Mercurial > repos > fubar > shrnaseq_test
changeset 7:2c6bcbc1e76a draft default tip
Uploaded
author | fubar |
---|---|
date | Mon, 19 Jan 2015 22:14:10 -0500 |
parents | c1b3da0fde4a |
children | |
files | hairpinTool.R hairpinTool.xml readme.rst test-data/shrnaseq_zuber_test.html test-data/zuber-count_matrix.txt test-data/zuber-sample_anno.txt test-data/zuber-target_anno.txt tool_dependencies.xml |
diffstat | 8 files changed, 4055 insertions(+), 0 deletions(-) [+] |
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--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/hairpinTool.R Mon Jan 19 22:14:10 2015 -0500 @@ -0,0 +1,1069 @@ +#!/usr/bin/env Rscript +# ARGS: 1.inputType -String specifying format of input (fastq or table) +# IF inputType is "fastq" or "pairedFastq: +# 2*.fastqPath -One or more strings specifying path to fastq files +# 2.annoPath -String specifying path to hairpin annotation table +# 3.samplePath -String specifying path to sample annotation table +# 4.barStart -Integer specifying starting position of barcode +# 5.barEnd -Integer specifying ending position of barcode +# ### +# IF inputType is "pairedFastq": +# 6.barStartRev -Integer specifying starting position of barcode +# on reverse end +# 7.barEndRev -Integer specifying ending position of barcode +# on reverse end +# ### +# 8.hpStart -Integer specifying startins position of hairpin +# unique region +# 9.hpEnd -Integer specifying ending position of hairpin +# unique region +# IF inputType is "counts": +# 2.countPath -String specifying path to count table +# 3.annoPath -String specifying path to hairpin annotation table +# 4.samplePath -String specifying path to sample annotation table +# ### +# 10.secFactName -String specifying name of secondary factor +# 11.cpmReq -Float specifying cpm requirement +# 12.sampleReq -Integer specifying cpm requirement +# 13.readReq -Integer specifying read requirement +# 14.fdrThresh -Float specifying the FDR requirement +# 15.lfcThresh -Float specifying the log-fold-change requirement +# 16.workMode -String specifying exact test or GLM usage +# 17.htmlPath -String specifying path to HTML file +# 18.folderPath -String specifying path to folder for output +# IF workMode is "classic" (exact test) +# 19.pairData[2] -String specifying first group for exact test +# 20.pairData[1] -String specifying second group for exact test +# ### +# IF workMode is "glm" +# 19.contrastData -String specifying contrasts to be made +# 20.roastOpt -String specifying usage of gene-wise tests +# 21.hairpinReq -String specifying hairpin requirement for gene- +# wise test +# 22.selectOpt -String specifying type of selection for barcode +# plots +# 23.selectVals -String specifying members selected for barcode +# plots +# ### +# +# OUT: Bar Plot of Counts Per Index +# Bar Plot of Counts Per Hairpin +# MDS Plot +# BCV Plot +# Smear Plot +# Barcode Plots (If Genewise testing was selected) +# Top Expression Table +# Feature Counts Table +# HTML file linking to the ouputs +# +# Author: Shian Su - registertonysu@gmail.com - Jan 2014 + +# Record starting time +timeStart <- as.character(Sys.time()) +options(bitmapType='cairo') +# needed to prevent missing x11 errors for png() +# Loading and checking required packages +library(methods, quietly=TRUE, warn.conflicts=FALSE) +library(statmod, quietly=TRUE, warn.conflicts=FALSE) +library(splines, quietly=TRUE, warn.conflicts=FALSE) +library(edgeR, quietly=TRUE, warn.conflicts=FALSE) +library(limma, quietly=TRUE, warn.conflicts=FALSE) + +if (packageVersion("edgeR") < "3.7.17") { + stop("Please update 'edgeR' to version >= 3.7.17 to run this tool") +} + +if (packageVersion("limma")<"3.21.16") { + message("Update 'limma' to version >= 3.21.16 to see updated barcode graphs") +} + +################################################################################ +### Function declarations +################################################################################ + +# Function to load libaries without messages +silentLibrary <- function(...) { + list <- c(...) + for (package in list){ + suppressPackageStartupMessages(library(package, character.only=TRUE)) + } +} + +# Function to sanitise contrast equations so there are no whitespaces +# surrounding the arithmetic operators, leading or trailing whitespace +sanitiseEquation <- function(equation) { + equation <- gsub(" *[+] *", "+", equation) + equation <- gsub(" *[-] *", "-", equation) + equation <- gsub(" *[/] *", "/", equation) + equation <- gsub(" *[*] *", "*", equation) + equation <- gsub("^\\s+|\\s+$", "", equation) + return(equation) +} + +# Function to sanitise group information +sanitiseGroups <- function(string) { + string <- gsub(" *[,] *", ",", string) + string <- gsub("^\\s+|\\s+$", "", string) + return(string) +} + +# Function to change periods to whitespace in a string +unmake.names <- function(string) { + string <- gsub(".", " ", string, fixed=TRUE) + return(string) +} + +# Function has string input and generates an output path string +makeOut <- function(filename) { + return(paste0(folderPath, "/", filename)) +} + +# Function has string input and generates both a pdf and png output strings +imgOut <- function(filename) { + assign(paste0(filename, "Png"), makeOut(paste0(filename,".png")), + envir=.GlobalEnv) + assign(paste0(filename, "Pdf"), makeOut(paste0(filename,".pdf")), + envir=.GlobalEnv) +} + +# Create cat function default path set, default seperator empty and appending +# true by default (Ripped straight from the cat function with altered argument +# defaults) +cata <- function(..., file=htmlPath, sep="", fill=FALSE, labels=NULL, + append=TRUE) { + if (is.character(file)) + if (file == "") + file <- stdout() + else if (substring(file, 1L, 1L) == "|") { + file <- pipe(substring(file, 2L), "w") + on.exit(close(file)) + } + else { + file <- file(file, ifelse(append, "a", "w")) + on.exit(close(file)) + } + .Internal(cat(list(...), file, sep, fill, labels, append)) +} + +# Function to write code for html head and title +HtmlHead <- function(title) { + cata("<head>\n") + cata("<title>", title, "</title>\n") + cata("</head>\n") +} + +# Function to write code for html links +HtmlLink <- function(address, label=address) { + cata("<a href=\"", address, "\" target=\"_blank\">", label, "</a><br />\n") +} + +# Function to write code for html images +HtmlImage <- function(source, label=source, height=600, width=600) { + cata("<img src=\"", source, "\" alt=\"", label, "\" height=\"", height) + cata("\" width=\"", width, "\"/>\n") +} + +# Function to write code for html list items +ListItem <- function(...) { + cata("<li>", ..., "</li>\n") +} + +TableItem <- function(...) { + cata("<td>", ..., "</td>\n") +} + +TableHeadItem <- function(...) { + cata("<th>", ..., "</th>\n") +} +################################################################################ +### Input Processing +################################################################################ + +# Grabbing arguments from command line +argv <- commandArgs(T) + +inputType <- as.character(argv[1]) +if (inputType == "fastq") { + + fastqPath <- as.character(gsub("fastq::", "", argv[grepl("fastq::", argv)], + fixed=TRUE)) + + # Remove fastq paths + argv <- argv[!grepl("fastq::", argv, fixed=TRUE)] + + fastqPathRev <- NULL + annoPath <- as.character(argv[2]) + samplePath <- as.character(argv[3]) + barStart <- as.numeric(argv[4]) + barEnd <- as.numeric(argv[5]) + barStartRev <- NULL + barStartRev <- NULL + hpStart <- as.numeric(argv[8]) + hpEnd <- as.numeric(argv[9]) +} else if (inputType=="pairedFastq") { + + fastqPath <- as.character(gsub("fastq::", "", argv[grepl("fastq::", argv)], + fixed=TRUE)) + + fastqPathRev <- as.character(gsub("fastqRev::", "", + argv[grepl("fastqRev::", argv)], fixed=TRUE)) + + # Remove fastq paths + argv <- argv[!grepl("fastq::", argv, fixed=TRUE)] + argv <- argv[!grepl("fastqRev::", argv, fixed=TRUE)] + + annoPath <- as.character(argv[2]) + samplePath <- as.character(argv[3]) + barStart <- as.numeric(argv[4]) + barEnd <- as.numeric(argv[5]) + barStartRev <- as.numeric(argv[6]) + barEndRev <- as.numeric(argv[7]) + hpStart <- as.numeric(argv[8]) + hpEnd <- as.numeric(argv[9]) +} else if (inputType == "counts") { + countPath <- as.character(argv[2]) + annoPath <- as.character(argv[3]) + samplePath <- as.character(argv[4]) +} + +secFactName <- as.character(argv[10]) +cpmReq <- as.numeric(argv[11]) +sampleReq <- as.numeric(argv[12]) +readReq <- as.numeric(argv[13]) +fdrThresh <- as.numeric(argv[14]) +lfcThresh <- as.numeric(argv[15]) +selectDirection <- as.character(argv[16]) +workMode <- as.character(argv[17]) +htmlPath <- as.character(argv[18]) +folderPath <- as.character(argv[19]) + +if (workMode == "classic") { + pairData <- character() + pairData[2] <- as.character(argv[20]) + pairData[1] <- as.character(argv[21]) +} else if (workMode == "glm") { + contrastData <- as.character(argv[20]) + roastOpt <- as.character(argv[21]) + hairpinReq <- as.numeric(argv[22]) + selectOpt <- as.character(argv[23]) + selectVals <- as.character(argv[24]) +} + +# Read in inputs + +samples <- read.table(samplePath, header=TRUE, sep="\t") + +anno <- read.table(annoPath, header=TRUE, sep="\t") + +if (inputType == "counts") { + counts <- read.table(countPath, header=TRUE, sep="\t") +} + +###################### Check inputs for correctness ############################ +samples$ID <- make.names(samples$ID) + +if ( !any(grepl("group", names(samples))) ) { + stop("'group' column not specified in sample annotation file") +} # Check if grouping variable has been specified + +if (secFactName != "none") { + if ( !any(grepl(secFactName, names(samples))) ) { + tempStr <- paste0("Second factor specified as \"", secFactName, "\" but ", + "no such column name found in sample annotation file") + stop(tempStr) + } # Check if specified secondary factor is present +} + + +if ( any(table(samples$ID) > 1) ){ + tab <- table(samples$ID) + offenders <- paste(names(tab[tab > 1]), collapse=", ") + offenders <- unmake.names(offenders) + stop("'ID' column of sample annotation must have unique values, values ", + offenders, " are repeated") +} # Check that IDs in sample annotation are unique + +if (inputType == "fastq" || inputType == "pairedFastq") { + + if ( any(table(anno$ID) > 1) ){ + tab <- table(anno$ID) + offenders <- paste(names(tab[tab>1]), collapse=", ") + stop("'ID' column of hairpin annotation must have unique values, values ", + offenders, " are repeated") + } # Check that IDs in hairpin annotation are unique + +} else if (inputType == "counts") { + # The first element of the colnames will be 'ID' and should not match + idFromSample <- samples$ID + idFromTable <- colnames(counts)[-1] + if (any(is.na(match(idFromTable, idFromSample)))) { + stop("not all samples have groups specified") + } # Check that a group has be specifed for each sample + + if ( any(table(counts$ID) > 1) ){ + tab <- table(counts$ID) + offenders <- paste(names(tab[tab>1]), collapse=", ") + stop("'ID' column of count table must have unique values, values ", + offenders, " are repeated") + } # Check that IDs in count table are unique +} +if (workMode == "glm") { + if (roastOpt == "yes") { + if (is.na(match("Gene", colnames(anno)))) { + tempStr <- paste("Gene-wise tests selected but'Gene' column not", + "specified in hairpin annotation file") + stop(tempStr) + } + } +} + +if (secFactName != "none") { + if (workMode != "glm") { + tempStr <- paste("only glm analysis type possible when secondary factor", + "used, please change appropriate option.") + } +} + +################################################################################ + +# Process arguments +if (workMode == "glm") { + if (roastOpt == "yes") { + wantRoast <- TRUE + } else { + wantRoast <- FALSE + } +} + +# Split up contrasts seperated by comma into a vector and replace spaces with +# periods +if (exists("contrastData")) { + contrastData <- unlist(strsplit(contrastData, split=",")) + contrastData <- sanitiseEquation(contrastData) + contrastData <- gsub(" ", ".", contrastData, fixed=TRUE) +} + +# Replace spaces with periods in pair data +if (exists("pairData")) { + pairData <- make.names(pairData) +} + +# Generate output folder and paths +dir.create(folderPath, showWarnings=FALSE) + +# Generate links for outputs +imgOut("barHairpin") +imgOut("barIndex") +imgOut("mds") +imgOut("bcv") +if (workMode == "classic") { + smearPng <- makeOut(paste0("smear(", pairData[2], "-", pairData[1],").png")) + smearPdf <- makeOut(paste0("smear(", pairData[2], "-", pairData[1],").pdf")) + topOut <- makeOut(paste0("toptag(", pairData[2], "-", pairData[1],").tsv")) +} else if (workMode == "glm") { + smearPng <- character() + smearPdf <- character() + topOut <- character() + roastOut <- character() + barcodePng <- character() + barcodePdf <- character() + for (i in 1:length(contrastData)) { + smearPng[i] <- makeOut(paste0("smear(", contrastData[i], ").png")) + smearPdf[i] <- makeOut(paste0("smear(", contrastData[i], ").pdf")) + topOut[i] <- makeOut(paste0("toptag(", contrastData[i], ").tsv")) + roastOut[i] <- makeOut(paste0("gene_level(", contrastData[i], ").tsv")) + barcodePng[i] <- makeOut(paste0("barcode(", contrastData[i], ").png")) + barcodePdf[i] <- makeOut(paste0("barcode(", contrastData[i], ").pdf")) + } +} +countsOut <- makeOut("counts.tsv") +sessionOut <- makeOut("session_info.txt") + +# Initialise data for html links and images, table with the link label and +# link address +linkData <- data.frame(Label=character(), Link=character(), + stringsAsFactors=FALSE) +imageData <- data.frame(Label=character(), Link=character(), + stringsAsFactors=FALSE) + +# Initialise vectors for storage of up/down/neutral regulated counts +upCount <- numeric() +downCount <- numeric() +flatCount <- numeric() + +################################################################################ +### Data Processing +################################################################################ + +# Transform gene selection from string into index values for mroast +if (workMode == "glm") { + if (selectOpt == "rank") { + selectVals <- gsub(" ", "", selectVals, fixed=TRUE) + selectVals <- unlist(strsplit(selectVals, ",")) + + for (i in 1:length(selectVals)) { + if (grepl(":", selectVals[i], fixed=TRUE)) { + temp <- unlist(strsplit(selectVals[i], ":")) + selectVals <- selectVals[-i] + a <- as.numeric(temp[1]) + b <- as.numeric(temp[2]) + selectVals <- c(selectVals, a:b) + } + } + selectVals <- as.numeric(unique(selectVals)) + } else { + selectVals <- gsub(" ", "", selectVals, fixed=TRUE) + selectVals <- unlist(strsplit(selectVals, ",")) + } +} + +if (inputType == "fastq" || inputType == "pairedFastq") { + # Use EdgeR hairpin process and capture outputs + + hpReadout <- capture.output( + data <- processAmplicons(readfile=fastqPath, readfile2=fastqPathRev, + barcodefile=samplePath, + hairpinfile=annoPath, + barcodeStart=barStart, barcodeEnd=barEnd, + barcodeStartRev=barStartRev, + barcodeEndRev=barEndRev, + hairpinStart=hpStart, hairpinEnd=hpEnd, + verbose=TRUE) + ) + + # Remove function output entries that show processing data or is empty + hpReadout <- hpReadout[hpReadout!=""] + hpReadout <- hpReadout[!grepl("Processing", hpReadout)] + hpReadout <- hpReadout[!grepl("in file", hpReadout)] + hpReadout <- gsub(" -- ", "", hpReadout, fixed=TRUE) + + # Make the names of groups syntactically valid (replace spaces with periods) + data$samples$group <- make.names(data$samples$group) + if (secFactName != "none") { + data$samples[[secFactName]] <- make.names(data$samples[[secFactName]]) + } +} else if (inputType == "counts") { + # Process counts information, set ID column to be row names + rownames(counts) <- counts$ID + counts <- counts[ , !(colnames(counts) == "ID")] + countsRows <- nrow(counts) + + # Process group information + sampleNames <- colnames(counts) + matchedIndex <- match(sampleNames, samples$ID) + factors <- samples$group[matchedIndex] + + if (secFactName != "none") { + secFactors <- samples[[secFactName]][matchedIndex] + } + + annoRows <- nrow(anno) + anno <- anno[match(rownames(counts), anno$ID), ] + annoMatched <- sum(!is.na(anno$ID)) + + if (any(is.na(anno$ID))) { + warningStr <- paste("count table contained more hairpins than", + "specified in hairpin annotation file") + warning(warningStr) + } + + # Filter out rows with zero counts + sel <- rowSums(counts)!=0 + counts <- counts[sel, ] + anno <- anno[sel, ] + + # Create DGEList + data <- DGEList(counts=counts, lib.size=colSums(counts), + norm.factors=rep(1,ncol(counts)), genes=anno, group=factors) + + # Make the names of groups syntactically valid (replace spaces with periods) + data$samples$group <- make.names(data$samples$group) +} + +# Filter out any samples with zero counts +if (any(data$samples$lib.size == 0)) { + sampleSel <- data$samples$lib.size != 0 + filteredSamples <- paste(data$samples$ID[!sampleSel], collapse=", ") + data$counts <- data$counts[, sampleSel] + data$samples <- data$samples[sampleSel, ] +} + +# Filter hairpins with low counts +preFilterCount <- nrow(data) +selRow <- rowSums(cpm(data$counts) > cpmReq) >= sampleReq +selCol <- colSums(data$counts) > readReq +data <- data[selRow, selCol] + +# Check if any data survived filtering +if (length(data$counts) == 0) { + stop("no data remains after filtering, consider relaxing filters") +} + +# Count number of filtered tags and samples +postFilterCount <- nrow(data) +filteredCount <- preFilterCount - postFilterCount +sampleFilterCount <- sum(!selCol) + +if (secFactName == "none") { + # Estimate dispersions + data <- estimateDisp(data) + commonBCV <- round(sqrt(data$common.dispersion), 4) +} else { + # Construct design + if (inputType == "counts") { + + sampleNames <- colnames(counts) + matchedIndex <- match(sampleNames, samples$ID) + factors <- factor(make.names(samples$group[matchedIndex])) + + secFactors <- factor(make.names(samples[[secFactName]][matchedIndex])) + + } else if (inputType == "fastq" || inputType == "pairedFastq") { + + factors <- factor(data$sample$group) + secFactors <- factor(data$sample[[secFactName]]) + + } + + design <- model.matrix(~0 + factors + secFactors) + + # Estimate dispersions + data <- estimateDisp(data, design=design) + commonBCV <- round(sqrt(data$common.dispersion), 4) +} + + +################################################################################ +### Output Processing +################################################################################ + +# Plot number of hairpins that could be matched per sample +png(barIndexPng, width=600, height=600) +barplot(height<-colSums(data$counts), las=2, main="Counts per index", + cex.names=1.0, cex.axis=0.8, ylim=c(0, max(height)*1.2)) +imageData[1, ] <- c("Counts per Index", "barIndex.png") +invisible(dev.off()) + +pdf(barIndexPdf) +barplot(height<-colSums(data$counts), las=2, main="Counts per index", + cex.names=1.0, cex.axis=0.8, ylim=c(0, max(height)*1.2)) +linkData[1, ] <- c("Counts per Index Barplot (.pdf)", "barIndex.pdf") +invisible(dev.off()) + +# Plot per hairpin totals across all samples +png(barHairpinPng, width=600, height=600) +if (nrow(data$counts)<50) { + barplot(height<-rowSums(data$counts), las=2, main="Counts per hairpin", + cex.names=0.8, cex.axis=0.8, ylim=c(0, max(height)*1.2)) +} else { + barplot(height<-rowSums(data$counts), las=2, main="Counts per hairpin", + cex.names=0.8, cex.axis=0.8, ylim=c(0, max(height)*1.2), + names.arg=FALSE) +} +imageData <- rbind(imageData, c("Counts per Hairpin", "barHairpin.png")) +invisible(dev.off()) + +pdf(barHairpinPdf) +if (nrow(data$counts)<50) { + barplot(height<-rowSums(data$counts), las=2, main="Counts per hairpin", + cex.names=0.8, cex.axis=0.8, ylim=c(0, max(height)*1.2)) +} else { + barplot(height<-rowSums(data$counts), las=2, main="Counts per hairpin", + cex.names=0.8, cex.axis=0.8, ylim=c(0, max(height)*1.2), + names.arg=FALSE) +} +newEntry <- c("Counts per Hairpin Barplot (.pdf)", "barHairpin.pdf") +linkData <- rbind(linkData, newEntry) +invisible(dev.off()) + +# Make an MDS plot to visualise relationships between replicate samples +png(mdsPng, width=600, height=600) +plotMDS(data, labels=data$samples$group, col=as.numeric(data$samples$group), main="MDS Plot") +imageData <- rbind(imageData, c("MDS Plot", "mds.png")) +invisible(dev.off()) + +pdf(mdsPdf) +plotMDS(data, labels=data$samples$group, col=as.numeric(data$samples$group),main="MDS Plot") +newEntry <- c("MDS Plot (.pdf)", "mds.pdf") +linkData <- rbind(linkData, newEntry) +invisible(dev.off()) + +# BCV Plot +png(bcvPng, width=600, height=600) +plotBCV(data, main="BCV Plot") +imageData <- rbind(imageData, c("BCV Plot", "bcv.png")) +invisible(dev.off()) + +pdf(bcvPdf) +plotBCV(data, main="BCV Plot") +newEntry <- c("BCV Plot (.pdf)", "bcv.pdf") +linkData <- rbind(linkData, newEntry) +invisible(dev.off()) + +if (workMode == "classic") { + # Assess differential representation using classic exact testing methodology + # in edgeR + testData <- exactTest(data, pair=pairData) + + top <- topTags(testData, n=Inf) + + if (selectDirection == "all") { + topIDs <- top$table[(top$table$FDR < fdrThresh) & + (abs(top$table$logFC) > lfcThresh), 1] + } else if (selectDirection == "up") { + topIDs <- top$table[(top$table$FDR < fdrThresh) & + (top$table$logFC > lfcThresh), 1] + } else if (selectDirection == "down") { + topIDs <- top$table[(top$table$FDR < fdrThresh) & + (top$table$logFC < -lfcThresh), 1] +} + + write.table(top, file=topOut, row.names=FALSE, sep="\t") + + linkName <- paste0("Top Tags Table(", pairData[2], "-", pairData[1], + ") (.tsv)") + linkAddr <- paste0("toptag(", pairData[2], "-", pairData[1], ").tsv") + linkData <- rbind(linkData, c(linkName, linkAddr)) + + upCount[1] <- sum(top$table$FDR < fdrThresh & top$table$logFC > lfcThresh) + + downCount[1] <- sum(top$table$FDR < fdrThresh & + top$table$logFC < -lfcThresh) + + flatCount[1] <- sum(top$table$FDR > fdrThresh | + abs(top$table$logFC) < lfcThresh) + + + + # Select hairpins with FDR < 0.05 to highlight on plot + png(smearPng, width=600, height=600) + plotTitle <- gsub(".", " ", + paste0("Smear Plot: ", pairData[2], "-", pairData[1]), + fixed=TRUE) + plotSmear(testData, de.tags=topIDs, + pch=20, cex=1.0, main=plotTitle) + abline(h=c(-1, 0, 1), col=c("dodgerblue", "yellow", "dodgerblue"), lty=2) + imgName <- paste0("Smear Plot(", pairData[2], "-", pairData[1], ")") + imgAddr <- paste0("smear(", pairData[2], "-", pairData[1],").png") + imageData <- rbind(imageData, c(imgName, imgAddr)) + invisible(dev.off()) + + pdf(smearPdf) + plotTitle <- gsub(".", " ", + paste0("Smear Plot: ", pairData[2], "-", pairData[1]), + fixed=TRUE) + plotSmear(testData, de.tags=topIDs, + pch=20, cex=1.0, main=plotTitle) + abline(h=c(-1, 0, 1), col=c("dodgerblue", "yellow", "dodgerblue"), lty=2) + imgName <- paste0("Smear Plot(", pairData[2], "-", pairData[1], ") (.pdf)") + imgAddr <- paste0("smear(", pairData[2], "-", pairData[1], ").pdf") + linkData <- rbind(linkData, c(imgName, imgAddr)) + invisible(dev.off()) + +} else if (workMode == "glm") { + # Generating design information + if (secFactName == "none") { + + factors <- factor(data$sample$group) + design <- model.matrix(~0 + factors) + + colnames(design) <- gsub("factors", "", colnames(design), fixed=TRUE) + + } else { + + factors <- factor(data$sample$group) + + if (inputType == "counts") { + + sampleNames <- colnames(counts) + matchedIndex <- match(sampleNames, samples$ID) + factors <- factor(samples$group[matchedIndex]) + + secFactors <- factor(samples[[secFactName]][matchedIndex]) + + } else if (inputType == "fastq" || inputType == "pairedFastq") { + + secFactors <- factor(data$sample[[secFactName]]) + + } + + design <- model.matrix(~0 + factors + secFactors) + + colnames(design) <- gsub("factors", "", colnames(design), fixed=TRUE) + colnames(design) <- gsub("secFactors", secFactName, colnames(design), + fixed=TRUE) + } + + + # Split up contrasts seperated by comma into a vector + contrastData <- unlist(strsplit(contrastData, split=",")) + + for (i in 1:length(contrastData)) { + # Generate contrasts information + contrasts <- makeContrasts(contrasts=contrastData[i], levels=design) + + # Fit negative bionomial GLM + fit <- glmFit(data, design) + # Carry out Likelihood ratio test + testData <- glmLRT(fit, contrast=contrasts) + + # Select hairpins with FDR < 0.05 to highlight on plot + top <- topTags(testData, n=Inf) + + if (selectDirection == "all") { + topIDs <- top$table[(top$table$FDR < fdrThresh) & + (abs(top$table$logFC) > lfcThresh), 1] + } else if (selectDirection == "up") { + topIDs <- top$table[(top$table$FDR < fdrThresh) & + (top$table$logFC > lfcThresh), 1] + } else if (selectDirection == "down") { + topIDs <- top$table[(top$table$FDR < fdrThresh) & + (top$table$logFC < -lfcThresh), 1] + } + + write.table(top, file=topOut[i], row.names=FALSE, sep="\t") + + linkName <- paste0("Top Tags Table(", contrastData[i], ") (.tsv)") + linkAddr <- paste0("toptag(", contrastData[i], ").tsv") + linkData <- rbind(linkData, c(linkName, linkAddr)) + + # Collect counts for differential representation + upCount[i] <- sum(top$table$FDR < fdrThresh & top$table$logFC > lfcThresh) + downCount[i] <- sum(top$table$FDR < fdrThresh & + top$table$logFC < -lfcThresh) + flatCount[i] <- sum(top$table$FDR > fdrThresh | + abs(top$table$logFC) < lfcThresh) + + # Make a plot of logFC versus logCPM + png(smearPng[i], height=600, width=600) + plotTitle <- paste("Smear Plot:", gsub(".", " ", contrastData[i], + fixed=TRUE)) + plotSmear(testData, de.tags=topIDs, pch=20, cex=0.8, main=plotTitle) + abline(h=c(-1, 0, 1), col=c("dodgerblue", "yellow", "dodgerblue"), lty=2) + + imgName <- paste0("Smear Plot(", contrastData[i], ")") + imgAddr <- paste0("smear(", contrastData[i], ").png") + imageData <- rbind(imageData, c(imgName, imgAddr)) + invisible(dev.off()) + + pdf(smearPdf[i]) + plotTitle <- paste("Smear Plot:", gsub(".", " ", contrastData[i], + fixed=TRUE)) + plotSmear(testData, de.tags=topIDs, pch=20, cex=0.8, main=plotTitle) + abline(h=c(-1, 0, 1), col=c("dodgerblue", "yellow", "dodgerblue"), lty=2) + + linkName <- paste0("Smear Plot(", contrastData[i], ") (.pdf)") + linkAddr <- paste0("smear(", contrastData[i], ").pdf") + linkData <- rbind(linkData, c(linkName, linkAddr)) + invisible(dev.off()) + + genes <- as.character(data$genes$Gene) + unq <- unique(genes) + unq <- unq[!is.na(unq)] + geneList <- list() + for (gene in unq) { + if (length(which(genes == gene)) >= hairpinReq) { + geneList[[gene]] <- which(genes == gene) + } + } + + if (wantRoast) { + # Input preparaton for roast + nrot <- 9999 + set.seed(602214129) + roastData <- mroast(data, index=geneList, design=design, + contrast=contrasts, nrot=nrot) + roastData <- cbind(GeneID=rownames(roastData), roastData) + write.table(roastData, file=roastOut[i], row.names=FALSE, sep="\t") + linkName <- paste0("Gene Level Analysis Table(", contrastData[i], + ") (.tsv)") + linkAddr <- paste0("gene_level(", contrastData[i], ").tsv") + linkData <- rbind(linkData, c(linkName, linkAddr)) + if (selectOpt == "rank") { + selectedGenes <- rownames(roastData)[selectVals] + } else { + selectedGenes <- selectVals + } + + if (packageVersion("limma")<"3.19.19") { + png(barcodePng[i], width=600, height=length(selectedGenes)*150) + } else { + png(barcodePng[i], width=600, height=length(selectedGenes)*300) + } + par(mfrow=c(length(selectedGenes), 1)) + for (gene in selectedGenes) { + barcodeplot(testData$table$logFC, index=geneList[[gene]], + main=paste("Barcode Plot for", gene, "(logFCs)", + gsub(".", " ", contrastData[i])), + labels=c("Positive logFC", "Negative logFC")) + } + imgName <- paste0("Barcode Plot(", contrastData[i], ")") + imgAddr <- paste0("barcode(", contrastData[i], ").png") + imageData <- rbind(imageData, c(imgName, imgAddr)) + dev.off() + if (packageVersion("limma")<"3.19.19") { + pdf(barcodePdf[i], width=8, height=2) + } else { + pdf(barcodePdf[i], width=8, height=4) + } + for (gene in selectedGenes) { + barcodeplot(testData$table$logFC, index=geneList[[gene]], + main=paste("Barcode Plot for", gene, "(logFCs)", + gsub(".", " ", contrastData[i])), + labels=c("Positive logFC", "Negative logFC")) + } + linkName <- paste0("Barcode Plot(", contrastData[i], ") (.pdf)") + linkAddr <- paste0("barcode(", contrastData[i], ").pdf") + linkData <- rbind(linkData, c(linkName, linkAddr)) + dev.off() + } + } +} + +# Generate data frame of the significant differences +sigDiff <- data.frame(Up=upCount, Flat=flatCount, Down=downCount) +if (workMode == "glm") { + + row.names(sigDiff) <- contrastData + +} else if (workMode == "classic") { + + row.names(sigDiff) <- paste0(pairData[2], "-", pairData[1]) + +} + +# Output table of summarised counts +ID <- rownames(data$counts) +outputCounts <- cbind(ID, data$counts) +write.table(outputCounts, file=countsOut, row.names=FALSE, sep="\t", + quote=FALSE) +linkName <- "Counts table (.tsv)" +linkAddr <- "counts.tsv" +linkData <- rbind(linkData, c(linkName, linkAddr)) + +# Record session info +writeLines(capture.output(sessionInfo()), sessionOut) +linkData <- rbind(linkData, c("Session Info", "session_info.txt")) + +# Record ending time and calculate total run time +timeEnd <- as.character(Sys.time()) +timeTaken <- capture.output(round(difftime(timeEnd,timeStart), digits=3)) +timeTaken <- gsub("Time difference of ", "", timeTaken, fixed=TRUE) +################################################################################ +### HTML Generation +################################################################################ +# Clear file +cat("", file=htmlPath) + +cata("<html>\n") +HtmlHead("EdgeR Output") + +cata("<body>\n") +cata("<h3>EdgeR Analysis Output:</h3>\n") +cata("<h4>Input Summary:</h4>\n") +if (inputType == "fastq" || inputType == "pairedFastq") { + + cata("<ul>\n") + ListItem(hpReadout[1]) + ListItem(hpReadout[2]) + cata("</ul>\n") + cata(hpReadout[3], "<br />\n") + cata("<ul>\n") + ListItem(hpReadout[4]) + ListItem(hpReadout[7]) + cata("</ul>\n") + cata(hpReadout[8:11], sep="<br />\n") + cata("<br />\n") + cata("<b>Please check that read percentages are consistent with ") + cata("expectations.</b><br >\n") + +} else if (inputType == "counts") { + + cata("<ul>\n") + ListItem("Number of Samples: ", ncol(data$counts)) + ListItem("Number of Hairpins: ", countsRows) + ListItem("Number of annotations provided: ", annoRows) + ListItem("Number of annotations matched to hairpin: ", annoMatched) + cata("</ul>\n") + +} + +cata("The estimated common biological coefficient of variation (BCV) is: ", + commonBCV, "<br />\n") + +if (secFactName == "none") { + + cata("No secondary factor specified.<br />\n") + +} else { + + cata("Secondary factor specified as: ", secFactName, "<br />\n") + +} + +cata("<h4>Output:</h4>\n") +cata("PDF copies of JPEGS available in 'Plots' section.<br />\n") +for (i in 1:nrow(imageData)) { + if (grepl("barcode", imageData$Link[i])) { + + if (packageVersion("limma")<"3.19.19") { + + HtmlImage(imageData$Link[i], imageData$Label[i], + height=length(selectedGenes)*150) + + } else { + + HtmlImage(imageData$Link[i], imageData$Label[i], + height=length(selectedGenes)*300) + + } + } else { + + HtmlImage(imageData$Link[i], imageData$Label[i]) + + } +} +cata("<br />\n") + +cata("<h4>Differential Representation Counts:</h4>\n") + +cata("<table border=\"1\" cellpadding=\"4\">\n") +cata("<tr>\n") +TableItem() +for (i in colnames(sigDiff)) { + TableHeadItem(i) +} +cata("</tr>\n") +for (i in 1:nrow(sigDiff)) { + cata("<tr>\n") + TableHeadItem(unmake.names(row.names(sigDiff)[i])) + for (j in 1:ncol(sigDiff)) { + TableItem(as.character(sigDiff[i, j])) + } + cata("</tr>\n") +} +cata("</table>") + +cata("<h4>Plots:</h4>\n") +for (i in 1:nrow(linkData)) { + if (grepl(".pdf", linkData$Link[i])) { + HtmlLink(linkData$Link[i], linkData$Label[i]) + } +} + +cata("<h4>Tables:</h4>\n") +for (i in 1:nrow(linkData)) { + if (grepl(".tsv", linkData$Link[i])) { + HtmlLink(linkData$Link[i], linkData$Label[i]) + } +} + +cata("<p>Alt-click links to download file.</p>\n") +cata("<p>Click floppy disc icon on associated history item to download ") +cata("all files.</p>\n") +cata("<p>.tsv files can be viewed in Excel or any spreadsheet program.</p>\n") + +cata("<h4>Additional Information:</h4>\n") + +if (inputType == "fastq") { + + ListItem("Data was gathered from fastq raw read file(s).") + +} else if (inputType == "counts") { + + ListItem("Data was gathered from a table of counts.") + +} + +if (cpmReq != 0 && sampleReq != 0) { + tempStr <- paste("Target sequences without more than", cpmReq, + "CPM in at least", sampleReq, "samples are insignificant", + "and filtered out.") + ListItem(tempStr) + + filterProp <- round(filteredCount/preFilterCount*100, digits=2) + tempStr <- paste0(filteredCount, " of ", preFilterCount," (", filterProp, + "%) target sequences were filtered out for low ", + "count-per-million.") + ListItem(tempStr) +} + +if (readReq != 0) { + tempStr <- paste("Samples that did not produce more than", readReq, + "counts were filtered out.") + ListItem(tempStr) + + tempStr <- paste0(sampleFilterCount, " samples were filtered out for low ", + "counts.") + ListItem(tempStr) +} + +if (exists("filteredSamples")) { + tempStr <- paste("The following samples were filtered out for having zero", + "library size: ", filteredSamples) + ListItem(tempStr) +} + +if (workMode == "classic") { + ListItem("An exact test was performed on each target sequence.") +} else if (workMode == "glm") { + ListItem("A generalised linear model was fitted to each target sequence.") +} + +cit <- character() +link <-character() +link[1] <- paste0("<a href=\"", + "http://www.bioconductor.org/packages/release/bioc/", + "vignettes/limma/inst/doc/usersguide.pdf", + "\">", "limma User's Guide", "</a>.") +link[2] <- paste0("<a href=\"", + "http://www.bioconductor.org/packages/release/bioc/", + "vignettes/edgeR/inst/doc/edgeRUsersGuide.pdf", + "\">", "edgeR User's Guide", "</a>") + +cit[1] <- paste("Robinson MD, McCarthy DJ and Smyth GK (2010).", + "edgeR: a Bioconductor package for differential", + "expression analysis of digital gene expression", + "data. Bioinformatics 26, 139-140") +cit[2] <- paste("Robinson MD and Smyth GK (2007). Moderated statistical tests", + "for assessing differences in tag abundance. Bioinformatics", + "23, 2881-2887") +cit[3] <- paste("Robinson MD and Smyth GK (2008). Small-sample estimation of", + "negative binomial dispersion, with applications to SAGE data.", + "Biostatistics, 9, 321-332") + +cit[4] <- paste("McCarthy DJ, Chen Y and Smyth GK (2012). Differential", + "expression analysis of multifactor RNA-Seq experiments with", + "respect to biological variation. Nucleic Acids Research 40,", + "4288-4297") + +cata("<h4>Citations</h4>") +cata("<ol>\n") +ListItem(cit[1]) +ListItem(cit[2]) +ListItem(cit[3]) +ListItem(cit[4]) +cata("</ol>\n") + +cata("<p>Report problems to: su.s@wehi.edu.au</p>\n") + +for (i in 1:nrow(linkData)) { + if (grepl("session_info", linkData$Link[i])) { + HtmlLink(linkData$Link[i], linkData$Label[i]) + } +} + +cata("<table border=\"0\">\n") +cata("<tr>\n") +TableItem("Task started at:"); TableItem(timeStart) +cata("</tr>\n") +cata("<tr>\n") +TableItem("Task ended at:"); TableItem(timeEnd) +cata("</tr>\n") +cata("<tr>\n") +TableItem("Task run time:"); TableItem(timeTaken) +cata("<tr>\n") +cata("</table>\n") + +cata("</body>\n") +cata("</html>")
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/hairpinTool.xml Mon Jan 19 22:14:10 2015 -0500 @@ -0,0 +1,645 @@ +<?xml version="1.0"?> +<tool id="shrnaseq" name="shRNAseq" version="1.2.1"> + <description> + for differential representation for shRNAseq and sgRNA + </description> + + <requirements> + <requirement type="package" version="3.1.2">R</requirement> + <requirement type="package" version="0.1">shrnaseq_r</requirement> + <requirement type="set_environment">HAIRPINTOOL_R_SOURCE</requirement> + </requirements> + + <stdio> + <exit_code range="1:" level="fatal" description="Tool exception" /> + </stdio> + + <command> + Rscript \$HAIRPINTOOL_R_SOURCE "$inputOpt.inputType" + #if $inputOpt.inputType=="fastq": + + #for $i, $fas in enumerate($inputOpt.fastq): + fastq::$fas.file + #end for + + "$inputOpt.hairpin" + "$inputOpt.samples" + + #if $inputOpt.positions.posOption=="yes": + $inputOpt.positions.barstart + $inputOpt.positions.barend + 0 + 0 + $inputOpt.positions.hpstart + $inputOpt.positions.hpend + #else: + 1 + 5 + 0 + 0 + 37 + 57 + #end if + #elif $inputOpt.inputType=="pairedFastq": + + #for $i, $fas in enumerate($inputOpt.fastq): + fastq::$fas.file + #end for + + #for $i, $fas in enumerate($inputOpt.fastq): + fastqRev::$fas.fileRev + #end for + + "$inputOpt.hairpin" + "$inputOpt.samples" + + #if $inputOpt.positions.posOption=="yes": + $inputOpt.positions.barstart + $inputOpt.positions.barend + $inputOpt.positions.barstartRev + $inputOpt.positions.barendRev + $inputOpt.positions.hpstart + $inputOpt.positions.hpend + #else: + 1 + 5 + 0 + 0 + 37 + 57 + #end if + + #elif $inputOpt.inputType=="counts": + "$inputOpt.counts" + "$inputOpt.hairpin" + "$inputOpt.samples" + 0 + 0 + 0 + 0 + 0 + #end if + + #if $inputOpt.secondaryFactor.secFactorOpt=="yes": + "$inputOpt.secondaryFactor.secFactName" + #else: + "none" + #end if + + #if $filterCPM.filtOption=="yes": + $filterCPM.cpmReq + $filterCPM.sampleReq + $filterCPM.readReq + #else: + -Inf + -Inf + -Inf + #end if + + "$fdr" + "$lfc" + "$direction" + "$workMode.mode" + "$outFile" + "$outFile.files_path" + + #if $workMode.mode=="classic": + "$workMode.pair1" + "$workMode.pair2" + #elif $workMode.mode=="glm": + "$workMode.contrast" + "$workMode.roast.roastOption" + + #if $workMode.roast.roastOption=="yes": + "$workMode.roast.hairpinReq" + "$workMode.roast.select.selOption" + "$workMode.roast.select.selection" + #else: + 0 + 0 + 0 + #end if + + #end if + </command> + + <inputs> + <conditional name="inputOpt"> + + <param name="inputType" type="select" label="Input File Type"> + <option value="fastq">FastQ File</option> + <option value="pairedFastq">Paired FastQ File</option> + <option value="counts">Table of Counts</option> + </param> + + <when value="fastq"> + <param name="hairpin" type="data" format="tabular" + label="Target Annotation"/> + + <param name="samples" type="data" format="tabular" + label="Sample Annotation"/> + + <repeat name="fastq" title="FastQ Files"> + <param name="file" type="data" format="fastq"/> + </repeat> + + <conditional name="secondaryFactor"> + + <param name="secFactorOpt" type="select" + label="Include Secondary Factor"> + + <option value="no" selected="True">No</option> + + <option value="yes">Yes</option> + + </param> + + <when value="yes"> + + <param name="secFactName" type="text" label="Secondary Factor Name" + size="80"/> + + </when> + + <when value="no"> + </when> + </conditional> + + <conditional name="positions"> + <param name="posOption" type="select" + label="Specify Sample Index and Target Sequence Locations?" + help="Default Positions: Index: 1 to 5, Target: 37 to 57."> + <option value="no" selected="True">No</option> + <option value="yes">Yes</option> + </param> + + <when value="yes"> + <param name="barstart" type="integer" value="1" + label="Index Starting Position"/> + <param name="barend" type="integer" value="5" + label="Index Ending Position"/> + + <param name="hpstart" type="integer" value="37" + label="Target Starting Position"/> + + <param name="hpend" type="integer" value="57" + label="Target Ending Position"/> + </when> + + <when value="no"/> + </conditional> + </when> + + <when value="pairedFastq"> + <param name="hairpin" type="data" format="tabular" + label="Target Sequence Annotation"/> + + <param name="samples" type="data" format="tabular" + label="Sample Annotation"/> + + <repeat name="fastq" title="FastQ Files"> + <param name="file" type="data" format="fastq"/> + <param name="fileRev" type="data" format="fastq"/> + </repeat> + + <conditional name="secondaryFactor"> + + <param name="secFactorOpt" type="select" + label="Include Secondary Factor"> + + <option value="no" selected="True">No</option> + + <option value="yes">Yes</option> + + </param> + + <when value="yes"> + + <param name="secFactName" type="text" label="Secondary Factor Name" + size="80"/> + + </when> + + <when value="no"> + </when> + </conditional> + + <conditional name="positions"> + + <param name="posOption" type="select" + label="Specify Sample Index and Target Sequence Locations?" + help="Default Positions: Index: 1 to 5, Input required for + reverse end, Target: 37 to 57."> + + <option value="no" selected="True">No</option> + + <option value="yes">Yes</option> + + </param> + + <when value="yes"> + <param name="barstart" type="integer" value="1" + label="Index Starting Position"/> + + <param name="barend" type="integer" value="5" + label="Index Ending Position"/> + + <param name="barstartRev" type="integer" value="0" + label="Reverse Index Starting Position"/> + + <param name="barendRev" type="integer" value="0" + label="Reverse Index Ending Position"/> + + <param name="hpstart" type="integer" value="37" + label="Target Starting Position"/> + + <param name="hpend" type="integer" value="57" + label="Target Ending Position"/> + </when> + + <when value="no"> + </when> + + </conditional> + + </when> + + <when value="counts"> + + <param name="counts" type="data" format="tabular" label="Counts Table"/> + + <param name="hairpin" type="data" format="tabular" + label="Target Sequence Annotation"/> + + <param name="samples" type="data" format="tabular" + label="Sample Annotation"/> + + <conditional name="secondaryFactor"> + + <param name="secFactorOpt" type="select" + label="Include Secondary Factor"> + + <option value="no" selected="True">No</option> + + <option value="yes">Yes</option> + + </param> + + <when value="yes"> + + <param name="secFactName" type="text" label="Secondary Factor Name" + size="80"/> + + </when> + + <when value="no"> + </when> + + </conditional> + + </when> + + </conditional> + + <conditional name="filterCPM"> + <param name="filtOption" type="select" label="Filter Low CPM?" + help="Ignore target sequences with very low representation when + performing analysis."> + <option value="yes">Yes</option> + <option value="no">No</option> + </param> + + <when value="yes"> + <param name="cpmReq" type="float" value="0.5" min="0" + label="Minimum CPM"/> + + <param name="sampleReq" type="integer" value="1" min="0" + label="Minimum Samples" + help="Filter out all the genes that do not meet the minimum + CPM in at least this many samples."/> + + <param name="readReq" type="integer" value="1000" min="0" + label="Minimum Reads" + help="Filter out all samples that do not have the minimum + number of reads."/> + + </when> + + <when value="no"/> + + </conditional> + + <conditional name="workMode"> + <param name="mode" type="select" label="Analysis Type" + help="Classic Exact Tests are useful for simple comparisons across + two sampling groups. Generalised linear models allow for more + complex contrasts and gene level analysis to be made."> + <option value="classic">Classic Exact Test</option> + <option value="glm">Generalised Linear Model</option> + </param> + + <when value="classic"> + <param name="pair1" type="text" label="Compare" size="40"/> + <param name="pair2" type="text" label="To" size="40" + help="The analysis will subtract values of this group from those + in the group above to establish the difference."/> + </when> + + <when value="glm"> + <param name="contrast" type="text" size="60" + label="Contrasts of interest" + help="Specify equations defining contrasts to be made. Eg. + KD-Control will result in positive fold change if KD has + greater expression and negative if Control has greater + expression."/> + + <conditional name="roast"> + <param name="roastOption" type="select" + label="Perform Gene Level Analysis?" + help="Analyse LogFC tendencies for target sequences belonging + to the same gene. NOTE: this is a slow procedure that + scales badly with the number of genes analysed."> + <option value="no">No</option> + <option value="yes">Yes</option> + </param> + + <when value="yes"> + <param name="hairpinReq" type="integer" value="2" min="2" + label="Minimum Targets Found" + help="Only genes with at least this many target sequences + found will be analysed."/> + + <conditional name="select"> + <param name="selOption" type="select" + label="Gene Selection Method"> + <option value="rank">By p-value Rank</option> + <option value="geneID">By Gene Identifier</option> + </param> + <when value="rank"> + <param name="selection" type="text" size="40" value="1:5" + label="Ranks of Top Genes to Plot" + help="Genes are ranked in ascending p-value for + differential representation, individual ranks can + be entered seperated by comma or a range seperated + by colon."/> + </when> + <when value="geneID"> + <param name="selection" type="text" size="80" value="" + label="Symbols of Genes to Plot" + help="Select genes based on their identifier in the + 'Gene' column of the sample information file. + Please ensure exact match with the values in input + file and separate selections with commas."/> + </when> + </conditional> + + + </when> + + <when value="no"/> + </conditional> + </when> + </conditional> + + <param name="fdr" type="float" value="0.05" min="0" max="1" + label="FDR Threshold" + help="All observations below this threshold will be highlighted + in the smear plot."/> + + <param name="lfc" type="float" value="0" min="0" + label="Absolute LogFC Threshold" + help="In additional to meeting the FDR requirement, the absolute + value of the log-fold-change of the observation must be above + this threshold to be highlighted."/> + + <param name="direction" type="select" label="Highlight Option" + help="Only hightlight positive or negative fold changes in smear plot?"> + <option value="all">Default</option> + <option value="up">Positive Only</option> + <option value="down">Negative Only</option> + </param> + </inputs> + + <outputs> + <data format="html" name="outFile" label="TagSeq Analysis"/> + </outputs> +<tests> +<test> + <param name='inputType' value="counts"/> + <param name='counts' value='zuber-count_matrix.txt' /> + <param name='hairpin' value='zuber-target_anno.txt' /> + <param name='samples' value='zuber-sample_anno.txt' /> + <param name="secFactorOpt" value='no' /> + <param name="filtOption" value='no' /> + <param name="fdr" value='0.05' /> + <param name="lfc" value="0" /> + <param name="direction" value="all" /> + <param name="mode" value="classic" /> + <param name="pair1" value="Day 0" /> + <param name="pair2" value="Day 14" /> + <output name='outFile' file='shrnaseq_zuber_test.html' compare='diff' lines_diff = '10'/> +</test> +</tests> + <help> + +.. class:: infomark + +**What it does** + +Given tables containing information about the hairpins/sgRNA and their +associated sample indices, information about the samples and fastq file +containing the sequencing reads. This tool will generate plots and tables for +the analysis of differential representation. + +.. class:: infomark + +A tutorial of how to use this tool is available at: +http://bioinf.wehi.edu.au/shRNAseq/galaxy.html + +----- + +.. class:: infomark + +**INPUTS** + +**Input File Type:** + +This tool is able to either generate counts from a raw FastQ file given the +information regarding the samples and hairpins/sgRNA. Alternatively if a table +of counts has already been generated it can also be used. + +**Counts Table (Counts Input):** + +A tab delimited text table of information regarding the counts of +hairpins/sgRNA. Should have a column 'ID' to denote the hairpins/sgRNA that +counts correspond to. Each additional column should have titles corresponding to +the label for the sample. + +Example:: + + ID Sample1 Sample2 Sample3 + Control1 49802 48014 40148 + Control2 12441 16352 14232 + Control3 9842 9148 9111 + Hairpin1 3300 3418 2914 + Hairpin2 91418 95812 93174 + Hairpin3 32985 31975 35104 + Hairpin4 12082 14081 14981 + Hairpin5 2491 2769 2691 + Hairpin6 1294 1486 1642 + Hairpin7 49501 49076 47611 + ... + +**Target Sequence Annotation:** + +A tab delimited text table of information regarding the targetted +hairpins/sgRNA sequence. Should have columns 'ID', 'Sequences' and 'Gene' to +uniquely identify the target, align it with the reads to produce counts and +identify which gene the target acts on. + +NOTE: the column names are case sensitive and should be input exactly as they +are shown here. + +Example:: + + ID Sequences Gene + Control1 TCTCGCTTGGGCGAGAGTAAG 2 + Control2 CCGCCTGAAGTCTCTGATTAA 2 + Control3 AGGAATTATAATGCTTATCTA 2 + Hairpin1 AAGGCAGAGACTGACCACCTA 4 + Hairpin2 GAGCGACCTGGTGTTACTCTA 4 + Hairpin3 ATGGTGTAAATAGAGCTGTTA 4 + Hairpin4 CAGCTCATCTTCTGTGAAGAA 4 + Hairpin5 CAGCTCTGTGGGTCAGAAGAA 4 + Hairpin6 CCAGGCACAGATCTCAAGATA 4 + Hairpin7 ATGACAAGAAAGACATCTCAA 7 + ... + +**Sample Annotation (FastQ Input):** + +A tab delimited text table of information regarding the samples. Should have +columns 'ID', 'Sequences' and 'group' to uniquely identify each sample, identify +the sample in the reads by its sample index sequence and correctly group +replicates for analysis. Additional columns may inserted for annotation purposes +and will not interfere with analysis as long as the necessary columns are +present. + +NOTE: With the exception of other_group, column names are case sensitive and +should be input exactly as they are shown here. The other_group column can be +named by the user and specified in the "Include Secondary Factor" option of the +tool. + +Example:: + + ID Sequences group other_group Replicate + 3 GAAAG Day 2 male 1 + 6 GAACC Day 10 female 1 + 9 GAAGA Day 5 GFP neg male 1 + 16 GAATT Day 5 GFP pos male 1 + 18 GACAC Day 2 female 2 + 21 GACCA Day 10 male 2 + 28 GACGT Day 5 GFP neg male 2 + 31 GACTG Day 5 GFP pos female 2 + 33 GAGAA Day 2 male 3 + 40 GAGCT Day 10 female 3 + ... + +**Include Secondary Factor** + +If there are two factors involved in the experiment (i.e. Age and Gender) then +then secondary factor should be included to improve the statistical analysis. +The secondary factor should be specified as a column in the sample annotation +file and the corresponding column name should be input exactly as it is into +the provided field in the tool. + +NOTE: Currently the secondary factor is used only to improve statistical +analysis, comparisons can only be made in the primary factor specified as +"group" in the sample annotation. + +**Specify Sample Index and Target Sequence Locations (FastQ Input):** + +It is assumed that in the sequencing reads that the first 5 bases are the +sample index sequence and that bases 37-57 are the hairpins/sgRNA. If this is +not the case then the values of the positions can be changed, however it still +requires the sample indices and hairpins/sgRNA to be in a consistent location an +in a continuous sequence. + +NOTE: position values start at 1 for the first base. + +**Filter Low CPM?:** + +Often in a large screen there may members with very low counts which are of no +interest in the experiment, these may be filtered out to speed up computations. +Filtering will be based on counts per million in a required number of samples. + +**Analysis Type:** + + * **Classic Exact Test:** This allows two experimental groups to be compared + and p-values for differential representation derivec for each target + sequence. Simple and fast for straightforward comparisons. In this option you + will have the option of "*Compare* x *To* y" which implicitly subtracts the + data from y from that of x to produce the comparison. + + * **Generalised Linear Model:** This allow for complex contrasts to be specified + and also gene level analysis to be performed. If this option is chosen then + contrasts must be explicitly stated in equations and multiple contrasts can + be made. In addition there will be the option to analyse hairpins/sgRNA on a + per-gene basis to see if hairpins/sgRNA belonging to a particular gene have + any overall tendencies for the direction of their log-fold-change. + +**FDR Threshold:** +The smear plot in the output will have hairpins/sgRNA highlighted to signify +significant differential representation. The significance is determined by +contorlling the false discovery rate, only those with a FDR lower than the +threshold will be highlighted in the plot. + + +----- + + +**Citations:** + +.. class:: infomark + +limma + +Please cite the paper below for the limma software itself. Please also try +to cite the appropriate methodology articles that describe the statistical +methods implemented in limma, depending on which limma functions you are +using. The methodology articles are listed in Section 2.1 of the limma +User's Guide. + + * Smyth, GK (2005). Limma: linear models for microarray data. In: + 'Bioinformatics and Computational Biology Solutions using R and + Bioconductor'. R. Gentleman, V. Carey, S. Dudoit, R. Irizarry, + W. Huber (eds), Springer, New York, pages 397-420. + +.. class:: infomark + +edgeR + +Please cite the first paper for the software itself and the other papers for +the various original statistical methods implemented in edgeR. See +Section 1.2 in the User's Guide for more detail. + + * Robinson MD, McCarthy DJ and Smyth GK (2010). edgeR: a Bioconductor + package for differential expression analysis of digital gene expression + data. Bioinformatics 26, 139-140 + + * Robinson MD and Smyth GK (2007). Moderated statistical tests for assessing + differences in tag abundance. Bioinformatics 23, 2881-2887 + + * Robinson MD and Smyth GK (2008). Small-sample estimation of negative + binomial dispersion, with applications to SAGE data. + Biostatistics, 9, 321-332 + + * McCarthy DJ, Chen Y and Smyth GK (2012). Differential expression analysis + of multifactor RNA-Seq experiments with respect to biological variation. + Nucleic Acids Research 40, 4288-4297 + +Report problems to: su.s@wehi.edu.au + +.. _edgeR: http://www.bioconductor.org/packages/release/bioc/html/edgeR.html +.. _limma: http://www.bioconductor.org/packages/release/bioc/html/limma.html + </help> +</tool>
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/readme.rst Mon Jan 19 22:14:10 2015 -0500 @@ -0,0 +1,15 @@ +shrnaseq wrapper +================ + +This is a self installing Galaxy tool exposing the shrnaseq_ R package which has excellent documentation at +shrnaseq_ Minimal details are provided in this wrapper - please RTM to get the best out of it. + + +.. _shrnaseq: http://bioinf.wehi.edu.au/shRNAseq/ + +Underlying R code written by Matt Ritchie. +Galaxy wrapper by Shian Su +Autoinstallation and fixin' : Ross Lazarus + +This version first passed tests on 18 Jan 2015 +
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/test-data/shrnaseq_zuber_test.html Mon Jan 19 22:14:10 2015 -0500 @@ -0,0 +1,80 @@ +<html> +<head> +<title>EdgeR Output</title> +</head> +<body> +<h3>EdgeR Analysis Output:</h3> +<h4>Input Summary:</h4> +<ul> +<li>Number of Samples: 4</li> +<li>Number of Hairpins: 1105</li> +<li>Number of annotations provided: 1105</li> +<li>Number of annotations matched to hairpin: 1105</li> +</ul> +The estimated common biological coefficient of variation (BCV) is: 0.9323<br /> +No secondary factor specified.<br /> +<h4>Output:</h4> +PDF copies of JPEGS available in 'Plots' section.<br /> +<img src="barIndex.png" alt="Counts per Index" height="600" width="600"/> +<img src="barHairpin.png" alt="Counts per Hairpin" height="600" width="600"/> +<img src="mds.png" alt="MDS Plot" height="600" width="600"/> +<img src="bcv.png" alt="BCV Plot" height="600" width="600"/> +<img src="smear(Day.0-Day.14).png" alt="Smear Plot(Day.0-Day.14)" height="600" width="600"/> +<br /> +<h4>Differential Representation Counts:</h4> +<table border="1" cellpadding="4"> +<tr> +<td></td> +<th>Up</th> +<th>Flat</th> +<th>Down</th> +</tr> +<tr> +<th>Day 0-Day 14</th> +<td>376</td> +<td>682</td> +<td>35</td> +</tr> +</table><h4>Plots:</h4> +<a href="barIndex.pdf" target="_blank">Counts per Index Barplot (.pdf)</a><br /> +<a href="barHairpin.pdf" target="_blank">Counts per Hairpin Barplot (.pdf)</a><br /> +<a href="mds.pdf" target="_blank">MDS Plot (.pdf)</a><br /> +<a href="bcv.pdf" target="_blank">BCV Plot (.pdf)</a><br /> +<a href="smear(Day.0-Day.14).pdf" target="_blank">Smear Plot(Day.0-Day.14) (.pdf)</a><br /> +<h4>Tables:</h4> +<a href="toptag(Day.0-Day.14).tsv" target="_blank">Top Tags Table(Day.0-Day.14) (.tsv)</a><br /> +<a href="counts.tsv" target="_blank">Counts table (.tsv)</a><br /> +<p>Alt-click links to download file.</p> +<p>Click floppy disc icon on associated history item to download all files.</p> +<p>.tsv files can be viewed in Excel or any spreadsheet program.</p> +<h4>Additional Information:</h4> +<li>Data was gathered from a table of counts.</li> +<li>Target sequences without more than 0.5 CPM in at least 1 samples are insignificant and filtered out.</li> +<li>1 of 1094 (0.09%) target sequences were filtered out for low count-per-million.</li> +<li>Samples that did not produce more than 1000 counts were filtered out.</li> +<li>0 samples were filtered out for low counts.</li> +<li>An exact test was performed on each target sequence.</li> +<h4>Citations</h4><ol> +<li>Robinson MD, McCarthy DJ and Smyth GK (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139-140</li> +<li>Robinson MD and Smyth GK (2007). Moderated statistical tests for assessing differences in tag abundance. Bioinformatics 23, 2881-2887</li> +<li>Robinson MD and Smyth GK (2008). Small-sample estimation of negative binomial dispersion, with applications to SAGE data. Biostatistics, 9, 321-332</li> +<li>McCarthy DJ, Chen Y and Smyth GK (2012). Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Research 40, 4288-4297</li> +</ol> +<p>Report problems to: su.s@wehi.edu.au</p> +<a href="session_info.txt" target="_blank">Session Info</a><br /> +<table border="0"> +<tr> +<td>Task started at:</td> +<td>2015-01-20 12:16:59</td> +</tr> +<tr> +<td>Task ended at:</td> +<td>2015-01-20 12:17:05</td> +</tr> +<tr> +<td>Task run time:</td> +<td>6 secs</td> +<tr> +</table> +</body> +</html> \ No newline at end of file
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/test-data/zuber-count_matrix.txt Mon Jan 19 22:14:10 2015 -0500 @@ -0,0 +1,1106 @@ +ID Reads_A_T0 Reads_A_T14 Reads_B_T0 Reads_B_T14 +100043305.2 34133 9171 31158 4111 +100043305.4 5589 1 4311 5737 +100043305.5 38651 7722 24711 15331 +2900092E17Rik.377 11759 24 9328 21 +2900092E17Rik.546 3581 30 3211 3 +2900092E17Rik.1051 11498 1907 10809 4116 +2900092E17Rik.1361 5590 1128 5753 1704 +Actl6b.379 8147 3520 8262 3861 +Actl6b.819 7152 5311 7080 2636 +Actl6b.917 3549 3 3326 9 +Actl6b.989 9928 3200 12207 1787 +Actl6b.1221 2449 173 4553 190 +Alkbh2.462 8219 0 8307 3674 +Alkbh2.557 17312 11788 16162 7253 +Alkbh2.590 908 0 3 0 +Alkbh2.640 29881 9982 32507 6981 +Alkbh2.641 30677 31067 21977 5244 +Alkbh3.552 7748 95 10813 628 +Alkbh3.843 7288 47 6161 1141 +Alkbh3.901 11464 10632 13945 986 +Alkbh3.1184 235478 121880 223886 199915 +Aof2.1869 4835 1 6635 18 +Aof2.1956 9168 74 11901 2001 +Aof2.2435 22561 5296 27662 4349 +Aof2.2857 12275 0 13761 0 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42465 +Wdr82.3705 25607 2390 22211 5706 +Wdr82.4023 47467 54245 35961 10951 +Whsc1.812 5301 8373 8384 1919 +Whsc1.818 21079 7170 18333 11013 +Whsc1.3055 20202 3 16818 4080 +Whsc1.3056 14848 3566 17067 3063 +Whsc1l1.276 8983 16 15356 86 +Whsc1l1.373 24335 1 18394 19 +Whsc1l1.524 25667 33492 20530 18722 +Whsc1l1.1307 36165 3810 43502 5067 +Whsc1l1.1653 1740 0 2619 0 +Wnt5a.2013 14662 2054 14921 8784 +Wnt5a.2659 31981 25084 30296 21463 +Wnt5a.2764 3508 402 4188 0 +Wnt5a.4154 24598 41766 25258 11415
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/test-data/zuber-sample_anno.txt Mon Jan 19 22:14:10 2015 -0500 @@ -0,0 +1,5 @@ +"ID" "group" +"Reads_A_T0" "Day 0" +"Reads_A_T14" "Day 14" +"Reads_B_T0" "Day 0" +"Reads_B_T14" "Day 14"
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/test-data/zuber-target_anno.txt Mon Jan 19 22:14:10 2015 -0500 @@ -0,0 +1,1106 @@ +ID Gene GeneID Pool shRNA_start Mean_T14.T0 T14.T0_A T14.T0_B +100043305.2 100043305 100043305 LIB 158 0.200312349 0.268684264 0.131940433 +100043305.4 100043305 100043305 LIB 369 0.665480322 0.000178923 1.330781721 +100043305.5 100043305 100043305 LIB 458 0.410099904 0.199787845 0.620411962 +2900092E17Rik.377 2900092E17Rik 67278 LIB 377 0.002146138 0.00204099 0.002251286 +2900092E17Rik.546 2900092E17Rik 67278 LIB 546 0.004655918 0.008377548 0.000934288 +2900092E17Rik.1051 2900092E17Rik 67278 LIB 1051 0.273324357 0.165854931 0.380793783 +2900092E17Rik.1361 2900092E17Rik 67278 LIB 1361 0.2489911 0.201788909 0.29619329 +Actl6b.379 Actl6b 83766 LIB 379 0.449690571 0.432060881 0.467320261 +Actl6b.819 Actl6b 83766 LIB 819 0.557452935 0.742589485 0.372316384 +Actl6b.917 Actl6b 83766 LIB 917 0.001775631 0.000845309 0.002705953 +Actl6b.989 Actl6b 83766 LIB 989 0.234356062 0.322320709 0.146391415 +Actl6b.1221 Actl6b 83766 LIB 1221 0.056185902 0.070641078 0.041730727 +Alkbh2.462 Alkbh2 231642 LIB 462 0.221138799 0 0.442277597 +Alkbh2.557 Alkbh2 231642 LIB 557 0.564841845 0.680914972 0.448768717 +Alkbh2.590 Alkbh2 231642 LIB 590 NA NA NA +Alkbh2.640 Alkbh2 231642 LIB 640 0.274406089 0.334058432 0.214753745 +Alkbh2.641 Alkbh2 231642 LIB 641 0.625663102 1.012713108 0.238613096 +Alkbh3.552 Alkbh3 69113 LIB 552 0.035169734 0.012261229 0.058078239 +Alkbh3.843 Alkbh3 69113 LIB 843 0.095823083 0.006448957 0.185197208 +Alkbh3.901 Alkbh3 69113 LIB 901 0.499065664 0.927424983 0.070706346 +Alkbh3.1184 Alkbh3 69113 LIB 1184 0.705258812 0.517585507 0.892932117 +Aof2.1869 Aof2 99982 LIB 1869 0.001459856 0.000206825 0.002712886 +Aof2.1956 Aof2 99982 LIB 1956 0.088104342 0.008071553 0.168137131 +Aof2.2435 Aof2 99982 LIB 2435 0.195980329 0.234741368 0.15721929 +Aof2.2857 Aof2 99982 LIB 2857 0 0 0 +Ash1l.1031 Ash1l 192195 LIB 1031 0.31947451 0.313544189 0.32540483 +Ash1l.1280 Ash1l 192195 LIB 1280 0.167683133 0.220091783 0.115274483 +Ash1l.3810 Ash1l 192195 LIB 3810 NA NA NA +Ash1l.8132 Ash1l 192195 LIB 8132 0.401332534 0.200805702 0.601859367 +Ash1l.9540 Ash1l 192195 LIB 9540 0.139184254 0.254215372 0.024153137 +Ash2l.586 Ash2l 23808 LIB 586 0.46112578 0.22570026 0.696551301 +Ash2l.805 Ash2l 23808 LIB 805 0.573261557 0.767245885 0.37927723 +Ash2l.948 Ash2l 23808 LIB 948 0.235730162 0.418269791 0.053190532 +Ash2l.1135 Ash2l 23808 LIB 1135 0.035165394 0.002115526 0.068215262 +Ash2l.2130 Ash2l 23808 LIB 2130 1.241609259 1.460589922 1.022628597 +Asxl1.2020 Asxl1 228790 LIB 2020 0.059766771 0.009568839 0.109964703 +Asxl1.3548 Asxl1 228790 LIB 3548 0.10962211 0.118078719 0.101165501 +Asxl1.3785 Asxl1 228790 LIB 3785 0.418395785 0.701615799 0.135175771 +Asxl1.4792 Asxl1 228790 LIB 4792 0.292975143 0.202313712 0.383636573 +Asxl1.5221 Asxl1 228790 LIB 5221 NA NA NA +Asxl1.6315 Asxl1 228790 LIB 6315 0.42809245 0.550629344 0.305555556 +Asxl2.11 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Prdm14 383491 LIB 530 0.405412499 0.39411959 0.416705408 +Prdm14.1756 Prdm14 383491 LIB 1756 0.005945122 0 0.011890244 +Prdm14.1934 Prdm14 383491 LIB 1934 0.293485392 0.527073733 0.059897052 +Prdm14.2271 Prdm14 383491 LIB 2271 0.111037316 0.097569355 0.124505277 +Prdm14.2348 Prdm14 383491 LIB 2348 0.492448295 0.042176871 0.94271972 +Prdm15.958 Prdm15 114604 LIB 958 0.98252896 1.932489451 0.032568468 +Prdm15.1034 Prdm15 114604 LIB 1034 0.004062756 0.003733333 0.004392179 +Prdm15.1690 Prdm15 114604 LIB 1690 0.524906994 0.429446747 0.620367241 +Prdm15.1796 Prdm15 114604 LIB 1796 0.174355348 0.183572542 0.165138154 +Prdm15.2734 Prdm15 114604 LIB 2734 0.038957255 0.003943577 0.073970934 +Prdm16.680 Prdm16 70673 LIB 680 0.533914395 0.660762545 0.407066246 +Prdm16.5683 Prdm16 70673 LIB 5683 0.096716361 0.180946552 0.01248617 +Prdm16.8212 Prdm16 70673 LIB 8212 NA NA NA +Prdm16.8363 Prdm16 70673 LIB 8363 0.430720448 0.248793956 0.61264694 +Prdm16.8571 Prdm16 70673 LIB 8571 2.329170058 2.550566982 2.107773134 +Prdm2.1020 Prdm2 110593 LIB 1020 0.153596644 0.259297344 0.047895945 +Prdm2.4191 Prdm2 110593 LIB 4191 0.559676498 0.502441293 0.616911704 +Prdm2.4655 Prdm2 110593 LIB 4655 NA NA NA +Prdm2.4956 Prdm2 110593 LIB 4956 0.090352843 0.160106435 0.020599251 +Prdm2.5781 Prdm2 110593 LIB 5781 0.2845147 0.353298153 0.215731247 +Prdm4.1943 Prdm4 72843 LIB 1943 0.115287664 0.047414054 0.183161274 +Prdm4.2535 Prdm4 72843 LIB 2535 0.085408022 0.000193199 0.170622844 +Prdm4.3139 Prdm4 72843 LIB 3139 0.001128668 0 0.002257336 +Prdm4.3391 Prdm4 72843 LIB 3391 0.069371374 0.05967095 0.079071797 +Prdm4.3667 Prdm4 72843 LIB 3667 NA NA NA +Prdm5.908 Prdm5 70779 LIB 908 0.149313429 0.128342246 0.170284612 +Prdm5.984 Prdm5 70779 LIB 984 0.413409055 0.622305737 0.204512373 +Prdm5.1353 Prdm5 70779 LIB 1353 1.643969827 2.094062103 1.193877551 +Prdm5.2148 Prdm5 70779 LIB 2148 0.648213784 0.715798492 0.580629076 +Prdm6.1018 Prdm6 225518 LIB 1018 0.009633756 0.011159403 0.008108108 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0.026339154 +Prkaa1.4596 Prkaa1 105787 LIB 4596 0.034088344 0.058964593 0.009212095 +Prkaa2.2299 Prkaa2 108079 LIB 2299 0.004331754 0.000484027 0.008179481 +Prkaa2.2748 Prkaa2 108079 LIB 2748 0.340786503 0.362280234 0.319292772 +Prkaa2.4825 Prkaa2 108079 LIB 4825 NA NA NA +Prkaa2.6172 Prkaa2 108079 LIB 6172 0.020265668 0 0.040531335 +Prkaa2.6849 Prkaa2 108079 LIB 6849 0.135065674 0.072316173 0.197815176 +Prkcd.161 Prkcd 18753 LIB 161 0.179448269 0.099882029 0.259014509 +Prkcd.441 Prkcd 18753 LIB 441 0.327507015 0.001459854 0.653554175 +Prkcd.707 Prkcd 18753 LIB 707 0.664294206 1.071999112 0.2565893 +Prkcd.1389 Prkcd 18753 LIB 1389 1.255652224 0.641311475 1.869992973 +Prmt1.243 Prmt1 15469 LIB 243 0.110664823 0.120254845 0.101074802 +Prmt1.264 Prmt1 15469 LIB 264 0.272777523 0.382384883 0.163170163 +Prmt1.630 Prmt1 15469 LIB 630 1.328852347 1.764027294 0.8936774 +Prmt1.1014 Prmt1 15469 LIB 1014 0.484140888 0.638219895 0.330061882 +Prmt2.151 Prmt2 15468 LIB 151 0.031631618 0.037750897 0.025512338 +Prmt2.154 Prmt2 15468 LIB 154 0.973162231 1.211030345 0.735294118 +Prmt2.1066 Prmt2 15468 LIB 1066 9.18E-05 0.000183688 0 +Prmt2.1067 Prmt2 15468 LIB 1067 0.815640774 0.654386731 0.976894818 +Prmt3.502 Prmt3 71974 LIB 502 0.157500935 0.315001871 0 +Prmt3.814 Prmt3 71974 LIB 814 4.89E-05 0 9.79E-05 +Prmt3.838 Prmt3 71974 LIB 838 3.559311442 4.211350585 2.907272299 +Prmt3.985 Prmt3 71974 LIB 985 0.274535697 0.423260006 0.125811388 +Prmt3.1839 Prmt3 71974 LIB 1839 0.113816928 0.148898678 0.078735178 +Prmt5.56 Prmt5 27374 LIB 56 0.778904793 0.453405018 1.104404568 +Prmt5.266 Prmt5 27374 LIB 266 0.201764199 0.173979378 0.22954902 +Prmt5.2152 Prmt5 27374 LIB 2152 0.126875652 0.076575692 0.177175612 +Prmt5.2293 Prmt5 27374 LIB 2293 2.188455879 0.469499821 3.907411937 +Prmt6.247 Prmt6 99890 LIB 247 0.033732942 0.000853242 0.066612642 +Prmt6.1493 Prmt6 99890 LIB 1493 0.134310673 0.232186732 0.036434613 +Prmt6.1983 Prmt6 99890 LIB 1983 0.154846836 0.167501928 0.142191745 +Prmt6.2355 Prmt6 99890 LIB 2355 0.50072786 0.541981695 0.459474025 +Prmt7.23 Prmt7 214572 LIB 23 0.009433344 0.004819277 0.01404741 +Prmt7.45 Prmt7 214572 LIB 45 0.543933929 0.518786922 0.569080936 +Prmt7.88 Prmt7 214572 LIB 88 0.080612502 0.04869904 0.112525964 +Prmt7.1284 Prmt7 214572 LIB 1284 0.06501016 0.126209284 0.003811036 +Prmt7.2135 Prmt7 214572 LIB 2135 0.23497578 0.386599091 0.083352468 +Prmt8.302 Prmt8 381813 LIB 302 0.244600451 0.198269736 0.290931167 +Prmt8.455 Prmt8 381813 LIB 455 0.59669119 0.802821189 0.390561191 +Prmt8.667 Prmt8 381813 LIB 667 0.045662786 0.087305569 0.004020002 +Prmt8.1854 Prmt8 381813 LIB 1854 0.108178455 0.06973876 0.146618151 +Psip1.763 Psip1 101739 LIB 763 0.008304892 0 0.016609784 +Psip1.1311 Psip1 101739 LIB 1311 0 0 0 +Psip1.1596 Psip1 101739 LIB 1596 0.059717843 0.087169563 0.032266123 +Psip1.2474 Psip1 101739 LIB 2474 0.006237689 0.012475378 0 +Psip1.2652 Psip1 101739 LIB 2652 0.06194056 0.10699453 0.016886591 +Rbbp4.2088 Rbbp4 19646 LIB 2088 0.193789327 0.126048165 0.261530489 +Rbbp4.2174 Rbbp4 19646 LIB 2174 0.005813953 0 0.011627907 +Rbbp4.2511 Rbbp4 19646 LIB 2511 0.928220759 1.004589667 0.851851852 +Rbbp4.3753 Rbbp4 19646 LIB 3753 0.073360623 0.135745027 0.010976218 +Rbbp5.1634 Rbbp5 213464 LIB 1634 1.015990856 1.156510409 0.875471303 +Rbbp5.2248 Rbbp5 213464 LIB 2248 0.000789875 0.00145743 0.00012232 +Rbbp5.2655 Rbbp5 213464 LIB 2655 0.121958567 0.170557717 0.073359417 +Rbbp5.2974 Rbbp5 213464 LIB 2974 1.399302063 1.445902998 1.352701127 +Renilla.713 Renilla Luciferase NC 713 1.666788004 1.533762916 1.799813091 +Ring1.260 Ring1 19763 LIB 260 0.286061741 0.026611473 0.54551201 +Ring1.669 Ring1 19763 LIB 669 0.165118469 0.170742026 0.159494912 +Ring1.1034 Ring1 19763 LIB 1034 0.180669238 0.016304348 0.345034127 +Rnf2.1856 Rnf2 19821 LIB 1856 0.673388184 0.833867094 0.512909274 +Rnf2.2203 Rnf2 19821 LIB 2203 0.954595303 0.850564925 1.05862568 +Rnf2.2538 Rnf2 19821 LIB 2538 0.837959761 0.541602515 1.134317006 +Rnf2.2809 Rnf2 19821 LIB 2809 0.187347204 0.081992104 0.292702304 +Rnf2.2875 Rnf2 19821 LIB 2875 0.546987675 0.692397599 0.401577752 +Rnf20.420 Rnf20 109331 LIB 420 0.063920224 0.054351697 0.073488751 +Rnf20.948 Rnf20 109331 LIB 948 0.278768206 0.423430663 0.134105749 +Rnf20.1295 Rnf20 109331 LIB 1295 0.000109004 0 0.000218007 +Rnf20.3244 Rnf20 109331 LIB 3244 NA NA NA +Rnf20.3277 Rnf20 109331 LIB 3277 0.459938908 0.803347787 0.11653003 +Rnf20.3718 Rnf20 109331 LIB 3718 1.136821022 1.414336918 0.859305126 +Rpa1.1620 Rpa1 68275 PC 1620 0.007210873 0.000351973 0.014069774 +Rpa3.276 Rpa3 68240 PC 278 7.32E-05 0.000111564 3.48E-05 +Rpa3.455 Rpa3 68240 PC 457 0.018174308 0.02069689 0.015651726 +Rpa3.561 Rpa3 68240 PC 561 0.00476048 0.002574918 0.006946042 +Satb1.710 Satb1 20230 LIB 710 0.161960667 0.323389702 0.000531632 +Satb1.1401 Satb1 20230 LIB 1401 0.392569667 0.3563288 0.428810533 +Satb1.1478 Satb1 20230 LIB 1478 0.24003152 0.473695747 0.006367292 +Satb1.1709 Satb1 20230 LIB 1709 0.193635318 0.075057818 0.312212817 +Satb1.2566 Satb1 20230 LIB 2566 0.193845463 0.035309973 0.352380952 +Setd1a.119 Setd1a 233904 LIB 119 0.107452217 0.170620438 0.044283995 +Setd1a.388 Setd1a 233904 LIB 388 0.608381477 0.653331355 0.5634316 +Setd1a.643 Setd1a 233904 LIB 643 1.521441453 1.691804708 1.351078197 +Setd1a.5859 Setd1a 233904 LIB 5859 0.797831182 0.900645478 0.695016886 +Setd1b.3553 Setd1b 208043 LIB 3553 8.23E-05 0 0.0001646 +Setd1b.3557 Setd1b 208043 LIB 3557 0.18069148 0.180119102 0.181263858 +Setd1b.4520 Setd1b 208043 LIB 4520 0.199934074 0.05129672 0.348571429 +Setd1b.4522 Setd1b 208043 LIB 4522 0.003342509 0 0.006685018 +Setd1b.4699 Setd1b 208043 LIB 4699 0.137405083 0.097939189 0.176870978 +Setd2.182 Setd2 235626 LIB 182 0.040179039 0.047492416 0.032865662 +Setd2.1467 Setd2 235626 LIB 1467 0.00017313 0 0.00034626 +Setd2.1785 Setd2 235626 LIB 1785 0.038294803 0.027488932 0.049100673 +Setd2.3632 Setd2 235626 LIB 3632 0.377349359 0.44562685 0.309071868 +Setd2.4051 Setd2 235626 LIB 4051 0 0 0 +Setd3.795 Setd3 52690 LIB 795 0.126767545 0.027322404 0.226212687 +Setd3.1496 Setd3 52690 LIB 1496 NA NA NA +Setd3.2395 Setd3 52690 LIB 2395 0.012222377 0 0.024444755 +Setd4.506 Setd4 224440 LIB 506 0.001721296 0.001794728 0.001647864 +Setd4.1308 Setd4 224440 LIB 1308 2.55E-05 5.10E-05 0 +Setd4.1517 Setd4 224440 LIB 1517 0 0 0 +Setd7.4328 Setd7 73251 LIB 4328 0.79628381 1.371309831 0.22125779 +Setd7.5317 Setd7 73251 LIB 5317 0.441117913 0.586184524 0.296051303 +Setd7.5342 Setd7 73251 LIB 5342 0.143929674 0.043706114 0.244153234 +Setd7.5940 Setd7 73251 LIB 5940 NA NA NA +Setd7.7009 Setd7 73251 LIB 7009 0.066151971 0.049381188 0.082922753 +Setd8.2578 Setd8 67956 LIB 2578 0.189731658 0.000972526 0.37849079 +Setd8.2622 Setd8 67956 LIB 2622 0.411566348 0.623313229 0.199819467 +Setd8.2632 Setd8 67956 LIB 2632 1.643161397 2.493088617 0.793234176 +Setdb1.1145 Setdb1 84505 LIB 1145 0.517510269 0.470614035 0.564406503 +Setdb1.1925 Setdb1 84505 LIB 1925 0.086579742 0 0.173159485 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353282 LIB 602 0.281181984 0.520202721 0.042161246 +Sfmbt2.3592 Sfmbt2 353282 LIB 3592 0.082878502 0.005644644 0.16011236 +Sfmbt2.5673 Sfmbt2 353282 LIB 5673 0.134401376 0.019676674 0.249126078 +Sin3a.531 Sin3a 20466 LIB 531 0 0 0 +Sin3a.3537 Sin3a 20466 LIB 3537 0.024340683 0 0.048681366 +Sin3a.3559 Sin3a 20466 LIB 3559 1.728939777 2.831898734 0.62598082 +Sin3a.4729 Sin3a 20466 LIB 4729 0 0 0 +Sin3b.188 Sin3b 20467 LIB 188 0.042293799 0.084587598 0 +Sin3b.326 Sin3b 20467 LIB 326 1.438726271 2.275190706 0.602261836 +Sin3b.338 Sin3b 20467 LIB 338 0.105590062 0 0.211180124 +Sin3b.381 Sin3b 20467 LIB 381 0.134926536 0.248162305 0.021690768 +Sin3b.475 Sin3b 20467 LIB 475 0.639579924 1.118636662 0.160523187 +Sirt1.688 Sirt1 93759 LIB 688 1.451209242 1.979805805 0.922612678 +Sirt1.1708 Sirt1 93759 LIB 1708 0.631165203 0.733237638 0.529092768 +Sirt1.1779 Sirt1 93759 LIB 1779 0.436869096 0.336818563 0.536919629 +Sirt1.2191 Sirt1 93759 LIB 2191 1.729713563 2.528467711 0.930959415 +Sirt2.735 Sirt2 64383 LIB 735 0.147707432 0.003062938 0.292351926 +Sirt2.1418 Sirt2 64383 LIB 1418 0.131476192 0.093366665 0.169585719 +Sirt2.1460 Sirt2 64383 LIB 1460 0.276894538 0.290329505 0.26345957 +Sirt2.1600 Sirt2 64383 LIB 1600 0.023729675 0.026015228 0.021444122 +Sirt3.462 Sirt3 64384 LIB 462 0.43015768 0.70883566 0.1514797 +Sirt3.869 Sirt3 64384 LIB 869 0.087373128 0.032567568 0.142178688 +Sirt3.993 Sirt3 64384 LIB 993 1.118453529 1.035942224 1.200964834 +Sirt3.1002 Sirt3 64384 LIB 1002 0.316055067 0.208823529 0.423286604 +Sirt3.1236 Sirt3 64384 LIB 1236 0.23257166 0.248811512 0.216331808 +Sirt4.105 Sirt4 75387 LIB 105 0.787448713 0.84617509 0.728722336 +Sirt4.633 Sirt4 75387 LIB 633 0.413766331 0.018150999 0.809381663 +Sirt4.1490 Sirt4 75387 LIB 1490 0.271724181 0.467032967 0.076415394 +Sirt4.1806 Sirt4 75387 LIB 1806 0.211146978 0.364117918 0.058176038 +Sirt5.586 Sirt5 68346 LIB 586 0.04315097 0.061098763 0.025203178 +Sirt5.1032 Sirt5 68346 LIB 1032 NA NA NA +Sirt5.1219 Sirt5 68346 LIB 1219 1.159282043 1.459821841 0.858742245 +Sirt5.1290 Sirt5 68346 LIB 1290 0.148949431 0.165555092 0.132343769 +Sirt6.83 Sirt6 50721 LIB 83 0.036022476 0.05303979 0.019005162 +Sirt6.421 Sirt6 50721 LIB 421 0.000122991 0 0.000245982 +Sirt6.937 Sirt6 50721 LIB 937 0.232027633 0.160600291 0.303454974 +Sirt6.1609 Sirt6 50721 LIB 1609 0.364602844 0.086260111 0.642945578 +Sirt7.841 Sirt7 209011 LIB 841 0 0 0 +Sirt7.844 Sirt7 209011 LIB 844 0.221531023 0.111507818 0.331554227 +Sirt7.1238 Sirt7 209011 LIB 1238 0.21552915 0.207286174 0.223772125 +Sirt7.1604 Sirt7 209011 LIB 1604 0.118405846 0.003893381 0.232918312 +Sirt7.1677 Sirt7 209011 LIB 1677 0.451595828 0.535251456 0.367940199 +Smarca1.1401 Smarca1 93761 LIB 1401 1.153548695 1.688711584 0.618385807 +Smarca1.1430 Smarca1 93761 LIB 1430 0.46036335 0.786028584 0.134698116 +Smarca1.1613 Smarca1 93761 LIB 1613 0.11066776 0.061904944 0.159430577 +Smarca1.1893 Smarca1 93761 LIB 1893 0.213806299 0.218937644 0.208674954 +Smarca1.3418 Smarca1 93761 LIB 3418 0.607874304 1.091527057 0.124221551 +Smarca2.263 Smarca2 67155 LIB 263 0.172543567 0.178539893 0.166547241 +Smarca2.274 Smarca2 67155 LIB 274 0 0 0 +Smarca2.712 Smarca2 67155 LIB 712 0.035563959 0.003878282 0.067249636 +Smarca2.1061 Smarca2 67155 LIB 1061 0.08050899 0.120002107 0.041015873 +Smarca4.3232 Smarca4 20586 LIB 3232 0 0 0 +Smarca4.3364 Smarca4 20586 LIB 3364 0.185891845 0 0.371783689 +Smarca4.3633 Smarca4 20586 LIB 3633 0.101871507 0.169502565 0.03424045 +Smarca4.4935 Smarca4 20586 LIB 4935 0.464028994 0.638482699 0.28957529 +Smarca4.5466 Smarca4 20586 LIB 5466 0 0 0 +Smarca5.1139 Smarca5 93762 LIB 1139 39.02044459 29.48150462 48.55938456 +Smarca5.1264 Smarca5 93762 LIB 1264 0.051361802 0.102723605 0 +Smarca5.3886 Smarca5 93762 LIB 3886 2.889445252 4.275900073 1.502990431 +Smarca5.4421 Smarca5 93762 LIB 4421 0.027038126 0.047147846 0.006928406 +Smarca5.4522 Smarca5 93762 LIB 4522 1.659875833 1.301821998 2.017929668 +Smarcc2.1398 Smarcc2 68094 LIB 1398 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--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/tool_dependencies.xml Mon Jan 19 22:14:10 2015 -0500 @@ -0,0 +1,29 @@ +<?xml version="1.0"?> +<tool_dependency> + <package name="R" version="3.1.2"> + <repository changeset_revision="f0626dac6765" name="package_r_3_1_2" owner="iuc" prior_installation_required="True" toolshed="https://testtoolshed.g2.bx.psu.edu" /> + </package> + <package name="shrnaseq_r" version="0.1"> + <install version="1.0"> + <actions> + <action type="setup_r_environment"> + <repository changeset_revision="f0626dac6765" name="package_r_3_1_2" owner="iuc" toolshed="https://testtoolshed.g2.bx.psu.edu"> + <package name="R" version="3.1.2" /> + </repository> + <package>https://github.com/fubar2/galaxy_tool_source/blob/master/RELEASE_2_14/locfit_1.5-9.1.tar.gz?raw=true</package> + <package>https://github.com/fubar2/galaxy_tool_source/blob/master/RELEASE_2_14/limma_3.22.4.tar.gz?raw=true</package> + <package>https://github.com/fubar2/galaxy_tool_source/blob/master/RELEASE_2_14/edgeR_3.8.5.tar.gz?raw=true</package> + <package>https://github.com/fubar2/galaxy_tool_source/blob/master/RELEASE_2_14/statmod_1.4.20.tar.gz?raw=true</package> + </action> + <action type="chmod"> + <file mode="755">$REPOSITORY_INSTALL_DIR/hairpinTool.R</file> + </action> + <action type="set_environment"> + <environment_variable action="set_to" name="HAIRPINTOOL_R_SOURCE">$REPOSITORY_INSTALL_DIR/hairpinTool.R</environment_variable> + </action> + </actions> + </install> + </package> + <readme> + </readme> +</tool_dependency>