output gene lists with associated p values. Also attached is the ## gene list "ErrorGenes" which gives genes showing aliassing. This ## can mess up the model calculations, so the user should be wary ## about results for these genes results <- list() results <- result.output(adjp, pCutoff, adjParamData, d, errorGenes, fileResults, MEorINT, param4INT, failedAnova) ## Info on errors temp <- NULL tmpNames <- NULL for (i in 1:length(errorGenes)) { if (!is.na(errorGenes[[i]][1])) { temp <- c(temp, i) tmpNames <- c(tmpNames, names(errorGenes)[i]) } } temp2 <- list() if (length(temp) > 0) { for (i in 1:length(temp)) temp2[[i]] <- errorGenes[[temp[i]]] names(temp2) <- names(errorGenes)[temp] results[[length(results)+1]] <- temp2 names(results)[length(results)] <- "errorGenes" } if (length(failedAnova) > 0) { results[[length(results)+1]] <- failedAnova names(results)[length(results)] <- "failedAnova" } } else if (MEorINT == "ME") { tempP <- modelCalcME(adjParamData, d, startingModel) pVal <- tempP$pValues failedAnova <- tempP$failedAnova errorGenes <- tempP$errorGenes ## Use multiple testing correction to limit false positives adjp <- mulTestCor(pVal, MTC, adjParamData, d, param4INT) ####################################################################### # 10 - RESULTS # ####################################################################### ## Output gene lists with associated p values. Also attached is the gene ## list "ErrorGenes" which gives genes showing aliassing. This can mess ## up the model calculations, so the user should be wary about results ## for these genes results <- list() results <- result.output(adjp, pCutoff, adjParamData, d, errorGenes, fileResults, MEorINT, param4INT, failedAnova) ## Info on errors temp <- NULL tmpNames <- NULL for (i in 1:length(errorGenes)) { if (!is.na(errorGenes[[i]][1])) { temp <- c(temp, i) tmpNames <- c(tmpNames, names(errorGenes)[i]) } } temp2 <- list() if (length(temp) > 0) { for (i in 1:length(temp)) temp2[[i]] <- errorGenes[[temp[i]]] names(temp2) <- names(errorGenes)[temp] results[[length(results)+1]] <- temp2 names(results)[length(results)] <- "errorGenes" } if (length(failedAnova) > 0) { results[[length(results)+1]] <- failedAnova names(results)[length(results)] <- "failedAnova" } } return(results) } } ############################################################################### ############################################################################### ## END - FUNCTION - envisage ## ############################################################################### ############################################################################### Envisage/R/alias.glmNA.R0000755003020100016370000000300611031122552015066 0ustar sr9signg00000000000000## Copyright (C) 2007 Sam Robson ## ## This file is part of Envisage. ## ## Envisage is free software; you can redistribute it and/or modify ## it under the terms of the GNU General Public License as published by ## the Free Software Foundation; either version 2 of the License, or ## (at your option) any later version. ## ## Envisage is distributed in the hope that it will be useful, ## but WITHOUT ANY WARRANTY; without even the implied warranty of ## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the ## GNU General Public License for more details. ## ## You should have received a copy of the GNU General Public License ## along with Envisage; if not, write to the Free Software ## Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA ############################################################################### # BEGIN - FUNCTION - alias.glmNA # ############################################################################### ## Create function for a new class of glm objects - glmNA. Anova ## function has trouble with aliased terms, this extension to the ## linear model class helps to overcome these problems alias.glmNA <- function(object, ...){list(Model=formula(object))} ############################################################################### # END - FUNCTION - alias.glmNA # ###############################################################################Envisage/R/labelParams.R0000755003020100016370000000325511031122552015231 0ustar sr9signg00000000000000## Copyright (C) 2007 Sam Robson ## ## This file is part of Envisage. ## ## Envisage is free software; you can redistribute it and/or modify ## it under the terms of the GNU General Public License as published by ## the Free Software Foundation; either version 2 of the License, or ## (at your option) any later version. ## ## Envisage is distributed in the hope that it will be useful, ## but WITHOUT ANY WARRANTY; without even the implied warranty of ## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the ## GNU General Public License for more details. ## ## You should have received a copy of the GNU General Public License ## along with Envisage; if not, write to the Free Software ## Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA ############################################################################### # BEGIN - FUNCTION - labelParams # ############################################################################### ## Remove any variables who have the same number of levels as there are samples ## (eg any labelling variables). These will prevent the modelling method from ## functioning correctly. labelParams <- function(pData) { list <- NULL for (i in 1:ncol(pData)) { if (length(levels(pData[[i]])) == nrow(pData)) { list <- c(list, i) } } rm(i) return(list) } ################################################################################# # END - FUNCTION - labelParams # #################################################################################Envisage/man/0000755003020100016370000000000011031122552013225 5ustar sr9signg00000000000000Envisage/man/alias.glmNA.Rd0000755003020100016370000000500711031122551015606 0ustar sr9signg00000000000000\name{alias.glmNA} \alias{alias.glmNA} \title{Find Aliases (Dependencies) in a Model} \description{Find aliases (linearly dependent terms) in a linear model specified by a formula. This extends the S3 method \code{\link[stats]{alias}} to objects of class \code{\link[Envisage:glmNA-class]{glmNA}} for package \pkg{Envisage}. } \usage{ \method{alias}{glmNA}(object, ...) } \arguments{ \item{object}{A fitted model object of class \code{\link[Envisage:glmNA-class]{glmNA}}.} \item{...}{Further arguments passed to or from other methods.} } \details{ The \pkg{Envisage} package contains methods allowing the use of linear models (LMs) for analysing significantly changing genes in experiments with a variety of sources of variation, be they experimentally controlled variables such as drug treatment or time, or non-controlled sources of confounding variation such as phenotypic or environmental differences. This allows all sources of variation to be considered when analysing for significant differential expression, ensuring resulting genes are biologically relevent to the experimental question. The function \code{\link[Envisage]{alias.glmNA}} extends the method \code{\link[stats]{alias}} to the S4 class \code{\link[Envisage:glmNA-class]{glmNA}} for package \pkg{Envisage}. This is an extension of classes \code{glm} and \code{lm} and deals appropriately with genes that produce NA calls in the modelling procedure. This allows use of the function \code{\link[car]{Anova}} for calculating significance for each gene. \code{\link[Envisage]{alias.glmNA}} is an internal function and is not designed to be accessed by users. } \value{ A list (of class "listof") containing components: \item{Model}{Description of the model; usually the formula.} \item{Complete}{A matrix with columns corresponding to effects that are linearly dependent on the rows.} \item{Partial}{The correlations of the estimable effects, with a zero diagonal. An object of class "mtable" which has its own print method.} } \references{ Robson, S. C., Hunter, E., Bird, H., Turner, H. (2008) \emph{Envisage: model-based significance analysis of microarray gene expression data}, manuscript in preparation. } \author{Sam Robson \email{S.C.Robson@warwick.ac.uk}} \seealso{ \code{\link[stats]{coef}}, \code{\link[Envisage:class.glmNA]{glmNA}. For the main \pkg{Envisage} method, see \code{\link[Envisage]{envisage}}.} } \keyword{models} Envisage/man/dataTransform.Rd0000755003020100016370000000370111031122551016324 0ustar sr9signg00000000000000\name{dataTransform} \alias{dataTransform} \title{Gene expression data formatting for Envisage} \description{Ensures that the gene expression data from the \code{\link[Biobase:class.ExpressionSet]{ExpressionSet}} object is in the correct format for package \pkg{Envisage}.} \usage{ dataTransform(expData) } \arguments{ \item{expData}{Object of class \code{\link[Biobase:class.ExpressionSet]{ExpressionSet}} containing gene expression data.} } \details{ The \pkg{Envisage} package contains methods allowing the use of linear models (LMs) for analysing significantly changing genes in experiments with a variety of sources of variation, be they experimentally controlled variables such as drug treatment or time, or non-controlled sources of confounding variation such as phenotypic or environmental differences. This allows all sources of variation to be considered when analysing for significant differential expression, ensuring resulting genes are biologically relevent to the experimental question. The function \code{\link[Envisage]{dataTransform}} is an internal function that ensures that the gene expression data (in the form of an object of class \code{\link[Biobase:class.ExpressionSet]{ExpressionSet}}) is in the correct format for future processing. In particular, this function ensures that data is in a logged format. \code{\link[Envisage]{dataTransform}} is an internal function and is not designed to be accessed by users. } \value{ Returns a matrix of processed expression values (this may be very large). } \references{ Robson, S. C., Hunter, E., Bird, H., Turner, H. (2008) \emph{Envisage: model-based significance analysis of microarray gene expression data}, manuscript in preparation } \author{Sam Robson \email{S.C.Robson@warwick.ac.uk}} \seealso{For the main \pkg{Envisage} method, see \code{\link[Envisage]{envisage}}.} \keyword{models} Envisage/man/mulTestCor.Rd0000755003020100016370000000573211031122552015627 0ustar sr9signg00000000000000\name{mulTestCor} \alias{mulTestCor} \title{Multiple testing corrections for package Envisage} \description{This function corrects the p-values obtained from package \pkg{Envisage} to account for problems with multiple testing. These corrected p-values give a measure of the significance of model terms on the effects on gene expression changes for individual genes.} \usage{ mulTestCor(pValues, MTC, pData, exprData, param4INT) } \arguments{ \item{pValues}{An object of class \code{data.frame} containing the p-values from the Type II sum of squares analysis for each model term for each gene.} \item{MTC}{A character string that defines which multiple testing correction method to use (if any). Available methods are \code{None}, \code{BH}, \code{BY}, \code{Bonferroni}, \code{Holm}, \code{Hochberg}, \code{SidakSS}, \code{SidakSD}. More information on these can be found in the package vignette. \code{BH} is selected by default.} \item{pData}{An object of class \code{\link[Biobase:class.phenoData]{phenoData}} containing the phenotypic information for the experiment.} \item{exprData}{A matrix of expression values for each gene for each sample. Usually calculated using \code{\link[Biobase]{exprs}} on an object of class \code{\link[Biobase:class.ExpressionSet]{ExpressionSet}}.} \item{param4INT}{Character vector of variables for which interaction terms should be calculated.} } \details{ The \pkg{Envisage} package contains methods allowing the use of linear models (LMs) for analysing significantly changing genes in experiments with a variety of sources of variation, be they experimentally controlled variables such as drug treatment or time, or non-controlled sources of confounding variation such as phenotypic or environmental differences. This allows all sources of variation to be considered when analysing for significant differential expression, ensuring resulting genes are biologically relevent to the experimental question. Due to the large number of genes analysed, gene expression analyses can suffer from problems due to multiple hypothesis testing. The function \code{\link[Envisage]{mulTestCor}} performs the selected multiple testing correction on the p-values indicating significance of each model term for each gene in the analysis. \code{\link[Envisage]{mulTestCor}} is an internal function and is not designed to be accessed by users. } \value{ An object of class \code{data.frame} is returned containing the adjusted p-values for each model term for each gene. } \references{ Robson, S. C., Hunter, E., Bird, H., Turner, H. (2008) \emph{Envisage: model-based significance analysis of microarray gene expression data}, manuscript in preparation } \author{Sam Robson \email{S.C.Robson@warwick.ac.uk}} \seealso{For the main \pkg{Envisage} method, see \code{\link[Envisage]{envisage}}.} \keyword{models} Envisage/man/corParams.Rd0000755003020100016370000000432611031122551015452 0ustar sr9signg00000000000000\name{corParams} \alias{corParams} \title{Flag highly correlated parameters} \description{Flags all highly correlated variables which model the same data to reduce the number of variables and interactions in the model calculation.} \usage{ corParams(pData) } \arguments{ \item{pData}{An object of class \code{\link[Biobase:class.phenoData]{phenoData}} containing the phenotypic information for the experiment.} } \details{ The \pkg{Envisage} package contains methods allowing the use of linear models (LMs) for analysing significantly changing genes in experiments with a variety of sources of variation, be they experimentally controlled variables such as drug treatment or time, or non-controlled sources of confounding variation such as phenotypic or environmental differences. This allows all sources of variation to be considered when analysing for significant differential expression, ensuring resulting genes are biologically relevent to the experimental question. The function \code{\link[Envisage]{corParams}} identifies variables within the study design that are highly correlated and flags them up for the user. These may represent different representations of the same data and will affect model fitting, so should be removed from the data set. Variables found to be highly correlated are suffixed with the flags '*', '**', '***' etc. \code{\link[Envisage]{corParams}} is an internal function and is not designed to be accessed by users. } \value{ A list is returned containing the following slots: \item{corParamNames}{A character vector of the names of the parameters found to have very high correlation, suffixed with flags '*', '**', '***' etc.} \item{corParams}{A list containing the indices for variables found to be highly correlated with one another.} } \references{ Robson, S. C., Hunter, E., Bird, H., Turner, H. (2008) \emph{Envisage: model-based significance analysis of microarray gene expression data}, manuscript in preparation } \author{Sam Robson \email{S.C.Robson@warwick.ac.uk}} \seealso{For the main \pkg{Envisage} method, see \code{\link[Envisage]{envisage}}.} \keyword{models} Envisage/man/result.output.Rd0000755003020100016370000001047611031122552016404 0ustar sr9signg00000000000000\name{result.output} \alias{result.output} \title{Output significant genes for package Envisage} \description{This function takes the adjusted p-values for the model terms for each gene and outputs those genes that are significant to the user.} \usage{ result.output(adjp, pCutoff, pData, exprData, errorGenes, fileResults, MEorINT, param4INT, failedAnova) } \arguments{ \item{adjp}{An object of class \code{data.frame} containing the adjusted p-values for each model term for each gene.} \item{pCutoff}{A number defining the p value cutoff to be used in significance analysis. Must be between 0 and 1 (default 0.05.)} \item{pData}{An object of class \code{\link[Biobase:class.phenoData]{phenoData}} containing the phenotypic information for the experiment.} \item{exprData}{A matrix of expression values for each gene for each sample. Usually calculated using \code{\link[Biobase]{exprs}} on an object of class \code{\link[Biobase:class.ExpressionSet]{ExpressionSet}}.} \item{errorGenes}{A list of the genes that failed and the model terms for which aliasing was detected.} \item{fileResults}{The folder in which to save the results of the modelling procedure. Defaults to the current working directory.} \item{MEorINT}{Character string defining whether to look at first order interactions ("INT") or only main effect terms ("ME") in the model.} \item{param4INT}{A character vector that defines the model variables for which interactions should be calculated.} \item{failedAnova}{A vector of genes for which the Anova Type II sum of squares failed, indicating model aliasing.} } \details{ The \pkg{Envisage} package contains methods allowing the use of linear models (LMs) for analysing significantly changing genes in experiments with a variety of sources of variation, be they experimentally controlled variables such as drug treatment or time, or non-controlled sources of confounding variation such as phenotypic or environmental differences. This allows all sources of variation to be considered when analysing for significant differential expression, ensuring resulting genes are biologically relevent to the experimental question. The function \code{\link[Envisage]{result.output}} outputs significant gene lists for each of the terms within the model. For each model term, a vector is created containing any genes for which the adjusted p-value for that terms is below \code{pCutoff} (along with the corresponding adjusted p-value). A list, \code{results}, is created containing all such data frames for all model terms. The resulting p-values for each model term are output in a tab delimitted text file within the directory \file{EnvisageResults/} which is created within the directory specified by \code{fileResults} (defaults to the working directory). If this directory already exists, existing files are appended with the suffix \file{OLD} to distingiush them from the most recent files. This avoids overwriting data, but please be aware that continually writing to the same file will result in an increase in the directory size. Also created are two output files pertaining to aliasing information. \file{AliasGenes.txt} gives the names of the genes that show problems due to model aliasing, and \file{AliasReport.txt} gives a more in depth report on the variable levels for which aliasing was detected. For more information, please see the package vignette. \code{\link[Envisage]{result.output}} is an internal function and is not designed to be accessed by users. } \value{ A list \code{results} is returned containing the following slots: \item{model term}{For each model term, significnat genes and their p-values are comntained within individual slots.} \item{AliasGenes}{A vector of genes which showed problems through the modelling procedure due to aliasing in the model.} } \references{ Robson, S. C., Hunter, E., Bird, H., Turner, H. (2008) \emph{Envisage: model-based significance analysis of microarray gene expression data}, manuscript in preparation } \author{Sam Robson \email{S.C.Robson@warwick.ac.uk}} \seealso{For the main \pkg{Envisage} method, see \code{\link[Envisage]{envisage}}.} \keyword{models} Envisage/man/coef.glmNA.Rd0000755003020100016370000000445311031122551015435 0ustar sr9signg00000000000000\name{coef.glmNA} \alias{coef.glmNA} \title{Extract Model Coefficients} \description{ \code{\link[stats]{coef}} is a generic function which extracts model coefficients from objects returned by modeling functions. \code{coefficients} is an \emph{alias} for it. This extends the S3 method \code{\link[stats]{coef}} to objects of class \code{\link[Envisage:glmNA-class]{glmNA}} for package \pkg{Envisage}. } \usage{ \method{coef}{glmNA}(object, ...) } \arguments{ \item{object}{A fitted model object of class \code{\link[Envisage:glmNA-class]{glmNA}}.} \item{...}{Further arguments passed to or from other methods.} } \details{ The \pkg{Envisage} package contains methods allowing the use of linear models (LMs) for analysing significantly changing genes in experiments with a variety of sources of variation, be they experimentally controlled variables such as drug treatment or time, or non-controlled sources of confounding variation such as phenotypic or environmental differences. This allows all sources of variation to be considered when analysing for significant differential expression, ensuring resulting genes are biologically relevent to the experimental question. The function \code{\link[Envisage]{coef.glmNA}} extends the method \code{\link[stats]{coef}} to the S4 class \code{\link[Envisage:glmNA-class]{glmNA}} for package \pkg{Envisage}. This is an extension of classes \code{glm} and \code{lm} and deals appropriately with the model output for genes that produce NA calls in the modelling procedure. This allows use of the function \code{\link[car]{Anova}} for calculating significance for each gene. \code{\link[Envisage]{alias.glmNA}} is an internal function and is not designed to be accessed by users. } \value{ Coefficients extracted from the model object \code{object}. } \references{ Robson, S. C., Hunter, E., Bird, H., Turner, H. (2008) \emph{Envisage: model-based significance analysis of microarray gene expression data}, manuscript in preparation. } \author{Sam Robson \email{S.C.Robson@warwick.ac.uk}} \seealso{\code{\link[stats]{alias}}, \code{\link[Envisage:glmNA-class]{glmNA}}. For the main \pkg{Envisage} method, see \code{\link[Envisage]{envisage}}.} \keyword{models} Envisage/man/modelCalcINT.Rd0000755003020100016370000001034511031122551015757 0ustar sr9signg00000000000000\name{modelCalcINT} \alias{modelCalcINT} \title{Fit a model with main effect and first order interaction terms for each gene and calculate significance} \description{This is the main calculation for the package \pkg{Envisage}. An optimised model, containing terms shown to have practical importance for explaining variation, is produced using a step-wise procedure. Terms may be main effect terms or first order interactions. The significance of each term is computed by using a type II sum of squares.} \usage{ modelCalcINT(paramData, exprData, param4INT, startingModel, progress) } \arguments{ \item{paramData}{An object of class \code{\link[Biobase:class.phenoData]{phenoData}} containing the phenotypic information for the experiment.} \item{exprData}{A matrix of expression values for each gene for each sample. Usually calculated using \code{\link[Biobase]{exprs}} on an object of class \code{\link[Biobase:class.ExpressionSet]{ExpressionSet}}.} \item{param4INT}{A character vector specifying the variables for which first order interactions should be calculated.} \item{startingModel}{An object of class \code{\link[stats]{formula}} containing the minimal model thta should be used in the step-wise model fitting process.} \item{progress}{Object of class \code{tclVar} indicating progress through the genes in the gene list. Used only if \code{widget=TRUE} for \code{\link[Envisage]{envisage}}.} } \details{ The \pkg{Envisage} package contains methods allowing the use of linear models (LMs) for analysing significantly changing genes in experiments with a variety of sources of variation, be they experimentally controlled variables such as drug treatment or time, or non-controlled sources of confounding variation such as phenotypic or environmental differences. This allows all sources of variation to be considered when analysing for significant differential expression, ensuring resulting genes are biologically relevent to the experimental question. The function \code{\link[Envisage]{modelCalcINT}} performs the main modelling step for the package \pkg{Envisage}. A stepwise procedure is used on a per-gene basis to calculate the optimum model for each gene. In comparison to \code{\link[Envisage]{modelCalcME}}, terms used in the model may be main effect terms or first-order interactions. The step-wise process begins by fitting the maximal model to the data, then removes terms using a leave-one-out approach based on the Akaike information criterion (AIC). Specification of a minimal model allows terms to be 'forced' into the final formula. To increase speed, the model fitting procedure is first performed using only main effect terms, and first order interactions are only calculated if the main effect term is thus shown to be significant. The significance of each term in the final model is estimated by using a Type II sum of squares to compare the model with and without each term. Also, if fitting the model results in aliased terms (either due to experimental design, insufficient data, or non-independence of model terms), this information is also returned. \code{\link[Envisage]{modelCalcINT}} is an internal function and is not designed to be accessed by users. } \value{ A list is returned with the following slots: \item{pValues}{An object of class \code{data.frame} containing the p-value from the Type II sum of squares analysis for each model term for each gene.} \item{failedAnova}{A vector of genes for which the Anova Type II sum of squares failed, indicating model aliasing.} \item{errorGenes}{A list of the genes that failed and the model terms for which aliasing was detected.} } \references{ Robson, S. C., Hunter, E., Bird, H., Turner, H. (2008) \emph{Envisage: model-based significance analysis of microarray gene expression data}, manuscript in preparation } \author{Sam Robson \email{S.C.Robson@warwick.ac.uk}} \seealso{For the main effect only version of this function, see \code{\link[Envisage]{modelCalcME}}. For the main \pkg{Envisage} method, see \code{\link[Envisage]{envisage}}.} \keyword{models} Envisage/man/modelEntry.Rd0000755003020100016370000000660111031122552015644 0ustar sr9signg00000000000000\name{modelEntry} \alias{modelEntry} \title{Specify a minimum model for step-wise model fitting} \description{This function allows specification of a minimum model for the step-wise model fitting procedure in the \pkg{Envisage} GUI. This can 'force' terms of interest to always be considered in the model for each gene.} \usage{ modelEntry(pData, Mentry, useParams, labParams, MEorINT, glm.formula) } \arguments{ \item{pData}{An object of class \code{\link[Biobase:class.phenoData]{phenoData}} containing the phenotypic information for the experiment.} \item{Mentry}{Object of class \code{tkentry} containing the model entry field from the \pkg{Envisage} GUI. This is used if the starting model is specified in the GUI.} \item{useParams}{Character vector containing the variable names to be used in the model fitting procedure.} \item{labParams}{Output from function \code{\link[Envisage]{labelParams}}, containing the indices of the labelling parameters for the \code{\link[Biobase:class.phenoData]{phenoData}} object \code{pData}.} \item{MEorINT}{Character string indicating whether to look at main effect terms only ('\code{ME}') or main effect and interaction terms ('\code{INT}').} \item{glm.formula}{Object of class \code{formula} containing the minimal model for the step-wise model fitting procedure.} } \details{ The \pkg{Envisage} package contains methods allowing the use of linear models (LMs) for analysing significantly changing genes in experiments with a variety of sources of variation, be they experimentally controlled variables such as drug treatment or time, or non-controlled sources of confounding variation such as phenotypic or environmental differences. This allows all sources of variation to be considered when analysing for significant differential expression, ensuring resulting genes are biologically relevent to the experimental question. The function \code{\link[Envisage]{modelEntry}} is used to set up the maximum and minimum models for the \pkg{Envisage} step-wise model fitting procedure. This can be done in one of two ways. If using the command line interface to \code{\link[Envisage]{envisage}}, the model can be explicitly stated in the command line in the form 'X ~ var1 + var2:var3 + etc.'. However, if using the GUI (\code{widget=TRUE}), users can specify the model in terms of the variable labels (of the form \code{p1}, \code{p2}, \code{p3}, etc. (e.g. \code{p1+p3+p5:p6}). This function processes these values to give the minimum model for the analysis. \code{\link[Envisage]{modelEntry}} is an internal function and is not designed to be accessed by users. } \value{ A list is returned with the followig slots: \item{startingModel}{An object of class \code{formula} giving the minimum model for the the step-wise model fitting procedure.} \item{intPos}{An internal object used to identify the location of interaction terms within the minimum model.} } \references{ Robson, S. C., Hunter, E., Bird, H., Turner, H. (2008) \emph{Envisage: model-based significance analysis of microarray gene expression data}, manuscript in preparation } \author{Sam Robson \email{S.C.Robson@warwick.ac.uk}} \seealso{For the main \pkg{Envisage} method, see \code{\link[Envisage]{envisage}}.} \keyword{models} Envisage/man/glmNA-class.Rd0000755003020100016370000000063311031122551015621 0ustar sr9signg00000000000000\name{glmNA-class} \alias{class:glmNA} \title{Class for Envisage Analysis} \description{Class that extends objects of class \code{glm} to allow use of function \code{\link[car]{Anova}} when NA calls are produced by the modelling procedure.} \author{Sam Robson \email{S.C.Robson@warwick.ac.uk}} \seealso{For the main \pkg{Envisage} method, see \code{\link[Envisage]{envisage}}.} \keyword{models} Envisage/man/GS2BC.Rd0000755003020100016370000000340611031122551014321 0ustar sr9signg00000000000000\name{GS2BC} \alias{GS2BC} \title{Convertor for GeneSpring GSint and Bioconductor ExpressionSet objects} \description{This function converts GeneSpring Experiment (Interpretation) objects into BioConductor microarray expression objects.} \usage{ GS2BC(interpretation, what = "nor") } \arguments{ \item{interpretation}{GeneSpring Experiment Interpretation object.} \item{what}{For GSload.intBC() only. Indicates what data from the GeneSpring Experiment Interpretation should be used. Valid values for what can be \code{nor} and \code{raw}, representing Normalized or Raw expression values. The control values cannot be used.} } \details{ The \pkg{Envisage} package contains methods allowing the use of linear models (LMs) for analysing significantly changing genes in experiments with a variety of sources of variation, be they experimentally controlled variables such as drug treatment or time, or non-controlled sources of confounding variation such as phenotypic or environmental differences. This allows all sources of variation to be considered when analysing for significant differential expression, ensuring resulting genes are biologically relevent to the experimental question. \code{\link[Envisage]{GS2BC}} is slightly changed from function \code{\link[GeneSpring]{GSint2BC}} from package \pkg{GeneSpring}, which currently only supports the defunct \code{exprSet} class of data. } \references{ Robson, S. C., Hunter, E., Bird, H., Turner, H. (2008) \emph{Envisage: model-based significance analysis of microarray gene expression data}, manuscript in preparation } \author{Sam Robson \email{S.C.Robson@warwick.ac.uk}} \seealso{\code{\link[GeneSpring]{GSint2BC}}} \keyword{models} Envisage/man/envisage.Rd0000755003020100016370000002077211031122551015327 0ustar sr9signg00000000000000\name{envisage} \alias{envisage} \title{Enables Numerous Variables In Significant Analysis of Gene Expression} \description{Linear model based analysis of significantly changing gene expression across multiple experiment variables.} \usage{ envisage(expData, widget=TRUE, MTC=NULL, pCutoff=NULL, useParams=NULL, param4INT=NULL, paramType=NULL, fileResults=getwd(), MEorINT="INT", startModel=NULL) } \arguments{ \item{expData}{An object of class \code{\link[Biobase:class.ExpressionSet]{ExpressionSet}}} \item{widget}{Logical value stating whether the interactive widget should be used. Set to \code{TRUE} by default.} \item{MTC}{A character string that defines which multiple testing correction method to use (if any). Available methods are \code{None}, \code{BH}, \code{BY}, \code{Bonferroni}, \code{Holm}, \code{Hochberg}, \code{SidakSS}, \code{SidakSD}. More information on these can be found in the package vignette. \code{BH} is selected by default.} \item{pCutoff}{A number defining the p value cutoff to be used in significance analysis. Must be between 0 and 1 (default 0.05.)} \item{useParams}{A character vector that defines which of the experiment variables to use in the model calculation.} \item{param4INT}{A character vector that defines the model variables for which interactions should be calculated.} \item{paramType}{A character vector specifying the classes (\code{numeric} or \code{categoric}) of the variables in the model.} \item{fileResults}{The folder in which to save the results of the modelling procedure. Defaults to the current working directory.} \item{MEorINT}{Character string defining whether to look at first order interactions ("INT") or only main effect terms ("ME") in the model.} \item{startModel}{Starting Model for analysis. Must be of the form "x ~ ...". Care must be taken to ensure that the model terms match the names of variables specified in \code{useParams}} } \details{ The \pkg{Envisage} package contains methods allowing the use of linear models (LMs) for analysing significantly changing genes in experiments with a variety of sources of variation, be they experimentally controlled variables such as drug treatment or time, or non-controlled sources of confounding variation such as phenotypic or environmental differences. This allows all sources of variation to be considered when analysing for significant differential expression, ensuring resulting genes are biologically relevent to the experimental question. The function \code{\link[Envisage]{envisage}} is the main function in the package and is run for an object \code{expData} of class \code{\link[Biobase:class.ExpressionSet]{ExpressionSet}}, which may represent 1-colour microarray data (log-intensity values) or 2-colour microarray data (as log-ratio values). This object contains gene expression values and 'phenodata' (experiment variable information) for each sample, along with annotation for the features contained in the array of interest. For further information on loading 1-colour microarray data into the \code{\link[Biobase:class.ExpressionSet]{ExpressionSet}} format see package \pkg{affy}, particularly \code{\link[affy]{read.affybatch}}. For information on loading 2-colour microarray data, see package \pkg{marray}. Arguments can be specified using a tcl/tk widget based GUI. The widget is used to avoid errors in variable entry; specifically variable class specification, errors in which can have a dramatic effect on the model outcome. For more information, see the \pkg{Envisage} vignette. Any labelling variables (i.e. any variable whereby every sample has a unique value) are removed before the GUI is displayed, as these should not be included in the model since they are irrelevant and will always result in a perfect fit. Also, any highly correlated variables that essentially contain identical information are flagged for the user. Only one of these should be chosen to avoid complications with overfitting of the data. Model selection is performed for each gene individually. A backwards stepwise procedure is used to select a model balanced for parsimony versus explanatory power by starting with a saturated model containing all specified variables and interactions and removing terms based on the Akaike information criterion (AIC). A primary model is fit for main effect terms initially, and a second model is fit for first order interaction terms for those variables found to be significant in the main effect model. This two-part test saves hugely on run-time due to the large number of variables and the huge number of genes for wich models are calculated. Further information on the modelling process can be found in the package vignette. The significance of each term in the selected model for each gene is calculated by using a Type II ANOVA F-test statistic, comparing the full selected model with a model with the term removed. This process produces a series of unadjusted p-value defining the significance of each model term. It is recommended to use a multiple testing correction, such as the Bonferroni & Hochberg false discovery rate method, to correct for the large number of statistical tests considered (one per gene). A gene is considered to change significantly for a particular model term if the resulting p value is less than \code{pCutoff}. After model fitting has been completed, a second GUI window is created which gives the user information on which variables and interactions show a significant effect on the genes. Variables and interactions can be selected for output gene lists by selecting the relevent check boxes. Lists of significantly changing genes for each experiment variable or interaction of interest and their associated p values are written to the newly created directory \file{\dots/EnvisageResults}, created in the current specified directory in a tab-delimited text format. These files can be read into analysis packages such as Genespring GX for further analysis. Two further report files may also be created; \file{errorGenes.txt} and \file{AliasReport.txt}. These files give information on aliasing in the ANOVA calculations which may occur due to modelling of highly correlated variables. More information on these files can be found in the package vignette. } \value{ A list \code{results} is returned with a slot for each of the variables or interactions specified in the results GUI. Each slot contains a vector of genes and their associated ANOVA p-values for genes that change significantly (i.e. have a p-value less than \code{pCutoff}) based on that variable or interaction. If no genes are shown to be significant for a particular variable or interaction, then that slot is removed to improve readability. If any genes fail the modelling procedure, the final slots in the list contain information on these genes that is then transferred to the files \file{errorGenes.txt} and \file{AliasReport.txt}. These lists are saved in the directory \file{\dots/EnvisageResults} contained in the current specified directory as tab-delimited text files for further analysis. } \references{ Robson, S. C., Hunter, E., Bird, H., Turner, H. (2008) \emph{Envisage: model-based significance analysis of microarray gene expression data}, manuscript in preparation } \author{Sam Robson \email{S.C.Robson@warwick.ac.uk}} \seealso{For information on loading 1-colour microarray data, see \code{\link[affy]{read.affybatch}}. For information on loading 2-colour microarray data, see \pkg{marray}. For more information on object classes, see \code{\link[Biobase:class.ExpressionSet]{ExpressionSet}}.} \examples{ ## Load dataset library(Envisage) data(SkinvsPancreas) ## Select which variables to use in analysis and their classes useParams <- c("RIN", "Tissue", "Time point", "Myc ON/OFF") paramType <- c("numeric", rep("categoric", 3)) ## We only want to look at the interactions for the 3 main variables param4INT <- c("Tissue", "Time point", "Myc ON/OFF") ## Run envisage results <- envisage(SkinvsPancreas, widget=FALSE, MTC="BH", pCutoff=0.05, useParams, param4INT, paramType, fileResults=getwd(), MEorINT="INT") ## Output results results } \keyword{models} Envisage/man/labelParams.Rd0000755003020100016370000000376311031122551015752 0ustar sr9signg00000000000000\name{labelParams} \alias{labelParams} \title{Remove labelling variables} \description{This function removes any categorical variables which have the same number of levels as there are samples (labelling variables).} \usage{ labelParams(pData) } \arguments{ \item{pData}{An object of class \code{\link[Biobase:class.phenoData]{phenoData}} containing the phenotypic information for the experiment.} } \details{ The \pkg{Envisage} package contains methods allowing the use of linear models (LMs) for analysing significantly changing genes in experiments with a variety of sources of variation, be they experimentally controlled variables such as drug treatment or time, or non-controlled sources of confounding variation such as phenotypic or environmental differences. This allows all sources of variation to be considered when analysing for significant differential expression, ensuring resulting genes are biologically relevent to the experimental question. The function \code{\link[Envisage]{labelParams}} ensures that any categorical variables included by the user for model fitting that have the same number of factor levels as there are samples are removed prior to fitting the model. Such labelling variables contain a unique value for each sample, and will this result in a perfect fit for the data. \code{\link[Envisage]{labelParams}} is an internal function and is not designed to be accessed by users. } \value{ A vector is returned containing the indices of the labelling parameters for the \code{\link[Biobase:class.phenoData]{phenoData}} object \code{pData}. } \references{ Robson, S. C., Hunter, E., Bird, H., Turner, H. (2008) \emph{Envisage: model-based significance analysis of microarray gene expression data}, manuscript in preparation } \author{Sam Robson \email{S.C.Robson@warwick.ac.uk}} \seealso{For the main \pkg{Envisage} method, see \code{\link[Envisage]{envisage}}.} \keyword{models} Envisage/man/SkinvsPancreas.Rd0000755003020100016370000000165511031122552016460 0ustar sr9signg00000000000000\name{SkinvsPancreas} \alias{SkinvsPancreas} \docType{data} \title{Comparison of Gene Expression in Skin and Pancreas} \description{ This data set is a small subset of the data of Robson et al. (2008), examining the differences in the changes in gene expression signature in transgenic models for c-Myc activation in the skin and the pancreas for 50 probe sets. } \usage{data(SkinvsPancreas)} \format{An object of class \code{ExpressionSet} containing 41 samples and normalised expression values for 50 probe sets.} \source{ Robson, S. C., Pelengaris, S., Khan, M. (2008), \emph{Life or cell death: Identifying c-Myc regulated genes in two distinct tissues}, manuscript in preparation } \references{ Robson, S. C., Hunter, E., Bird, H., Turner, H. (2008), \emph{Envisage: model-based significance analysis of microarray gene expression data}, manuscript in preparation } \keyword{datasets} Envisage/man/modelCalcME.Rd0000755003020100016370000000746111031122551015633 0ustar sr9signg00000000000000\name{modelCalcME} \alias{modelCalcME} \title{Fit a model with main effect terms only for each gene and calculate significance} \description{This is the main calculation for the package \pkg{Envisage}. An optimised model, containing terms shown to have practical importance for explaining variation, is produced using a step-wise procedure. Terms may be main effect terms only. The significnace of each term is computed by using a type II sum of squares.} \usage{ modelCalcME(paramData, exprData, startingModel, progress) } \arguments{ \item{paramData}{An object of class \code{\link[Biobase:class.phenoData]{phenoData}} containing the phenotypic information for the experiment.} \item{exprData}{A matrix of expression values for each gene for each sample. Usually calculated using \code{\link[Biobase]{exprs}} on an object of class \code{\link[Biobase:class.ExpressionSet]{ExpressionSet}}.} \item{startingModel}{An object of class \code{\link[stats]{formula}} containing the minimal model thta should be used in the step-wise model fitting process.} \item{progress}{Object of class \code{tclVar} indicating progress through the genes in the gene list. Used only if \code{widget=TRUE} for \code{\link[Envisage]{envisage}}.} } \details{ The \pkg{Envisage} package contains methods allowing the use of linear models (LMs) for analysing significantly changing genes in experiments with a variety of sources of variation, be they experimentally controlled variables such as drug treatment or time, or non-controlled sources of confounding variation such as phenotypic or environmental differences. This allows all sources of variation to be considered when analysing for significant differential expression, ensuring resulting genes are biologically relevent to the experimental question. The function \code{\link[Envisage]{modelCalcME}} performs the main modelling step for the package \pkg{Envisage}. A stepwise procedure is used on a per-gene basis to calculate the optimum model for each gene. In comparison to \code{\link[Envisage]{modelCalcINT}}, terms used in the model may be main effect terms only. The step-wise procedure begins by fitting the maximal model to the data, then removes terms in a leave-one-out approach based on the Akaike information criterion (AIC). Specification of a minimal model allows terms to be 'forced' into the final formula. The significance of each term in the final model is estimated by using a Type II sum of squares to compare the model with and without each term. Also, if fitting the model results in aliased terms (either due to experimental design, insufficient data, or non-independence of model terms), this information is also returned. \code{\link[Envisage]{modelCalcME}} is an internal function and is not designed to be accessed by users. } \value{ A list is returned with the following slots: \item{pValues}{An object of class \code{data.frame} containing the p-value from the Type II sum of squares analysis for each model term for each gene.} \item{failedAnova}{A vector of genes for which the Anova Type II sum of squares failed, indicating model aliasing.} \item{errorGenes}{A list of the genes that failed and the model terms for which aliasing was detected.} } \references{ Robson, S. C., Hunter, E., Bird, H., Turner, H. (2008) \emph{Envisage: model-based significance analysis of microarray gene expression data}, manuscript in preparation } \author{Sam Robson \email{S.C.Robson@warwick.ac.uk}} \seealso{For the interaction version of this function, see \code{\link[Envisage]{modelCalcINT}}. For the main \pkg{Envisage} method, see \code{\link[Envisage]{envisage}}.} \keyword{models} Envisage/man/INTlevels.Rd0000755003020100016370000000471111031122551015366 0ustar sr9signg00000000000000\name{INTlevels} \alias{INTlevels} \title{Calculate the total number of levels within a model for package Envisage} \description{This function calculates the total number of levels of all variables and interactions within the maximal model considered in the stepwise modelling procedure of \pkg{Envisage}.} \usage{ INTlevels(pData) } \arguments{ \item{pData}{An object of class \code{\link[Biobase:class.phenoData]{phenoData}} containing the phenotypic information for the experiment.} } \details{ The \pkg{Envisage} package contains methods allowing the use of linear models (LMs) for analysing significantly changing genes in experiments with a variety of sources of variation, be they experimentally controlled variables such as drug treatment or time, or non-controlled sources of confounding variation such as phenotypic or environmental differences. This allows all sources of variation to be considered when analysing for significant differential expression, ensuring resulting genes are biologically relevent to the experimental question. The function \code{\link[Envisage]{INTlevels}} calculates the total number of levels of all main effect and interaction terms in the maximal model considered within the stepwise modelling procedure of \pkg{Envisage}. If too many variables are included in the model, with a greater total of levels than there are data points, this may result in incorrect fitting of the model. This should be avoided by being aware of how many degrees of freedom each variable that you want to include will take up. If a variable of interest has a large number of possible levels, consider combining some of these to avoid over-fitting the data or only use this as a main effect term instead of considering all interactions. \code{\link[Envisage]{INTlevels}} is an internal function and is not designed to be accessed by users. } \value{ A vector is returned containing the total number of levels required for each main effect and first order interation of variables within the analysis. } \references{ Robson, S. C., Hunter, E., Bird, H., Turner, H. 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Such high-throughput data require suitably advanced methods of statistical hypothesis testing, such as analysis of variance, to ensure that measured changes in gene expression represent true biological events. Noise, due to a combination of technical, environmental, biological and random variation, can become a major problem in any microarray analysis and, whilst this can be reduced somewhat with careful experiment planning and implementation, statistical methods must be used to isolate real changes in gene expression from those occurring by random chance. To increase the power of statistical hypothesis testing techniques, replicate data must be collected~\cite{LEE}. It is all but impossible to design an experiment such that the variable(s) under the direct control of the experimenter (`parameter(s)') are the sole sources of variation within the experiment. Unavoidable environmental effects, nuisance variables such as batching effects, and phenotypic differences in \emph{in vivo} experimental samples will effect the response of the gene expression to the parameters of interest. These `covariates' must be considered when testing for significant changes in gene expression for the experiment parameters to ensure the biological relevence of the results. \subsection{Envisage} \textbf{Envisage} (Enable Numerous Variables In Significance Analysis of Gene Expression) is a package developed for the Bioconductor project in R~\cite{Bioconductor} that utilises an automated linear model (LM) selection process to extend ANOVA to a wide range of predictor variables, including experimental parameters (drug treatment, disease state, time point, etc.), phenotypic covariates (gender, age, height, weight, etc.) and nuisance covariates (such as batch effects). This allows the inclusion of a wider range of variables, be they interesting experiment parameters or unavoidable covariates, when testing for significance. The inclusion of such confounding variables in the significance tests provides a more accurate representation of the true biology of the system under study. This process would be particularly useful for clinical studies, where the between-sample variation can be very high within each condition due to the difficulties in complete patient matching in a clinical setting. The \textbf{Envisage} package has been designed to be user friendly, with a simple to use graphical user interface (GUI), described in section \ref{sec:GUI}, for those that don't want to use the command line interface of R. Also, \textbf{Envisage} can be run on an object of class \emph{ExpressionSet} within the R environment, or alternatively can be run directly from the gene expression analysis suite GeneSpring GX\footnote{Agilent Technologies, Santa Clara, CA}. Use of \textbf{Envisage} within Bioconductor is described in \ref{sec:programBC}, and through the GeneSpring GX plug-in external program interface in section \ref{sec:programGS}. \subsection{The Envisage Modelling Procedure} \label{sec:model} This section gives a brief overview of the steps involved in the \textbf{Envisage} model selection procedure. The input file for \textbf{Envisage} is an object of class \emph{ExpressionSet}, created within Bioconductor (section \ref{sec:programBC}) or created automatically using the GeneSpring EPI plugin (section \ref{sec:programGS}). This object contains information about probe level signals for the array, details on experimental variables for each sample, and annotation lists that can be used to link probes to their respective gene targets. The model fitting procedure is a two step operation. First, a model is fit containing only main effect variables. Next, a more complex model is fit based on the first order interactions of variables found to be significant in the first step. This model is selected by first fitting a saturated model containing all available terms, and then removing terms based on the Akaike information criterion. This is essentially a penalised log-likelihood function that determines the benefit of keeping terms in the model with respect to its explanatory power; if the addition of the term has no clear benefit to the fit of the model, it is deemed unnecessary and removed. This two-step method was chosen to reduce run-time at the risk of missing important interaction information. A type II ANOVA test is used to test the significance of each of the model terms for each gene. The model for each gene is compared with and without each of the terms, and a p-value is calculated using an F-test statistic. A table of unadjusted p-values is created with genes as rows and all model terms as columns. It is recommended that a multiple testing correction procedure such as the Benjamini \& Hochberg false discovery rate~\citep{BH} adjustment be applied to the p-values to correct the matrix of p-values to take the large number of individual statistical test performed into account. Available multiple testing corrections can be found in section \ref{sec:ArgInfo}). Corrected p-values are compared to the specified p-value cutoff to test for significant differential expression. Genes showing a significant ANOVA F-test p-value for a particular term are considered to be changing significantly based on that variable. A list of genes with significant p-values is output to the user for each term as a tab-delimited text files. For more details on the model selection procedure, please see Robson \emph{et al.}(manuscript in preparation). %%% %%% %%% Chapter 2: Getting Started %%% %%% \newpage \section{Getting Started} The following description of the \textbf{Envisage} package is focused on the analysis of single-channel microarray data such as Affymetrix GeneChips, but the process can easily be extended to two-colour microarray and even other high-throughput data such as mass spectrometry data. The primary input file for the package is an object of class \emph{ExpressionSet}, a class of objects in R for representing microarray data. The object must contain all necessary gene expression information and phenodata (experiment variable information) for each sample, and also annotation information for the genome of interest. Relevant information for MIAME (Minimum Information About a Microarray Experiment) compliance can also be entered~\cite{MIAME}. \textbf{Envisage} can be run within the R environment using an \emph{ExpressionSet} object created using the appropriate package in Bioconductor (\textbf{affy} for 1-colour data), or the package can be run directly through the Genespring GX analysis suite, which will automatically create the required object from the current Genespring GX experiment interpretation. This chapter gives information on setting up the R environment, installing the Bioconductor packages, installing the \textbf{Envisage} package, and creating the required Bioconductor object (both in Bioconductor itself and Genespring GX). Information for running the program itself can be found in section \ref{sec:program}. %%% %%% %%% \subsection{Getting Envisage Up and Running} \subsubsection{Installing R and Bioconductor} \label{sec:Bioc} \textbf{Envisage} is a package available from Bioconductor, a publicly available collection of packages for the statistical programming language R, allowing statistical and graphical methods for analysis of gene expression data. The R programming environment can be downloaded from the comprehensive R network archive (\emph{CRAN}) at \url{www.cran.r-project.org}. The current release, R-2.6-0, was released on 2007-10-03. The latest release of Bioconductor (Bioconductor 2.1 -- 2007-10-08) can be found at \url{www.bioconductor.org}. The simplest way to set up Bioconductor is to run it from the R environment, using the \emph{biocLite.R} installation script. This will install the basic packages required for gene expression analysis; \textbf{affy, affydata, affyPLM, annaffy, annotate, Biobase, Biostrings, DynDoc, gcrma, genefilter, geneplotter, hgu95av2, limma, marray, matchprobes, multtest, reposTools, ROC, vsn} and \textbf{xtable}. To do this, open the R environment, and type the following in the command line: <>= source('http://bioconductor.org/biocLite.R') biocLite() @ This will establish a connection to the Bioconductor website and initialise installation. To install additional packages, use the package name as an argument in the biocLite.R method. e.g. To install packages \textbf{affy} and \textbf{marray}, type the following in the command line: <>= biocLite(c('affy','marray')) @ The \emph{getBioC.R} installation script can also be used, which contains a larger number of default packages than the \emph{biocLite.R} script. To install this, follow the above steps, replacing \emph{`biocLite'} with \emph{`getBioC'}: <>= source('http://bioconductor.org/getBioC.R') getBioC() @ Once packages have been installed, they must be loaded into the R environment to allow use of the methods contained within the package. This must be done at the start of every session. To do this, use the \texttt{library()} method. e.g. To load package \textbf{affy}, type the following into the command line: <>= library('affy') @ Packages can also be downloaded, installed and loaded from the `\emph{Packages}' tab at the top of the R shell. Selecting `\emph{Install package(s)}' brings up a list of available mirrors for the CRAN depository. Select the most suitable mirror, and a list of available packages is displayed. Simply select the package of interest and choose '\emph{OK}'. The package will be downloaded and installed automatically. Selecting `\emph{Load package\ldots}' brings up a list of packages available to be loaded. Selecting the required package and selecting `\emph{OK}' has the same effect as using the \texttt{library()} function. Bioconductor packages are well documented, with help files available for all user-level functions. Every package contains at least one \emph{vignette}, an executable document consisting of a collection of code and documentation text, providing information regarding usage of the package. These vignettes can be found in the \emph{doc} subdirectory of an installed package, or can be called from the R command line using the \texttt{openVignette()} method from the Biobase package. To view documentation for package \textbf{affy}, type the following into the command line: <>= library('Biobase', 'affy') ##Ensure that both packages are loaded openVignette(package='affy') ##Bring up list of vignettes @ Help files for particular methods can be called internally within R by using the following methods, replacing `\texttt{affy}' with the name of the required method: <>= help.start() help.search('affy') help(affy) ?affy apropos(affy) example(affy) demo() demo(affy) @ \subsubsection{Installing Additional Required Packages in R} \textbf{Envisage} requires the following additional packages which can be obtained from Bioconductor as previously described: \begin{description} \item [\textbf{multtest}] Multiple testing procedures for controlling the family-wise error rate (FWER) and false discovery rate (FDR) \item [\textbf{GeneSpring (OPTIONAL)}] Package for creating \emph{ExpressionSet} object from GeneSpring GX experiment interpretations \item [\textbf{tcltk}] Package for integration of Tcl/Tk libraries and widgets within the R environment \item[\textbf{tkWidgets}] Package containing R based widgets from the Tcl/Tk libraries \end{description} \textbf{Envisage} also requires the following additional packages which can be obtained from the comprehensive R archive network (CRAN) by going to the CRAN website at \href{http://www.r-project.org/}{http://www.r-project.org/}, selecting ``\texttt{CRAN}'' under `downloads', and selecting a CRAN mirror site from the ones available. Available packages can be obtained by clicking on ``\texttt{Contributed extension packages}'': \begin{description} \item [\textbf{car}] Package accompanying J. Fox, \emph{An R and S-PLUS Companion to Applied Regression} (2002) and contains functions for applied regression, linear models, and generalized linear models, including the method \texttt{Anova()} which allows Type II ANOVA testing. \end{description} %%% %%% %%% \subsubsection{Installing Envisage in R} \textbf{Envisage} is available as a package within Bioconductor and can be installed directly through the R shell from the `\emph{Packages}' tab as described previously in section \ref{sec:Bioc}. Alternatively, the package source files can be obtained from the author's website at \href{http://www2.warwick.ac.uk/fac/sci/moac/currentstudents/2003/sam\_robson/linear\_models/downloads/}{www2.warwick.ac.uk\-/fac\-/sci\-/moac\-/currentstudents\-/2003\-/sam\_robson\-/linear\_models\-/downloads/}. To install the package in R, please do one of the following: \begin{description} \item[UNIX/LINUX] To install the \textbf{Envisage} source file (\texttt{.tar.gz}), first ensure that your \texttt{.../R/bin/} path is included in your PATH environment variable, and type the following into the terminal: ``R CMD INSTALL [full path to source tar.gz file]''. \item[WINDOWS] To install the \textbf{Envisage} binary file on a machine running windows (\texttt{.zip})go to the `\emph{Packages}' tab and select `\emph{Install package(s) from local zip files...}'. Navigate to the destination folder to which the \textbf{Envisage} .zip file has been saved and select it. R will install the package automatically. \end{description} To load the package in R, simply use the \texttt{library()} method: <>= library(Envisage) @ %%% %%% %%% \subsubsection{Installing Tcl/Tk} The graphical interface for \textbf{Envisage} has been developed using the R based integration of Tcl/Tk developed by Peter Dalgaard in the \textbf{tcltk} package. This package can be installed directly through the R shell from the `\emph{Packages}' tab as described previously in section \ref{sec:Bioc}. The \textbf{Envisage} GUI also uses widgets that are currently only implemented through the \textbf{BWidgets} and \textbf{Iwidgets} Tcl/Tk packages that are currently not supported by \textbf{tcltk}. To allow integration of R with these packages, users must install a current version of Tcl/Tk, such as the ActiveTcl distribution from ActiveState\footnote{\href{http://www.activestate.com}{www.activestate.com}}. The following is a step-by-step guide to installing ActiveTcl and loading the \textbf{BWidgets} and \textbf{Iwidgets}package for use in R: \begin{enumerate} \item Download the current distribution of ActiveTcl (release 8.4.16) free from the developer's website at \href{http://www.activestate.com/Products/activetcl/}{www.activestate.com\-/Products\-/activetcl/}. \item Install ActiveTcl as instructed. \item \textbf{For Windows} Go to your computers advanced system settings and select `\emph{Environmental Variables}'. Under \texttt{System Variables}, select `\emph{New...}' to create a new environment variable called \texttt{MY\_TCLTK} with value `\texttt{yes}'. \textbf{For LINUX} Create a new global environment variable called \texttt{MY\_TCLTK} with value `\texttt{yes}' by updating your profile file (eg .bash\_profile) by typing the following into the shell: export MY\_TCLTK = ``yes'' \item Repeat the above to create a new environment variable called \texttt{TCL\_LIBRARY} and set the value to the library path of your installation of ActiveTcl (e.g. ``\texttt{C:/Tcl/lib}'' if you keep the default settings). \item In R, use the \texttt{addTclPath(path)} method to tell R where to find the ActiveTcl library files, where \texttt{path} is the library path of your installation of ActiveTcl (e.g. ``\texttt{addTclPath(`C:/Tcl/lib')}'' if you keep the default settings). \end{enumerate} You will now be able to use widgets from all packages contained in the full version of Tcl/Tk. %%% %%% %%% \subsubsection{(OPTIONAL) Installing GeneSpring-R Integration External Program Interface} \label{sec:GSepi} A script is available for those using Genespring GX for analysis of their microarray data, which allows use of the \textbf{Envisage} package from within the Genespring GX environment. This script will export the selected experiment interpretation and gene list into an R environment, and use these to create a Bioconductor \emph{ExpressionSet} object. Genespring GX uses an External Program Interface (EPI) to allow interaction between the Genespring GX environment and the R environment. A file, \texttt{GS\_R\_in.txt}, is created in the Genespring GX \emph{`Data'} folder, and the batch file \texttt{GS\_exec\_R\_GUI.bat} opens the R environment directly. The function \texttt{GSload.int} in package \textbf{GeneSpring} can be called on the input file \texttt{GS\_R\_in.txt} to load the Genespring GX experiment interpretation as an object of class \texttt{GSint}. This can be converted directly to an object of class \emph{ExpressionSet} using the \textbf{GeneSpring} function \texttt{GSint2BC()}. Note: The current \texttt{GSint2BC()} is defunct since it still uses the old \texttt{exprSet} class for Bioconductor objects. The \textbf{Envisage} package contains a modified function, \texttt{GS2BC}, which has been updated to create a Bioconductor object of class \emph{ExpressionSet}. The R script \texttt{Envisage.R} runs the function \texttt{Envisage()} on the GeneSpring \texttt{ExpressionSet} object. Output genelists are returned in a tab-delimited text format as described in section \ref{sec:output} that can be read into the GeneSpring environment using the `\emph{Load Gene List From File}' and `\emph{Load Gene List With Numbers From File}' JAVA class external plugins. These are included in the Genespring-R integration package, and can be found in the \emph{`External Programs'} folder to the left of the main Genespring GX window). The following is a step-by-step guide to installing the GeneSpring-R Integration EPI to allow analysis of microarray data using \textbf{Envisage} directly through GeneSpring GX: \begin{enumerate} \item Download the \textbf{Genespring-R} integration package \emph{.zip} file from the Agilent website using one of the following links: \begin{itemize} \item For WINDOWS including R program (2.1.0) and BioConductor \& GeneSpring library -- \href{http://www.chem.agilent.com/cag/bsp/SiG/Downloads/zip/gs\_r\_winall.zip}{www.\-chem.\-agilent.\-com\-/cag\-/bsp\-/SiG\-/Down\-loads\-/zip\-/gs\_r\_winall.zip} \item For WINDOWS NOT including R program or any library -- \href{http://www.chem.agilent.com/cag/bsp/SiG/Downloads/zip/gs\_r\_win.zip}{www.\-chem.\-agilent.\-com\-/cag\-/bsp\-/SiG\-/Downloads\-/zip\-/gs\_r\_win.zip} \item For UNIX NOT including R program or any library -- \href{http:///www.chem.agilent.com/cag/bsp/SiG/Downloads/zip/gs\_r\_unix.zip}{www.\-chem.\-agilent.\-com\-/cag\-/bsp\-/SiG\-/Downloads\-/zip\-/gs\_r\_unix.zip} \end{itemize} \item Unzip the \textbf{Genespring-R} integration package directly in Genespring GX by dragging the \emph{.zip} file into any active Genespring GX window. Follow the setup procedure as instructed. \item Download and install the \textbf{GeneSpring} Bioconductor package from the Bioconductor website as described in section \ref{sec:Bioc}. \item Download the files \texttt{Envisage.programdef} and \texttt{Envisage.R} from the author's website at \href{http://www2.warwick.ac.uk/fac/sci/moac/currentstudents/2003/sam\_robson/linear\_models/downloads/}{www2.warwick.ac.uk\-/fac\-/sci\-/moac\-/currentstudents\-/2003\-/sam\_robson\-/linear\-\_models\-/downloads/}. The \texttt{Envisage.programdef} file should be added to the \emph{``\ldots/Agilent/\-Genespring GX/\-data/\-Programs''} folder, and the \texttt{Envisage.R} file should be added to the \emph{``\ldots/Agilent/\-Genespring GX/\-data''} folder. Upon loading Genespring GX, the external program \textbf{Envisage} will be available from the \emph{``External Programs''} folder (see figure \ref{fig:GS}). \end{enumerate} \begin{figure} \centerline{ \includegraphics[width=100mm]{GS.png} } \caption{Selecting Envisage from Genespring GX.} \label{fig:GS} \end{figure} %%% %%% %%% \subsection{Requirements} The following are required for package \textbf{Envisage}: %%% %%% %%% \begin{itemize} \item R version 2.6.1 or higher \item Bioconductor version 1.9 or higher \item tkWidgets version 1.16.0 or higher \item R packages \textbf{tcltk, car} \item Bioconductor packages \textbf{affy, multtest} \item Bioconductor package \textbf{GeneSpring} (if pre-processing data using Genespring GX) \item tcl packages \textbf{BWidgets} and \textbf{Iwidgets} (available as part of \href{http://www.activestate.com/products/activetcl/}{ActiveTcl}.) \end{itemize} %%% %%% %%% \subsection{Data Pre-Processing} Pre-processing and sample quality control must be carried out before using \textbf{Envisage} to avoid biasing the modelling procedures. This can be performed in Bioconductor, or within the gene expression analysis suite GeneSpring GX. The modelling procedure uses log-transformed gene expression data. In this way, the additive linear model corresponds to a multiplicative model on the original non-logged expression values. \textbf{Envisage} will automatically adjust the gene expression data if it is not log-transformed, but users are encouraged to perform this step before beginning the procedure. It is also recommended that in the quality control step, appropriate normalisation steps are used to limit the effects of outlying samples. It is left to the user to decide the level to which this quality control is performed. Quality control of genes should also be carried out across the samples, firstly to limit the number of genes used in the calculation (thus saving on run-time), and secondly to ensure that erroneous genes do not elicit a significant effect. For instance, non-changing genes should be removed from the experiment interpretation to avoid biasing in the multiple testing correction stage. The \emph{ExpressionSet} class is the basic container of microarray data within Bioconductor and contains the following slots: \begin{description} \item[\texttt{phenoData}] Variable information describing the phenotype for a microarray sample (such as age, weight, treatment, etc.). This is stored as an object of class \texttt{AnnotatedDataFrame}, which itself contains two slots; the \texttt{phenoData} slot containing the phenotype data, and the \texttt{varMetaData} slot containing information about the phenotype variables. See \texttt{help(class:AnnotatedDataFrame)} for more details. \item[\texttt{experimentData}] Experimental information required for MIAME compliance \citep[see][]{MIAME}. See \texttt{help(class:MIAME)} for more details. \item[\texttt{annotation}] Label associated with experiment annotation. \item[\texttt{exprs}] A matrix of gene expression values for each sample. \end{description} These slots can be filled either directly in Bioconductor, using the \textbf{affy} package for 1-colour data, or the \textbf{marray} package for 2-colour data (for further assistance on data pre-processing in Bioconductor, please see the appropriate package vignette), or by using the GeneSpring-R integration EPI as described previously in section \ref{sec:GSepi}. %%% %%% %%% Chapter 3: Running Envisage %%% %%% \newpage \section{Running Envisage} \label{sec:program} \subsection{Initialisation of Envisage} \subsubsection{Initialising Envisage from Bioconductor} \label{sec:programBC} To run \textbf{Envisage} from Bioconductor, follow these steps: \begin{enumerate} \item Load the R environment as normal. Create an object of class \emph{ExpressionSet} containing all of your experiment data (for instance, for Affymetrix data use the \texttt{ReadAffy()} function from package \textbf{affy} to load in information from the experiment \texttt{.CEL} files). \item Ensure that package \textbf{Envisage} has been correctly installed and load it as described in section \ref{sec:Bioc}. \item Either load the \textbf{Envisage} GUI by using the function call \texttt{envisage()} with argument \texttt{widget} set to TRUE: <>= library(Envisage) data(SkinvsPancreas) @ <>= envisage(expData, widget=TRUE) @ Or run \texttt{envisage()} through the command line by setting the argument \texttt{widget} to FALSE and providing further arguments as described in section \ref{sec:ArgInfo}. \emph{`expData'} is the name of the experiment \emph{ExpressionSet} object for analysis. Use of the \textbf{Envisage} GUI is described in section \ref{sec:GUI}. If the GUI is used, the results will be assigned to a global variable `\texttt{results}' so please be sure that this will not overwrite any other objects within the R environment. If using \texttt{envisage()} through the command line, users can assign the results to an object of their choice. \end{enumerate} % % % \subsubsection{Initialising Envisage from Genespring} \label{sec:programGS} To run \textbf{Envisage} from GeneSpring GX, follow these steps: \begin{enumerate} \item Ensure that the \textbf{GeneSpring} Bioconductor package has been downloaded and installed as described in section \ref{sec:Bioc}. \item Ensure that the \texttt{Envisage.programdef} and \texttt{Envisage.R} files have been downloaded and installed as described in section \ref{sec:GSepi}. \item Ensure that the Genespring-R integration package has been downloaded and installed as described in section \ref{sec:GSepi}. \item Load the Genespring GX environment as normal. Ensure that \emph{`Envisage'} is available from the \emph{`External Programs'} folder (figure \ref{fig:GS}). \item Select your normalised experiment interpretation and curated gene list and ensure that the \emph{`All Samples'} experiment interpretation is selected. Selecting the \emph{`All Samples'} interpretation ensures that samples are not grouped. \item Finally, select the \emph{Envisage} icon in the \emph{`External Programs'} drop-down menu to the left of the interpretation view window. This will load the \textbf{Envisage} GUI, as described in section \ref{sec:GUI}. \end{enumerate} \subsection{Envisage Graphical User Interface} \label{sec:GUI} \textbf{Envisage} utilises a graphical user interface (GUI) for input of arguments to the \texttt{envisage()} method. This prevents errors in argument input. \begin{figure} \centerline{ \includegraphics[width=100mm]{EnvisageGUI.png} } \caption{The Envisage GUI.} \label{fig:GUI} \end{figure} \subsubsection{Selection of Variables Classes} The main GUI window (figure \ref{fig:GUI}) lists the experiment interpretation variables, and allows the user to specify which of these to use in the model calculations, and what class of variable they are. The experiment interpretation may contain several variables that contain identical information (for instance, one variable may contain a range of values, and another may contain the averages of those ranges). Any such correlated variables are flagged with star symbols (`*', `**', `***', etc) which should not be confused with similar flags used for significance in ANOVA tables. Only one of these highly correlated variables should be used in the modelling procedure to avoid overfitting of the data, so users should be sure to select \texttt{`None'} for all but at most one variable flagged with the same number of stars. Selecting more than one may result in overfitting of the data and errors in the hypothesis tests. Users must specify the class of each of the variables to be used in the model by using the variable class radio buttons. By default, \texttt{None} is selected for all variables. Selecting \texttt{None} prevents a variable from being included in the modelling procedure. The two available classes are \texttt{numeric}, for variables where each value has a numerical relevance (e.g. weight, height, etc.), and \texttt{categoric}, for variables that have a small number of specific factor levels (e.g. gender, disease state, etc.). Choosing the correct class for each of the variables is essential for the modelling procedure. Selecting an incorrect class can result in the modelling procedure failing. The automated model selection process occurs in two steps. The first step optimises a model with only main effect terms to find those variables that have the greatest effect on the data. The second step calculates a model including first order interactions for the variables from the main effect model. However, the user can choose to consider only main effect terms if necessary. This may be done as a preliminary observation to see which variables are of interest, or possibly because the number of samples is low compared to the number of variables of interest, providing few degrees of freedom for the model. If the user chooses to look at interaction terms, but there are too many variable terms compared to the number of samples, the user will be prompted to remove some variables from the first order interaction calculations (figure \ref{fig:INTselect}). The number of degrees of freedom for the fitted model is dependent on the number of samples, so the size of the experiment will determine the variables that can be included. If some categoric variables have a particularly large number of levels, it is recommended to remove these from the interaction calculations to avoid over-fitting of the data. To ensure that variable names do not cause an internal error within R, they may be renamed by the program to ensure compatability. For instance, R does not like objects to have names beginning with a numeric character (e.g. `\texttt{2-Cycle batch}'). If such a name is discovered, it will be prefixed with the letter `\texttt{X}' to prevent errors. Also, any hyphens, slash characters or spaces will be removed and replaced with the character '\texttt{\_}' for the same reason. \begin{figure} \centerline{ \includegraphics[width=50mm]{INTselect.png} } \caption{Selection of variables for which to calculate interactions.} \label{fig:INTselect} \end{figure} \subsubsection{Envisage GUI Arguments} \begin{description} \item [Multiple Testing Correction] A number of multiple testing correction algorithms are available for use with the \textbf{Envisage} package. They are called through the Bioconductor package \textbf{multtest}. More information can be obtained by reading the \textbf{multtest} package vignette. It is suggested that a multiple testing correction procedure is used due to the large number of genes being analysed. A list of available multiple testing corrections can be found in section \ref{sec:ArgInfo}). A false discovery rate correction is recommended, and the method of Benjamini and Hochberg is selected by default ~\cite{BH}. \item [Model Terms] The user can choose to model only main effect terms with no interactions. If there are a large number of variables to model compared to the number of samples, this may be a useful preliminary analysis to find the most significant variables which may then be used with interactions. \item [Starting Model] If users have some idea of the variables that they would like to see in the per-gene model, they can specify the minimum model to force these terms to be present for all genes. In this way, the effect of variables of interest to the user can be studied whilst allowing for leeway in the selection of further terms for each gene. Variables are identified by a number with the prefix `p'. These identifiers are used to specify the model. For instance, if you want to force variables `p1' and `p2' into the per-gene model, type `p1 + p2' into the appropriate field. The syntax for this field follow the usual standards for model entry, with `+' indicating inclusion of a variable, `:' indicating an interation between two or more variables, and `*' indicating the inclusion of two or more variables and all possible interactions (e.g. ``A*B'' = ``A + B + A:B''). \item [P-Value Cutoff] The \emph{p-Value Cutoff} defines the significance cutoff for the type II ANOVA tests. A default of 0.05 is used by convention. \item [Results folder] Finally, the folder in which to save the output files (described in section \ref{sec:output}) can be specified. A new folder named \emph{EnvisageResults} is created within the specified directory, containing all of the \textbf{Envisage} output files. \end{description} % % % \subsection{Envisage Output} \label{sec:output} Once the modelling procedure is completed, results are output for significantly changing genes for each term within the analysis. If the GUI is used, a new window is created (figure \ref{fig:output}) allowing the user to specify for which of the variables (and subsequent first-order interactions) to output the results. If the output directory \emph{EnvisageResults} contains files from a previous analysis, these will be suffixed with the letters \texttt{\_OLD} to avoid overwriting data. \begin{figure} \centerline{ \includegraphics[width=80mm]{EnvisageOutput.png} } \caption{The Envisage output selection GUI.} \label{fig:output} \end{figure} For each model term, the column \emph{`All Changing Genes'} gives the percentage of the genes that show the term as significant in the model. The column \emph{`Significantly Changing Genes'} gives the percentage of all genes that change significantly and also have a corrected p-value less than the specified cutoff range. These values give the user a brief idea as to which of the model terms have a significant effect on the gene expression. Tick the boxes for all model terms that you require results for and press \emph{OK} (selecting `\emph{Check All}' will output all significant results). This will save the gene names and corresponding p-values into a tab-delimited text format in the folder \emph{EnvisageResults} in the specified folder. These files are named based on the variable names. Any spaces in the variable names are replaced by `\_' to ensure file name compatability. Due to the convention of specifying the interaction of two variables using a colon (e.g. \texttt{param1:param2}), which is not a valid character for specifying filenames, it is replaced by the characters \emph{`\_INT\_'}. The \emph{EnvisageResults} folder also contains information regarding genes that show problems in the ANOVA tests. We can see aliasing errors in the ANOVA calculation for interactions due to one of two reasons: \begin{enumerate} \item Extrinsic aliasing is due to the structure of the data -- There is no relevant data for a particular interaction group (e.g. there are no diseased patients in a particular age group, so the effects for the interaction \emph{diseased:age} cannot be estimated). \item Intrinsic aliasing is due to the structure of the data -- Particular effects cannot be separated adequately (e.g. all diseased individuals in a particular age group also have the same blood group. In this case we cannot separate the effects in \emph{diseased:age} and \emph{diseased:blood group}). \end{enumerate} Extrinsic aliasing will generally occur due to the use of an unbalanced data set, but will not cause a problem with the significance calculations. Intrinsic aliasing is indicative of correlation between explanatory variables. So the model will fit one variable and then try to fit the same information again with the second variable. This may then pose a problem since the significance results will depend on the order in which the terms are fitted. The two files produced, \emph{AliasGenes} and \emph{AliasReport}, give information on the genes showing intrinsic aliasing. \emph{AliasGenes} is a list of the aliased genes in a tab-delimited text file. \emph{AliasReport} gives more detailed information showing exactly which of the variable levels are aliased. This may be useful for users to spot correlation between their variables that may be unexpected. Users may wish to run the program a second time with one of the two aliased variables removed. If for some reason a gene gives a model for which the ANOVA calculation fails, the name of the gene is output to a file called \emph{FailedAnova}. This file should generally be absent. %%% %%% %%% Chapter 4: Program Specifications %%% %%% \newpage \section{Program Specifications} % \subsection{User Methods} The following methods are available for users: \begin{description} \item[\textbf{envisage}] This is the main function for the \textbf{Envisage} package. It takes in microarray gene expression data and variable phenodata in the form of a Bioconductor object of class \emph{ExpressionSet}, and returns a list of genes that vary significantly based on the experiment variables and their first-order interactions. \item[\textbf{GS2BC}] Updated version of the \texttt{GSint2BC()} function from package \textbf{GeneSpring} which converts GeneSpring experiment interpretation data into an object of class \emph{Express\-ionSet} for use with \textbf{Envisage}. \end{description} %%% %%% %%% \subsection{envisage() Argument Definition} \label{sec:ArgDef} The following arguments are required for the user-level function \texttt{envisage()}: \begin{description} \item[\textbf{expData}] The data construct, of class \emph{ExpressionSet}, containing all gene expression and phenotypic data for the experiment. \item[\textbf{widget}] A logical value specifying whether or not to use the widget based GUI for argument specification. This is set to \texttt{TRUE} by default. \item[\textbf{MTC}] Multiple testing correction to use for correcting calculated p-values (see section \ref{sec:ArgInfo}). \item[\textbf{pCutoff}] A number defining the p-value cutoff to be used in significance analysis. Must be between 0 and 1. \item[\textbf{useParams}] A character vector that defines which of the experiment variables to use in the model calculation. \item[\textbf{param4INT}] A character vector that defines the model variables for which interactions should be calculated. \item[\textbf{paramType}] A character vector specifying the classes (``\emph{numeric}'' or ``\emph{categoric}'') of the variables in the model. \item[\textbf{fileResults}] A character string defining the folder in which to save the results of the modelling procedure. \item[\textbf{MEorINT}] A character string defining whether to consider only main effect terms in the model(``\emph{ME}''), or to look at first order interactions(``\emph{INT}''). \item[\textbf{startModel}] A character string representing the starting model for analysis. Must be of the form ``$x \sim term1 + term2 + term1:term2 + $ \ldots''. Care must be taken to ensure that the model terms match the names of variables specified in \texttt{useParams}. \end{description} The following are the default argument terms: <>= envisage(expData, widget=TRUE, MTC=NULL, pCutoff=NULL, useParams=NULL, param4INT=NULL, paramType=NULL, fileResults=getwd(), MEorINT="INT", startModel=NULL) @ The following function call is sufficient if you want to run \textbf{Envisage} using the GUI: <>= envisage(expData) @ \subsection{envisage() Argument Information} \label{sec:ArgInfo} The following information is necessary when defining the arguments for \texttt{envisage()}: \begin{description} \item[\textbf{expData}] Try to ensure that explanatory variables are labelled sensibly. Avoid beginning variable names with a numeric character and using special characters in variable names. \textbf{Envisage} contains several basic internal functions for renaming variables to avoid these problems, but this should be considered by the user. Spaces are acceptable in variable names, but will be replaced by an underscore in output files. \item[\textbf{widget}] Default is `\texttt{TRUE}'. If set to \texttt{FALSE}, great care must be taken to ensure that other variables are correctly implemented. \item[\textbf{MTC}] Multiple testing corresction procedures are implemented through the package \textbf{multtest}. Available multiple testing procedures are as follows: \begin{enumerate} \item ``\textbf{NA}'' -- No MTC selected \item ``\textbf{BH}'' -- (Default) Benjamini \& Hochberg step-up false discovery rate (1995)~\cite{BH} \item ``\textbf{BY}'' -- Benjamini \& Yekutieli step-up false discovery rate (2001)~\cite{BY} \item ``\textbf{Bonferroni}'' -- Bonferroni single-step family wise error rate \item ``\textbf{Holm}'' -- Holm step-down family wise error rate (1979)~\cite{Holm} \item ``\textbf{Hochberg}'' -- Hochberg step-up family wise error rate (1988)~\cite{Hochberg} \item ``\textbf{SidakSS}'' -- Sidak single-step family wise error rate \item ``\textbf{SidakSD}'' -- Sidak step-down family wise error rate \end{enumerate} \item[\textbf{pCutoff}] Must be between $0$ and $1$. Default is $p=0.05$. \item[\textbf{useParams}] Users must ensure that no labelling variables are included in the list of variables to use. Users must also ensure that only one of any group of highly correlated variables is used, so be careful to select variables for modelling that represent different aspects of the data. Also be careful to ensure constant naming for all variables. This problem can be avoided by using the \textbf{Envisage} GUI. \item[\textbf{param4INT}] Be careful to ensure constant naming with variables specified in the \texttt{useParams} argument. This problem can be avoided by using the \textbf{Envisage} GUI. \item[\textbf{paramType}] Variable class must be one of ``numeric'' or ``categoric''. Users must be sure that the order of the variable classes is correct to ensure that variables are assigned the correct class. Assigning the wrong class to a variable can have a dramatic impact on the modelling procedure. This problem can be avoided by using the \textbf{Envisage} GUI. \item[\textbf{fileResults}] Default is the working directory for R. Be sure to choose a directory that can be accessed. \item[\textbf{MEorINT}] Default is ``\texttt{INT}''. \item[\textbf{startModel}] This argument allows the user to specify a minimum model for use in the modelling procedure to force terms into the selected model. This model must be specified as a character string of the form ``$x \sim term1 + term2 + term1:term2 + $ \ldots'', which will be converted internally to an object of class formula. Care must be taken to ensure that the model terms match the names of variables specified in \texttt{useParams}. \end{description} %%% %%% %%% \subsection{Non-User Methods} The following methods are called by the user-level function \texttt{envisage()}, and should not be directly called by the user: \begin{description} \item[\textbf{envisage.data}] This function takes the experiment data and extracts all relevant information (gene names, variable names, gene expression levels, etc) and assigns them to individual objects in R for future use. This function also ensures that the gene expression data is logged, so \texttt{envisage()} can take both logged and non-logged data as its argument. \item[\textbf{alias.glmNA}] This function directly extends the function \texttt{alias()} to objects of class \texttt{glmNA}. Class \texttt{glmNA} directly extend the classes \texttt{lm} and \texttt{glm} to objects that show aliasing in the modeling procedure. This allows the use of the type II ANOVA function. \item[\textbf{coef.glmNA}] This function directly extends the function \texttt{coef()} to objects of class \texttt{glmNA}. Class \texttt{glmNA} directly extend the classes \texttt{lm} and \texttt{glm} to objects that show aliasing in the modeling procedure. This allows the use of the type II ANOVA function. \item[\textbf{corParams}] This function flags highly correlated variables that may model the same information in the model. Parameters showing a correlation greater than 0.98 are flagged. \item[\textbf{labelParams}] This function removes any labelling variables (variables whereby each sample has a unique value) from the experiment interpretation. \item[\textbf{modelCalcME}] This function uses a stepwise method to fit a linear model for each gene based on main effect variables. \item[\textbf{modelCalcINT}] This function takes the main effect model of \texttt{modelCalcME} and uses only the significant terms to fit a linear model including first-order interaction terms in a stepwise process for each gene. A type II ANOVA calculation is used to calculate significance p-values for each model term based on an F-statistic. \item[\textbf{mulTestCor}] This function corrects the p-values for the type II ANOVA based on the specified multiple testing correction. \item[\textbf{result.output}] This function compares the significance p-values for each gene and variable with the specified p-value cutoff. If a gene has a p-value within the limit for a particular variable or interaction, then that gene is added to a gene list for the variable. These gene lists are output to a folder specified by the user. \item[\textbf{INTlevels}] This function calculates the number of levels for each main effect and first-order interaction term in the linear model. This is used to check model degrees of freedom against available degrees of freedom. \item[\textbf{modelEntry}] This function allows users to specify a minimum model for the model selection process. \end{description} %% %% %% \subsection{S3 Classes} The following classes are defined in the package \textbf{Envisage}: \begin{description} \item[\textbf{class-glmNA}] Class \texttt{glmNA} directly extend the classes \texttt{lm} and \texttt{glm} to objects that show aliasing in the linear model. \end{description} %%% %%% %%% %%% %%% %%% Chapter 5: Example Envisage session %%% %%% \newpage \section{An Example Envisage Session} \label{sec:example} We now demonstrate an example session using an example data set, \texttt{SkinvsPancreas}, available as part of the \textbf{Envisage} package. The example data set is an object of class \emph{ExpressionSet} made up of gene expression levels for 50 probes over 48 individual Affymetrix MOE430 Plus 2 mouse GeneChips. This is a subset of a larger data set for which a large number of experiment variables were recorded (Robson et al., In Preparation). The experiment design is multi-factorial, with 3 main explanatory variables; tissue type (2 levels - skin and pancreas), time point after activation of the Myc protein (4 levels - 4, 8, 16 and 32 hours) and status of the Myc transgene (2 levels - Myc ON or Myc OFF). Three batching variables correspond to batching of samples at the tissue extraction stage, RNA extraction stage and the microarray hybridisation stage. Further experiment information, such as RNA quality and yield, was collected providing a total of 12 variables. This data set is thus well suited for use with \textbf{Envisage}. The first step is to load the package into R, then load the example data set: <>= library('Envisage') data(SkinvsPancreas) @ This will load the example \emph{ExpressionSet} object \texttt{SkinvsPancreas} into the R environment: <<>>= SkinvsPancreas @ Parameter names can be seen by looking at the \texttt{phenoData} slot of \texttt{SkinvsPancreas}: <<>>= names(pData(SkinvsPancreas)) @ \subsection{Using the GUI} First, we will look at an example of how to run \texttt{envisage()} using the GUI: <>= envisage(SkinvsPancreas) @ The GUI should appear as in figure \ref{fig:GUI}. It is worth noting that several of the variable names have been renamed from their original names: \begin{enumerate} \item \textbf{2-Cycle batch} -> X2\_Cycle\_batch \item \textbf{Myc ON/OFF} -> Myc\_ON\_OFF \end{enumerate} The variable \texttt{Sample name} has unique values for each sample as shown below, so is classed as a labelling variable. Any such variables should be removed from the modelling process to avoid overfitting of the data, or alternatively users must be sure that the variables are selected as numeric, and not categoric. Selecting any of these variables for use in the model as a categorical variable results in an error message to limit accidental assignment by the user. <<>>= nlevels(pData(SkinvsPancreas)$'Sample name') length(sampleNames(SkinvsPancreas)) @ We make the following class selections for each variable: \begin{description} \item[\textbf{Sample name}] \texttt{None} \item[\textbf{Treatment}] \texttt{None} \item[\textbf{RIN}] \texttt{numeric} \item[\textbf{Nanodrop Conc.}] \texttt{None} \item[\textbf{Tissue batch}] \texttt{categoric} \item[\textbf{RNA batch}] \texttt{categoric} \item[\textbf{2-Cycle batch}] \texttt{categoric} \item[\textbf{Starting cRNA}] \texttt{numeric} \item[\textbf{Tissue}] \texttt{categoric} \item[\textbf{Time point}] \texttt{categoric} \item[\textbf{Replicate Number}] \texttt{None} \item[\textbf{Myc ON/OFF}] \texttt{categoric} \end{description} The default arguments are kept, which uses a Benjamini \& Hochberg false discovery rate multiple testing correction with a P-value cutoff of 0.05. Results will be saved to a folder in the working directory unless otherwise stated. Pressing `\texttt{OK}' brings up another window confirming the variable classes selected. Click `\texttt{Yes}' to continue. The next window that appears allows users to specify which variables to calculate interaction terms for (figure \ref{fig:INTselect}). For now we leave all variables selected, meaning that all of these will be used to calculate interaction terms. Pressing `\texttt{Ok}' brings up an error message that informs us that we have too many variable terms in our maximum model. We can choose to continue, and risk seeing errors in the analysis due to aliasing, or alternatively we can choose to exclude some variables from the interaction term calculations. The 3 batching variables each have a large number of levels which will have a large effect on the number of residual degrees of freedom. We can safely ignore the interaction terms for these variables, since randomisation of batches should remove significant effects. So press `\texttt{Cancel}', deselect the batch variables and press `\texttt{Ok}'. This will start the modelling process. Once the program has finished running, the output window shown in figure \ref{fig:output} will appear. By default, all terms are selected which will output results for all variables and interactions to the file selected (the R working directory by default). Alternatively, users can select which terms are of interest to them and output only these gene lists. \subsection{Using the command line} The same output can be seen by running the \texttt{envisage()} method through the command line: <>= ## Load dataset library(Envisage) data(SkinvsPancreas) @ <<>>= ## Select which variables to use in analysis and ## their classes (notice the useParams <- c("RIN", "Tissue batch", "RNA batch", "2-Cycle batch", "Starting cRNA", "Tissue", "Time point", "Myc ON/OFF") paramType <- c("numeric", rep("categoric", 3), "numeric", rep("categoric", 3)) ## We don't need to look at interactions for batch effects param4INT <- c("RIN", "Starting cRNA", "Tissue", "Time point", "Myc ON/OFF") ## Run envisage results1 <- envisage(SkinvsPancreas, widget=FALSE, MTC="BH", pCutoff=0.05, useParams, param4INT, paramType, fileResults=getwd(), MEorINT="INT") @ \subsection{Envisage output} The results folder \texttt{EnvisageResults} contains the resulting gene lists for the analysis, as well as the 2 alias outputs \texttt{AliasGenes.txt} and \texttt{AliasReport.txt}. These files contain information on genes that show aliasing in the modeling procedure. As we can see, the majority of the 50 genes (34 genes) have shown some problem due to aliasing of the variables used in the analysis, and this may mean that the results of the modeling procedure are affected. By looking at the alias report, we can see that aliasing is seen between the same two variables, \texttt{Tissue} and \texttt{Tissue batch}, for all genes: <<>>= names(results1$errorGenes)[1] results1$errorGenes[[1]] @ Batching for tissue extraction was not randomised, and skin samples were all identified with the tissue batch identifier \texttt{1}. Thus the variable \texttt{Tissue} is in fact a subset of the variable \texttt{Tissue\_batch} which explains the intrinsic aliasing seen. We can therefore repeat the process with the \texttt{Tissue batch} variable removed. After repeating analysis with the \texttt{Tissue batch} variable removed, you will find that the result output file contains only significantly changing lists of genes for several variables in the analysis with no problems due to aliasing. The resulting gene lists can be used for further analysis. \newpage \bibliographystyle{plainnat} \bibliography{EnvisageBib} \end{document} Envisage/inst/doc/GS.png0000755003020100016370000064655611031122551015243 0ustar sr9signg00000000000000PNG  IHDRg`_4sRGB@} pHYsgRtEXtSoftwareMicrosoft Office5qIDATx]|TP.+m)@qA,@;-@pHH%wf]8B{pw|wvvd&F2d$#HFz:H Cr^jpʤIMw+:ge(ʤmIh"4OX|7F2≒dGHr~/5bgDN=WFD#~TWjZMO!YF2ҿ~m$#7{#?B"s 0n:TM|0g ;U aҳ]Gir̛3!8+ItHBx}2#* Kˁ_WzQ@5w!/F2Hn$#ۉ0NUDo~% @ De DE(BSs W 6 x@I"4GvX`mlÙ|T&`2҈RQ Cp5^UEo\^W(^SH(zv4=g$#E#B#H'O5BG)zs 0-<'z)\OJ=;zgl/"tDDSPΒpVwg xP? s\ӭHI-7t I I(IdM 5Y0M2\(4CT03HFP k#onDO?Yd1Hˊsÿ߁))nO"kC=R`1&`b &Dc`ҐpS}Ջ$",U?> [ Imj0}6^0OgTlUev %ڍ2BPZޟ*ڟM2z3=K%HFu. 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B 57}, Pages = {289-300}, Year = {1995} } @article{ BY, Author = {Benjamini, Y. and Yekutieli, D.}, Title = {The control of the false discovery rate in multiple testing under dependency}, Journal = {Ann. Stat.}, Volume = {29}, Number = {4}, Pages = {1165–1188}, Year = {2001} } @article{ MIAME, Author = {Brazma, A. and Hingamp, P. and Quackenbush, J. and Sherlock, G. and Spellman, P. and Stoeckert, C. and Aach, J. and Ansorge, W. and Ball, C. A. and Causton, H. C. and Gaasterland, T. and Glenisson, P. and Holstege, F. C. and Kim, I. F. and Markowitz, V. and Matese, J. C. and Parkinson, H. and Robinson, A. and Sarkans, U. and Schulze-Kremer, S. and Stewart, J. and Taylor, R. and Vilo, J. and Vingron, M.}, Title = {Minimum information about a microarray experiment (MIAME)-toward standards for microarray data}, Journal = {Nat Genet}, Volume = {29}, Number = {4}, Pages = {365-71}, Note = {1061-4036 (Print) Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.}, Abstract = {Microarray analysis has become a widely used tool for the generation of gene expression data on a genomic scale. Although many significant results have been derived from microarray studies, one limitation has been the lack of standards for presenting and exchanging such data. Here we present a proposal, the Minimum Information About a Microarray Experiment (MIAME), that describes the minimum information required to ensure that microarray data can be easily interpreted and that results derived from its analysis can be independently verified. The ultimate goal of this work is to establish a standard for recording and reporting microarray-based gene expression data, which will in turn facilitate the establishment of databases and public repositories and enable the development of data analysis tools. With respect to MIAME, we concentrate on defining the content and structure of the necessary information rather than the technical format for capturing it.}, Keywords = {*Computational Biology Gene Expression Profiling/methods Oligonucleotide Array Sequence Analysis/*standards}, Year = {2001} } @article{ Bioconductor, Author = {Gentleman, R. C. and Carey, V. J. and Bates, D. M. and Bolstad, B. and Dettling, M. and Dudoit, S. and Ellis, B. and Gautier, L. and Ge, Y. and Gentry, J. and Hornik, K. and Hothorn, T. and Huber, W. and Iacus, S. and Irizarry, R. and Leisch, F. and Li, C. and Maechler, M. and Rossini, A. J. and Sawitzki, G. and Smith, C. and Smyth, G. and Tierney, L. and Yang, J. Y. and Zhang, J.}, Title = {Bioconductor: open software development for computational biology and bioinformatics}, Journal = {Genome Biol}, Volume = {5}, Number = {10}, Pages = {R80}, Note = {1465-6914 Journal Article}, Abstract = {The Bioconductor project is an initiative for the collaborative creation of extensible software for computational biology and bioinformatics. The goals of the project include: fostering collaborative development and widespread use of innovative software, reducing barriers to entry into interdisciplinary scientific research, and promoting the achievement of remote reproducibility of research results. We describe details of our aims and methods, identify current challenges, compare Bioconductor to other open bioinformatics projects, and provide working examples.}, Keywords = {Computational Biology/*instrumentation/*methods Internet Reproducibility of Results *Software}, Year = {2004} } @article{ Hochberg, Author = {Hochberg, Y.}, Title = {A sharper Bonferroni procedure for multiple tests of significance}, Journal = {Biometrika}, Volume = {75}, Pages = {800-802}, Year = {1988} } @article{ Holm, Author = {Holm, S.}, Title = {A simple sequentially rejective multiple test procedure}, Journal = {Scandinavian Journal of Statistics}, Volume = {6}, Pages = {65–70}, Year = {1979} } @article{ LEE, Author = {Lee, M. L. and Kuo, F. C. and Whitmore, G. A. and Sklar, J.}, Title = {Importance of replication in microarray gene expression studies: statistical methods and evidence from repetitive cDNA hybridizations}, Journal = {Proc Natl Acad Sci U S A}, Volume = {97}, Number = {18}, Pages = {9834-9}, Note = {0027-8424 (Print) Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.}, Abstract = {We present statistical methods for analyzing replicated cDNA microarray expression data and report the results of a controlled experiment. The study was conducted to investigate inherent variability in gene expression data and the extent to which replication in an experiment produces more consistent and reliable findings. We introduce a statistical model to describe the probability that mRNA is contained in the target sample tissue, converted to probe, and ultimately detected on the slide. We also introduce a method to analyze the combined data from all replicates. Of the 288 genes considered in this controlled experiment, 32 would be expected to produce strong hybridization signals because of the known presence of repetitive sequences within them. Results based on individual replicates, however, show that there are 55, 36, and 58 highly expressed genes in replicates 1, 2, and 3, respectively. On the other hand, an analysis by using the combined data from all 3 replicates reveals that only 2 of the 288 genes are incorrectly classified as expressed. Our experiment shows that any single microarray output is subject to substantial variability. By pooling data from replicates, we can provide a more reliable analysis of gene expression data. Therefore, we conclude that designing experiments with replications will greatly reduce misclassification rates. We recommend that at least three replicates be used in designing experiments by using cDNA microarrays, particularly when gene expression data from single specimens are being analyzed.}, Keywords = {DNA, Complementary/*genetics Gene Expression Profiling/*methods *Gene Expression Regulation *Models, Statistical *Oligonucleotide Array Sequence Analysis Probability RNA, Messenger/*genetics Reproducibility of Results Statistics/methods}, Year = {2000} } Envisage/inst/doc/EnvisageOutput.png0000755003020100016370000031571111031122551017676 0ustar sr9signg00000000000000PNG  IHDRd#sRGB@} pHYsgRtEXtSoftwareMicrosoft Office5qIDATx(Bpwwwww=pw nr ǹ ~WU\ $_ieoggg{S]]e JJJJJJJJJ??_(Gl<'>D10} a| `Q~!b`tCC~T8~IL#Q'F!hz%M4Qoiǯb?*)}ܢ~Ӹ(%\=>qL 25j4im"Eaw&eCnP)F 5=_ZE!uh$CH[tp{=b%L@ E8b(% ^#8 d /@@y@@@@@u@76%Fm[RU3z8%Zϕ>&USR\QeZxaYeb ++J5ZC*L_˒Jqň,.T&O4Ft pN(\̀Msa C}'$BlP0R '#jɱ~J2NO@OFP>ғί"🛸Oգxڎ9ʦߋf))ʴ9qH7.oૡ1;ɥ(]糢("C_?]?X H{dC|XREQq0E H>")G#9mK62Fŋ,o ڞIAyÈ8mFѹ|xdC=(NIè/&N!HFaAa1ȫR~QH7 y70 {0 ]I]n o%k)䯩+R_m[@mahoT':Ӷ04"*?KPGܸ[߇AY[کk:sz0 }ap(AZDFŐCi:* ɬ՟ QRRRRe꣤(˒ԋR;?SƲzS9$$IlWN P^C0!&"jQ0pԜxkM_dC×jbG۞P1 ٯ WӉ@v3a lRBDmDTH7#YY`f@pE_ji`od E͒E-Io"J%_Է*)))}R2tլ;:ꤝ+ON3,`љS[ؚ=MH 2\"ЪGTNS]jt$jFUDC ]ŗO%ٯGia(}NxM¢ 0NNtt=@FЭe"iլl==جg$Ү>鴡J>ٺޞ·aͷWJNͯ6,=5YѶv2±$j5ܴNh17Ob5巵~WR室 )}w Z "a2qe<r) 2ORwtm(\x+#T460k4X!JV0,EϒH^bLs#ZΈ@)MTah BױhsX;] g\{~[ގ(&(6 Vj`3K#XHtmM@IZyP/o)f[}S63s [VO17gxO}|w7j uڹ"^4-cRѸm_i;0#}lCR a0T}C=[~ C{NF*2AaU`nSl"+ Co`h~QbILQҧeJJԻgnHM p8^eGh4„-QG FF(bc(j?3fU8N GGUG/k] dgK7\z6qt,=LFa([^ :̊D"Ќ@1Oc֥2j GqyCJ@H@E[}>^ R2[ 8d/5Kp(\9gweߖ0GL)Ƙ ~&n@ׂjȫ*}0ȼNܵx( |4574W]%%O\46)@vf:ΉQ; j*+bxpV&F?ZPTmd* Cxdi/qہ~lKhX<}| Nu,ފš8p=`_/j~![`(6G4~Cbj܃tN*L @J0$`㧾^h0$QeZ kAAS0]fF'ʠ/8{߿zjXPd\҇{-qh5#c7Eap#jN$""PبpTS`FFȀ"Me2[h@vvFmЦD9<``cyd98[h4yr_?- E=o Ң(|Crz\ ^7/Ytxv$Ï"%ЇUh͚;i>䕐] X~ZD۶O`\/uݱvA;b- a n"Xu pv s[O|S-H[{Νkؖ7J;aHA 6<yGèF ?xᅓ`{y&& ^_ls FV+$)}%'Bg"'XtD rykυ!&Xpkb/OIC Y ^C)F \>}n:"`|jG84ekVU8  BQ=GoN@Lqy bVibw^y (N-'T;6xhB 6~sdGؕHĿ pNUֱ\Zp =u2e6AY+0dܘ.P iF 3NvdBc}B \`zhG6kpzL v-в~N,Uk2 ʒH@Wi5vv!G(Pn%r]47¢9]W{_;5M26n\c3P~' kw3ysZxyk1Z[;ѻ Oz[v?WFmFrZ֭d5yO} jMZJZ̦{ ]{pA$6_ Emӟ[^ݿFStGQuNo M_QY>o6\R\ՁI4v[ ך{4:,4`kGkOIIICRsm(+Ylc4 _lv$5!8 ŨpTM CQp]v k2piY>S,K|n$+ "dqwڔKzNRqBs R$ +QfC.~Z0mdc֤rލM:`8~Q9k )AXLJt0ck.+5  G+ P.-$_[hH'l- txH;UL;?cn*&L[&-mԁ}Iu'UgXoFHa\gOQg-[ϫCV c}/uyj_ꩥGb]A=q?Lkm#q  _OkhP16s#]xau'T߷i6}W{5%CP(pHWQ-( ) X;H+w5婤᪅]E-v8J^Yh==&kֱ:cx`j4Y1B` %`( A!HvYk xT/(GgQk04rMĀ,Gߑm2#x{'@fcO@< YB[yO1rd_J7'=֪0kH@.fpTZ8تސ[@y524Z8E؁@F03^HÑ]q<hۡIDX w4Iɺ3 +OjγYY] =3bԎP=wb1~W( s5FaX|<k΅ <({X?,?Ef6R#=7Nh8.Fb/sPdH߳- 'aS^687BYXO51fGB!Y8W&ay%%:7f2Eh@6A[EYm4ep$(; 4% +`i|dqd<+ϣqZ.spO^XTMVW+>bOZ!C0$o beuc>dHy& k- ;ݪ{L sX[$hhgz4\vR;5fyҢ$Y]0攕Oz݉@rY$}]#h t|~8't5¢'NpGa}v6鏡j9.rVlC#H'{iP&`6~Gm2X#lq?opCE !Ț" qH1:/ugB' fVߴ~_Go]7leGhhwKu,lp nn5M|PrBM6ؾ6^hTC :3v}gb3A>ö-N.L( EGڔl{ƾ(6:Z2o`%m'?#J@kuqw.·PpD⍝l[TQꎎ֮hmu@J 2u+^aKZ7N g]q8T3L?ƈݏ$S["}!B)/qKX-~4m]FsWopǚ-N`p Y'2.n/sBq84n>ϸE+.j2k嵟ű")Ģ4꼣8_9#6崥-N󼐂9ܱPjʹ#;oѤ벹/{k"73⸿$Ϋi%遾=@a-$C״Oc\"M3+oK@68o:O"^ l% {AȎ,s|pSOd^8lkYyx<<|V#xǂĩKoWI'8 |n5q U٨LPFe"?O@ͺ d=Xw X>H֏oM35|&uÌ50h` v7R A>Q,"'GfBu6_ui- 2Z-u0\| ۶g?E>xfU; 6QchDG"2"ϽQz(+ fҎy @TEAam8z@z ?\OnǦcZ=]S=`K`Cx: ƟBڬs1iu+CǜD'VwaQk>8+B8X @F6 <͜yT=KNup'B{oAr ޼d v|t A@q7͉5Y3:FŢHE@|--.l& ;[`5*FJd`U3 +H@tڇ!s Xd^! 4lAOS[ W748:uہ[KaiRs9|5Z?#xJ+6o2OUYZH"uķqVSȨ!yhz 6(Sc-|,~[j-6RT_C~j4bT>]G0%C>{ GWi݉h 89&Lwo;8!Y8"$]'mNreM0FP#‘cּ#S֯:˸pe<6BʚRy%EhS /3%&C"dȾFԴ-$f0q[v ^sG yn5%+| 5eYW&N_9ލ3p-Y@*уu7o{90p" #kCR o =Ba; }c| =2MPb^ G\#u>HR CAc*&H%sZF6]P-OdtC @wD꾑d f@AHέR4n#+bum9;DyB;ϣGn[f%CHΙTB].Bh׌E'N"G \ {n2F?m"+}v;</@Ti ߖލ΢ (RֽKڒ`'}߮ݪ;T"3lj޽ E92oAՙQ2xS-6M@)1& W[{ Λ`f![ r70\ +\CH=RDh8_wAiΦo46z 0nDڲ{вYs^}ɻ =Vl%V &O;Gvg1`9y ij^CE]^yj}r`GᮓvEÐW8XmZMNz*}wxmImL>2;ުӰ%>'#Łܶ,?t"ҷɳղ۵;dóWp & [<ZC[u R~I@N/Yh( ƒJ2 H ;#eV@%(JW"gClsGē␚bP\rX$CEhxrSbh^@ 4@됾_0܌-9]Sx\/'}ܼѮNο^%he9:`UOHWP"0+Qfr7:m6xV=ab2h}?>|#p'н:."uL>vr:4-oIu+ZSh|IP0Hp, ~{>d ?,.#2:9b 0K~ڋpXzePw KX?}]Hr Y /5*#ؾ-EC_0[C iWw=_jO3A5)e{*8ORS]z;`wh2Y[=ӏƋ3P>4Y@OWxLA;U6^麺!P,8 {/:i{bq8y?^KʧtZv>UqDsDCzǵZ Z^Αw%6w/#P z?){vdm,_ڭm:m D޲To#())t[c[ Xq8ۅH#Qg\8lG|{x% J s|2BQH;8_ CPfm&}F.op{g%48N:e%Y'b0ƖQc=屮$b ~H@FAcIpk0c/U}xt+ ջ:O?LP=GNmoz ¥{Ҷo8rUͷ G`hV!ֵ(Zz!,Z/=pvt]+/QH#T /pKٓyX!fUoҲ:[_:/! xC0V*ëW9xw/.;Ol@ Y84 kKX.qV5rn0d?drEt^04pA& cKNirz$K>8h`N=*" &oԓh;C(_I e 8$ڀdAr2-J^4E⻉@א14mߏmO#OSded LwCCݳz$o}6*L0U%H釆#g3Sb 2ZT7!yG;Ѱtd*i+-JT8ARd) mzO _M@i#s'j;]TI]deAbJqv|9RA쐫nd/iD򊧐BVd͟+L=_!sE*BhO7>_ɂ=Jz=?%YCAa4/YrKRhsΈ@_"QT`,H,"pECaXgo㗍/`L 2ueՑhiNY]^h͉gߤf"xvώDQJ8+ eB)2Z//X5{T^JNq8ξȻf±L87ֹ" Z洭lh,$Uc5pA獛9- tnJ:*_@ht<K[?o\:.H¨zNjʫZP=7qk$w\6N Y;Oonu4~/CNВyjǵr Ҿ?wxIϼQrVq C9O Y@~ZN%Ĺ49uLj :Q_26jdR} ܆<5l{o :i M~ܑW{Dj鐸5wEGO$c:o=x>/Ø<=C˹9/Ci:$^v.m:zs4 v.H/B]ުj.C|94.½tߪ݃QoJAqz-J*B*?BQ ER!a9" Y#pW=K6C똱l\ذx ,?ͬ][t /q9pp%^F3 F5*\o]{${{\C|9"u=Re&y"Ga1ά^k~~'}kw]o>cyum{yX|9hҶ[0N o^cJJJ%KﯾڮrvF>(l鋢}|PT/ C~~5{YIO,?@sza(q>/2~J%`پ (/vy6=3/@_pO+gmx(?^4|EyKy!5Hy$^5ɎW 3é~Fj~ ibJؕ:~2C Y=5X"][ 9f%l%Eʁ+ii&haҀ lQI, &!֖T싩%rW0oetz !6ld>VYH^E$R`K! }*AqH^ʷe/j[ԲF:6HQ.վO@ ! kuz_^+wa( ıXu%N29CޠG*~y)]C7q+\Cr p(a`=-sp@ .~SEHHEHS|dXȀK ^pŀ?p^ov( 3\ tYLY)K緀0%.*Ԧ/ R"0c(GFqKJEDJIu54! 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X&W#(?s?eX);{)}̯kOq-uK&"&4h+/MɛQ8*/_%|2ltOmk}>&1'cΖqͩVN.~t.pngEnvisage/inst/doc/EnvisageResults/0000755003020100016370000000000011031122660017317 5ustar sr9signg00000000000000Envisage/inst/doc/EnvisageResults/RIN.txt0000644003020100016370000000026711031122660020515 0ustar sr9signg000000000000001416773_at 0.0237892674122923 1416747_at 0.00959178186960008 1416712_at 0.0031192599236507 1416708_a_at 0.0154880265568913 1416699_at 0.0154880265568913 1416697_at 0.0031192599236507 Envisage/inst/doc/EnvisageResults/X2_Cycle_batch.txt0000644003020100016370000000124511031122660022633 0ustar sr9signg000000000000001416778_at 0.0336932285218928 1416775_at 0.0336932285218928 1416773_at 0.0350513917237831 1416770_at 0.00265299687174092 1416769_s_at 0.00224129884009883 1416763_at 0.000291342163069586 1416747_at 0.00486031303501624 1416738_at 0.000291342163069586 1416737_at 0.0131223693936522 1416736_at 0.00540381833594419 1416733_at 0.0430000303995981 1416730_at 0.00486031303501624 1416728_at 0.0193978573081863 1416721_s_at 0.0482987924219827 1416708_a_at 0.000257781365972654 1416703_at 0.0350513917237831 1416699_at 0.049676643252011 1416696_at 0.0336932285218928 1416692_at 0.000257781365972654 1416688_at 0.0394153103919921 1416687_at 0.049676643252011 1416686_at 0.0394153103919921 Envisage/inst/doc/EnvisageResults/AliasGenes.txt0000644003020100016370000000060211031122660022071 0ustar sr9signg000000000000001416780_at 1416778_at 1416776_at 1416775_at 1416773_at 1416770_at 1416768_at 1416766_at 1416756_at 1416753_at 1416751_a_at 1416750_at 1416748_a_at 1416745_x_at 1416744_at 1416743_at 1416742_at 1416738_at 1416733_at 1416728_at 1416723_at 1416721_s_at 1416713_at 1416712_at 1416708_a_at 1416706_at 1416704_at 1416701_at 1416699_at 1416691_at 1416690_at 1416688_at 1416687_at 1416685_s_at Envisage/inst/doc/EnvisageResults/RIN_INT_Time_point.txt0000644003020100016370000000027011031122660023410 0ustar sr9signg000000000000001416773_at 0.0386503411586849 1416769_s_at 0.0386503411586849 1416750_at 0.0295182727584664 1416733_at 0.0386503411586849 1416730_at 0.0378781958857063 1416708_a_at 0.0285211655424833 Envisage/inst/doc/EnvisageResults/Starting_cRNA_INT_Tissue.txt0000644003020100016370000000042111031122660024561 0ustar sr9signg000000000000001416780_at 0.0201070611530851 1416778_at 0.0184198724594209 1416769_s_at 0.0184198724594209 1416768_at 0.0213945442865719 1416730_at 0.0206664585751179 1416706_at 0.0184198724594209 1416699_at 0.00292996906293141 1416692_at 0.0184198724594209 1416688_at 0.0206664585751179 Envisage/inst/doc/EnvisageResults/Time_point_INT_Myc_ON_OFF.txt0000644003020100016370000000013211031122660024573 0ustar sr9signg000000000000001416730_at 0.0141132953866104 1416699_at 0.0141132953866104 1416692_at 0.0141132953866104 Envisage/inst/doc/EnvisageResults/RIN_INT_Tissue.txt0000644003020100016370000000007411031122660022557 0ustar sr9signg000000000000001416773_at 0.0361189287564678 1416703_at 0.0361189287564678 Envisage/inst/doc/EnvisageResults/AliasReport.txt0000644003020100016370000001145411031122660022312 0ustar sr9signg000000000000001416780_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416778_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416776_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416775_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416773_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416770_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416768_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416766_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416756_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416753_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416751_a_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416750_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416748_a_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416745_x_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416744_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416743_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416742_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416738_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416733_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416728_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416723_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416721_s_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416713_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416712_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416708_a_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416706_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416704_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416701_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416699_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416691_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416690_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416688_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416687_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 1416685_s_at Aliasing is seen across the following conditions: TissueSkin & (Intercept) TissueSkin & Tissue_batch2 TissueSkin & Tissue_batch3 Envisage/inst/doc/EnvisageResults/Starting_cRNA_INT_Time_point.txt0000644003020100016370000000042111031122660025414 0ustar sr9signg000000000000001416770_at 0.0402789247472089 1416763_at 0.0196971371575039 1416750_at 0.0418581973934999 1416747_at 0.0260044683355026 1416730_at 0.0260044683355026 1416708_a_at 0.00323845900051523 1416703_at 0.0402789247472089 1416688_at 0.0418581973934999 1416687_at 0.0402789247472089 Envisage/inst/doc/EnvisageResults/Tissue_INT_Time_point.txt0000644003020100016370000000070711031122660024241 0ustar sr9signg000000000000001416783_at 0.00230254024111143 1416773_at 0.0188757659433764 1416763_at 0.0188757659433764 1416756_at 0.0447343261470398 1416753_at 0.0447343261470398 1416750_at 0.0165297988047197 1416743_at 0.0467304241008508 1416733_at 0.0270917097825415 1416712_at 0.0165297988047197 1416708_a_at 0.00530486972122879 1416706_at 0.0188757659433764 1416703_at 0.0188757659433764 1416699_at 0.0214764863590011 1416688_at 0.00692514297423452 1416687_at 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Njٲ%{,#:P+ !ҏSѡݳeʔƓO>y$E\JLXLȐ! z 9s?2:9_ƅ#BHzCLT0"¶⇞гgGY\Jc߷+|ݻS_߷v, !n6rw |BqT G)ğ Al~i!MKVaR!B! -yAhC0Ztm( Cah3EB!B4rh](Z;.ۺkHT.m9U!B!LZ;?4UN!Cz!Bqgc^p ʖ_{Qz4YB٨XeB!w<E^W!B$/RIENDB`Envisage/DESCRIPTION0000755003020100016370000000272411031122667014177 0ustar sr9signg00000000000000Package: Envisage Version: 1.0-2 Date: 2008-06-26 Title: Enable Numerous Variables In Significance Analysis of Gene Expression Author: Sam Robson , Heather Turner Helen Brown Ewan Hunter Maintainer: Sam Robson Depends: R (>= 2.6.1), multtest, tcltk, tkWidgets (>= 1.16.0), car, Biobase Suggests: GeneSpring, affy, marray Description: Model-based analysis of significantly changing gene expression across a variety of experiment variables. Gene expression is modelled using experiment variables as explanatory variables. Variables can be experiment variables of primary interest, or phenotypic, environmental or technical variables that may influence their effect on gene expression. Stepwise model selection, based on the Akaike information criterion, identifies a candidate model for each gene. Model terms are tested for significance using a Type II SS F-test statistic, which is compared to a user defined threshold value to determine statistical significance of selected model terms. Lists of genes found to be significant for each model term are found and submitted to the user. URL: http://www2.warwick.ac.uk/fac/sci/moac/currentstudents/2003/sam_robson/linear_models/downloads/ biocViews: Microarray, OneChannel, Statistics License: GPL version 2 or newer Packaged: Fri Jun 27 09:32:55 2008; sr9 Envisage/NAMESPACE0000755003020100016370000000021711031122552013674 0ustar sr9signg00000000000000export(envisage, GS2BC) import(tcltk, tkWidgets, car, multtest) importFrom(stats, alias, coef) S3method(alias, glmNA) S3method(coef, glmNA)