annotate hairpinTool.R @ 3:17fee0726221

Deleted selected files
author shians
date Thu, 20 Feb 2014 20:55:28 -0500
parents 076ca575208f
children f8af57d6f60b
Ignore whitespace changes - Everywhere: Within whitespace: At end of lines:
rev   line source
2
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1 # Record starting time
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2 timeStart <- as.character(Sys.time())
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3
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4 # Loading and checking required packages
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5 library(methods, quietly=TRUE, warn.conflicts=FALSE)
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6 library(statmod, quietly=TRUE, warn.conflicts=FALSE)
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7 library(splines, quietly=TRUE, warn.conflicts=FALSE)
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8 library(edgeR, quietly=TRUE, warn.conflicts=FALSE)
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9 library(limma, quietly=TRUE, warn.conflicts=FALSE)
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10
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11 if (packageVersion("edgeR") < "3.5.23") {
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12 message("Please update 'edgeR' to version >= 3.5.23 to run this script")
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13 }
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14
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15 ################################################################################
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16 ### Function declarations
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17 ################################################################################
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18
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19 # Function to sanitise contrast equations so there are no whitespaces
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20 # surrounding the arithmetic operators, leading or trailing whitespace
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21 sanitiseEquation <- function(equation) {
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22 equation <- gsub(" *[+] *", "+", equation)
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23 equation <- gsub(" *[-] *", "-", equation)
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24 equation <- gsub(" *[/] *", "/", equation)
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25 equation <- gsub(" *[*] *", "*", equation)
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26 equation <- gsub("^\\s+|\\s+$", "", equation)
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27 return(equation)
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28 }
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29
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30 # Function to sanitise group information
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31 sanitiseGroups <- function(string) {
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32 string <- gsub(" *[,] *", ",", string)
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33 string <- gsub("^\\s+|\\s+$", "", string)
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34 return(string)
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35 }
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36
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37 # Function to change periods to whitespace in a string
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38 unmake.names <- function(string) {
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39 string <- gsub(".", " ", string, fixed=TRUE)
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40 return(string)
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41 }
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42
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43 # Function has string input and generates an output path string
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44 makeOut <- function(filename) {
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45 return(paste0(folderPath, "/", filename))
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46 }
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47
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48 # Function has string input and generates both a pdf and png output strings
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49 imgOut <- function(filename) {
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50 assign(paste0(filename, "Png"), makeOut(paste0(filename,".png")),
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51 envir = .GlobalEnv)
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52 assign(paste0(filename, "Pdf"), makeOut(paste0(filename,".pdf")),
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53 envir = .GlobalEnv)
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54 }
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55
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56 # Create cat function default path set, default seperator empty and appending
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57 # true by default (Ripped straight from the cat function with altered argument
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58 # defaults)
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59 cata <- function(..., file = htmlPath, sep = "", fill = FALSE, labels = NULL,
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60 append = TRUE) {
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61 if (is.character(file))
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62 if (file == "")
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63 file <- stdout()
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64 else if (substring(file, 1L, 1L) == "|") {
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65 file <- pipe(substring(file, 2L), "w")
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66 on.exit(close(file))
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67 }
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68 else {
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69 file <- file(file, ifelse(append, "a", "w"))
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70 on.exit(close(file))
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71 }
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72 .Internal(cat(list(...), file, sep, fill, labels, append))
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73 }
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74
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75 # Function to write code for html head and title
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76 HtmlHead <- function(title) {
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77 cata("<head>\n")
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78 cata("<title>", title, "</title>\n")
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79 cata("</head>\n")
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80 }
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81
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82 # Function to write code for html links
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83 HtmlLink <- function(address, label=address) {
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84 cata("<a href=\"", address, "\" target=\"_blank\">", label, "</a><br />\n")
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85 }
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86
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87 # Function to write code for html images
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88 HtmlImage <- function(source, label=source, height=600, width=600) {
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89 cata("<img src=\"", source, "\" alt=\"", label, "\" height=\"", height)
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90 cata("\" width=\"", width, "\"/>\n")
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91 }
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92
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93 # Function to write code for html list items
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94 ListItem <- function(...) {
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95 cata("<li>", ..., "</li>\n")
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96 }
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97
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98 TableItem <- function(...) {
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99 cata("<td>", ..., "</td>\n")
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100 }
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101
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102 TableHeadItem <- function(...) {
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103 cata("<th>", ..., "</th>\n")
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104 }
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105 ################################################################################
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106 ### Input Processing
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107 ################################################################################
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108
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109 # Grabbing arguments from command line
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110 argv <- commandArgs(TRUE)
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111
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112 # Remove fastq file paths after collecting from argument vector
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113 inputType <- as.character(argv[1])
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114 if (inputType=="fastq") {
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115 fastqPath <- as.character(gsub("fastq::", "", argv[grepl("fastq::", argv)],
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116 fixed=TRUE))
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117 argv <- argv[!grepl("fastq::", argv, fixed=TRUE)]
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118 hairpinPath <- as.character(argv[2])
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119 samplePath <- as.character(argv[3])
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120 barStart <- as.numeric(argv[4])
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121 barEnd <- as.numeric(argv[5])
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122 hpStart <- as.numeric(argv[6])
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123 hpEnd <- as.numeric(argv[7])
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124 } else if (inputType=="counts") {
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125 countPath <- as.character(argv[2])
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126 annoPath <- as.character(argv[3])
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127 samplePath <- as.character(argv[4])
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128 }
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129
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130 cpmReq <- as.numeric(argv[8])
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131 sampleReq <- as.numeric(argv[9])
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132 fdrThresh <- as.numeric(argv[10])
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133 lfcThresh <- as.numeric(argv[11])
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134 workMode <- as.character(argv[12])
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135 htmlPath <- as.character(argv[13])
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136 folderPath <- as.character(argv[14])
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137 if (workMode=="classic") {
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138 pairData <- character()
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139 pairData[2] <- as.character(argv[15])
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140 pairData[1] <- as.character(argv[16])
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141 } else if (workMode=="glm") {
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142 contrastData <- as.character(argv[15])
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143 roastOpt <- as.character(argv[16])
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144 hairpinReq <- as.numeric(argv[17])
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145 selectOpt <- as.character(argv[18])
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146 selectVals <- as.character(argv[19])
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147 }
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148
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149 # Read in inputs
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150 if (inputType=="fastq") {
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151 samples <- read.table(samplePath, header=TRUE, sep="\t")
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152 hairpins <- read.table(hairpinPath, header=TRUE, sep="\t")
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153 } else if (inputType=="counts") {
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154 samples <- read.table(samplePath, header=TRUE, sep="\t")
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155 counts <- read.table(countPath, header=TRUE, sep="\t")
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156 anno <- read.table(annoPath, header=TRUE, sep="\t")
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157 }
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158 ###################### Check inputs for correctness ############################
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159 samples$ID <- make.names(samples$ID)
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160
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161 if (!any(grepl("group", names(samples)))) {
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162 stop("'group' column not specified in sample annotation file")
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163 } # Check if grouping variable has been specified
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164
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165 if (any(table(samples$ID)>1)){
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166 tab <- table(samples$ID)
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167 offenders <- paste(names(tab[tab>1]), collapse=", ")
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168 offenders <- unmake.names(offenders)
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169 stop("ID column of sample annotation must have unique values, values ",
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170 offenders, " are repeated")
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171 } # Check that IDs in sample annotation are unique
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172
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173 if (any(is.na(match(samples$ID, colnames(counts))))) {
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174 stop("not all samples have groups specified")
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175 } # Check that a group has be specifed for each sample
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176
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177 if (inputType=="fastq") {
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178
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179 if (any(table(hairpins$ID)>1)){
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180 tab <- table(hairpins$ID)
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181 offenders <- paste(names(tab[tab>1]), collapse=", ")
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182 stop("ID column of hairpin annotation must have unique values, values ",
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183 offenders, " are repeated")
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184 } # Check that IDs in hairpin annotation are unique
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185
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186 } else if (inputType=="counts") {
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187
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188 if (any(table(counts$ID)>1)){
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189 tab <- table(counts$ID)
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190 offenders <- paste(names(tab[tab>1]), collapse=", ")
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191 stop("ID column of count table must have unique values, values ",
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192 offenders, " are repeated")
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193 } # Check that IDs in count table are unique
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194 }
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195 ################################################################################
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196
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197 # Process arguments
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198 if (workMode=="glm") {
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199 if (roastOpt=="yes") {
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200 wantRoast <- TRUE
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201 } else {
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202 wantRoast <- FALSE
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203 }
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204 }
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205
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206 # Split up contrasts seperated by comma into a vector and replace spaces with
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207 # periods
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208 if (exists("contrastData")) {
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209 contrastData <- unlist(strsplit(contrastData, split=","))
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210 contrastData <- sanitiseEquation(contrastData)
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211 contrastData <- gsub(" ", ".", contrastData, fixed=TRUE)
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212 }
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213
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214 # Replace spaces with periods in pair data
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215 if (exists("pairData")) {
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216 pairData <- make.names(pairData)
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217 }
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218
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219 # Generate output folder and paths
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220 dir.create(folderPath)
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221
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222 # Generate links for outputs
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223 imgOut("barHairpin")
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224 imgOut("barIndex")
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225 imgOut("mds")
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226 imgOut("bcv")
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227 if (workMode == "classic") {
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228 smearPng <- makeOut(paste0("smear(", pairData[2], "-", pairData[1],").png"))
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229 smearPdf <- makeOut(paste0("smear(", pairData[2], "-", pairData[1],").pdf"))
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230 topOut <- makeOut(paste0("toptag(", pairData[2], "-", pairData[1],").tsv"))
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231 } else if (workMode=="glm") {
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232 smearPng <- character()
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233 smearPdf <- character()
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234 topOut <- character()
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235 roastOut <- character()
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236 barcodePng <- character()
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237 barcodePdf <- character()
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238 for (i in 1:length(contrastData)) {
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239 smearPng[i] <- makeOut(paste0("smear(", contrastData[i], ").png"))
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240 smearPdf[i] <- makeOut(paste0("smear(", contrastData[i], ").pdf"))
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241 topOut[i] <- makeOut(paste0("toptag(", contrastData[i], ").tsv"))
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242 roastOut[i] <- makeOut(paste0("roast(", contrastData[i], ").tsv"))
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243 barcodePng[i] <- makeOut(paste0("barcode(", contrastData[i], ").png"))
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244 barcodePdf[i] <- makeOut(paste0("barcode(", contrastData[i], ").pdf"))
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245 }
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246 }
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247 # Initialise data for html links and images, table with the link label and
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248 # link address
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249 linkData <- data.frame(Label=character(), Link=character(),
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250 stringsAsFactors=FALSE)
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251 imageData <- data.frame(Label=character(), Link=character(),
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252 stringsAsFactors=FALSE)
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253 ################################################################################
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254 ### Data Processing
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255 ################################################################################
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256
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257 # Transform gene selection from string into index values for mroast
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258 if (workMode=="glm") {
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259 if (selectOpt=="rank") {
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260 selectVals <- gsub(" ", "", selectVals, fixed=TRUE)
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261 selectVals <- unlist(strsplit(selectVals, ","))
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262
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263 for (i in 1:length(selectVals)) {
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264 if (grepl(":", selectVals[i], fixed=TRUE)) {
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265 temp <- unlist(strsplit(selectVals[i], ":"))
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266 selectVals <- selectVals[-i]
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267 a <- as.numeric(temp[1])
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268 b <- as.numeric(temp[2])
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269 selectVals <- c(selectVals, a:b)
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270 }
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271 }
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272 selectVals <- as.numeric(unique(selectVals))
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273 } else {
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274 selectVals <- gsub(" ", "", selectVals, fixed=TRUE)
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275 selectVals <- unlist(strsplit(selectVals, " "))
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276 }
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277 }
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278
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279 if (inputType=="fastq") {
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280 # Use EdgeR hairpin process and capture outputs
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281 hpReadout <- capture.output(
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282 data <- processHairpinReads(fastqPath, samplePath, hairpinPath,
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283 hairpinStart=hpStart, hairpinEnd=hpEnd,
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284 verbose=TRUE)
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285 )
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286
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287 # Remove function output entries that show processing data or is empty
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288 hpReadout <- hpReadout[hpReadout!=""]
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289 hpReadout <- hpReadout[!grepl("Processing", hpReadout)]
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290 hpReadout <- hpReadout[!grepl("in file", hpReadout)]
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291 hpReadout <- gsub(" -- ", "", hpReadout, fixed=TRUE)
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292
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293
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294 # Make the names of groups syntactically valid (replace spaces with periods)
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295 data$samples$group <- make.names(data$samples$group)
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296 } else {
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297 # Process counts information, set ID column to be row names
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298 rownames(counts) <- counts$ID
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299 counts <- counts[ , !(colnames(counts)=="ID")]
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300 countsRows <- nrow(counts)
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301
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302 # Process group information
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303 factors <- samples$group[match(samples$ID, colnames(counts))]
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304 annoRows <- nrow(anno)
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305 anno <- anno[match(rownames(counts), anno$ID), ]
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306 annoMatched <- sum(!is.na(anno$ID))
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307
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308 if (any(is.na(anno$ID))) {
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309 warningStr <- paste("count table contained more hairpins than",
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310 "specified in hairpin annotation file")
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311 warning(warningStr)
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312 }
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313
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314 # Filter out rows with zero counts
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315 sel <- rowSums(counts)!=0
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316 counts <- counts[sel, ]
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317 anno <- anno[sel, ]
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318
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319 # Create DGEList
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320 data <- DGEList(counts=counts, lib.size=colSums(counts),
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321 norm.factors=rep(1,ncol(counts)), genes=anno, group=factors)
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322
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323 # Make the names of groups syntactically valid (replace spaces with periods)
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324 data$samples$group <- make.names(data$samples$group)
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325 }
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326
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327 # Filter hairpins with low counts
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328 sel <- rowSums(cpm(data$counts) > cpmReq) >= sampleReq
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329 data <- data[sel, ]
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330
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331 # Estimate dispersions
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332 data <- estimateDisp(data)
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333 commonBCV <- sqrt(data$common.dispersion)
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334
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335 ################################################################################
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336 ### Output Processing
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337 ################################################################################
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338
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339 # Plot number of hairpins that could be matched per sample
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340 png(barIndexPng, width=600, height=600)
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341 barplot(height<-colSums(data$counts), las=2, main="Counts per index",
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342 cex.names=1.0, cex.axis=0.8, ylim=c(0, max(height)*1.2))
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343 imageData[1, ] <- c("Counts per Index", "barIndex.png")
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344 invisible(dev.off())
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345
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346 pdf(barIndexPdf)
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347 barplot(height<-colSums(data$counts), las=2, main="Counts per index",
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348 cex.names=1.0, cex.axis=0.8, ylim=c(0, max(height)*1.2))
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349 linkData[1, ] <- c("Counts per Index Barplot (.pdf)", "barIndex.pdf")
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350 invisible(dev.off())
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351
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
352 # Plot per hairpin totals across all samples
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
353 png(barHairpinPng, width=600, height=600)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
354 if (nrow(data$counts)<50) {
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
355 barplot(height<-rowSums(data$counts), las=2, main="Counts per hairpin",
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
356 cex.names=0.8, cex.axis=0.8, ylim=c(0, max(height)*1.2))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
357 } else {
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
358 barplot(height<-rowSums(data$counts), las=2, main="Counts per hairpin",
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
359 cex.names=0.8, cex.axis=0.8, ylim=c(0, max(height)*1.2),
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
360 names.arg=FALSE)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
361 }
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
362 imageData <- rbind(imageData, c("Counts per Hairpin", "barHairpin.png"))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
363 invisible(dev.off())
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
364
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
365 pdf(barHairpinPdf)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
366 if (nrow(data$counts)<50) {
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
367 barplot(height<-rowSums(data$counts), las=2, main="Counts per hairpin",
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
368 cex.names=0.8, cex.axis=0.8, ylim=c(0, max(height)*1.2))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
369 } else {
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
370 barplot(height<-rowSums(data$counts), las=2, main="Counts per hairpin",
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
371 cex.names=0.8, cex.axis=0.8, ylim=c(0, max(height)*1.2),
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
372 names.arg=FALSE)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
373 }
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
374 newEntry <- c("Counts per Hairpin Barplot (.pdf)", "barHairpin.pdf")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
375 linkData <- rbind(linkData, newEntry)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
376 invisible(dev.off())
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
377
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
378 # Make an MDS plot to visualise relationships between replicate samples
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
379 png(mdsPng, width=600, height=600)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
380 plotMDS(data, labels=data$samples$group, col=as.numeric(data$samples$group),
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
381 main="MDS Plot")
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shian_su <registertonysu@gmail.com>
parents:
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382 imageData <- rbind(imageData, c("MDS Plot", "mds.png"))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
383 invisible(dev.off())
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
384
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
385 pdf(mdsPdf)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
386 plotMDS(data, labels=data$samples$group, col=as.numeric(data$samples$group),
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
387 main="MDS Plot")
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shian_su <registertonysu@gmail.com>
parents:
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388 newEntry <- c("MDS Plot (.pdf)", "mds.pdf")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
389 linkData <- rbind(linkData, newEntry)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
390 invisible(dev.off())
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
391
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
392 if (workMode=="classic") {
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
393 # Assess differential representation using classic exact testing methodology
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
394 # in edgeR
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
395 testData <- exactTest(data, pair=pairData)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
396
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
397 top <- topTags(testData, n=Inf)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
398 topIDs <- top$table[(top$table$FDR < fdrThresh) &
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
399 (abs(top$table$logFC) > lfcThresh), 1]
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
400 write.table(top, file=topOut, row.names=FALSE, sep="\t")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
401 linkName <- paste0("Top Tags Table(", pairData[2], "-", pairData[1],
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
402 ") (.tsv)")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
403 linkAddr <- paste0("toptag(", pairData[2], "-", pairData[1], ").tsv")
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shian_su <registertonysu@gmail.com>
parents:
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404 linkData <- rbind(linkData, c(linkName, linkAddr))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
405
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
406 # Select hairpins with FDR < 0.05 to highlight on plot
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
407 png(smearPng, width=600, height=600)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
408 plotTitle <- gsub(".", " ",
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
409 paste0("Smear Plot: ", pairData[2], "-", pairData[1]),
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
410 fixed = TRUE)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
411 plotSmear(testData, de.tags=topIDs,
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
412 pch=20, cex=1.0, main=plotTitle)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
413 abline(h = c(-1, 0, 1), col = c("dodgerblue", "yellow", "dodgerblue"), lty=2)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
414 imgName <- paste0("Smear Plot(", pairData[2], "-", pairData[1], ")")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
415 imgAddr <- paste0("smear(", pairData[2], "-", pairData[1],").png")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
416 imageData <- rbind(imageData, c(imgName, imgAddr))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
417 invisible(dev.off())
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
418
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
419 pdf(smearPdf)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
420 plotTitle <- gsub(".", " ",
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
421 paste0("Smear Plot: ", pairData[2], "-", pairData[1]),
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
422 fixed = TRUE)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
423 plotSmear(testData, de.tags=topIDs,
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
424 pch=20, cex=1.0, main=plotTitle)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
425 abline(h = c(-1, 0, 1), col = c("dodgerblue", "yellow", "dodgerblue"), lty=2)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
426 imgName <- paste0("Smear Plot(", pairData[2], "-", pairData[1], ") (.pdf)")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
427 imgAddr <- paste0("smear(", pairData[2], "-", pairData[1], ").pdf")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
428 linkData <- rbind(linkData, c(imgName, imgAddr))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
429 invisible(dev.off())
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
430 } else if (workMode=="glm") {
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
431 # Generating design information
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
432 factors <- factor(data$sample$group)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
433 design <- model.matrix(~0+factors)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
434
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
435 colnames(design) <- gsub("factors", "", colnames(design), fixed=TRUE)
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shian_su <registertonysu@gmail.com>
parents:
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436
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
437 # Split up contrasts seperated by comma into a vector
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shian_su <registertonysu@gmail.com>
parents:
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438 contrastData <- unlist(strsplit(contrastData, split=","))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
439 for (i in 1:length(contrastData)) {
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
440 # Generate contrasts information
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shian_su <registertonysu@gmail.com>
parents:
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441 contrasts <- makeContrasts(contrasts=contrastData[i], levels=design)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
442
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
443 # Fit negative bionomial GLM
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shian_su <registertonysu@gmail.com>
parents:
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444 fit = glmFit(data, design)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
445 # Carry out Likelihood ratio test
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shian_su <registertonysu@gmail.com>
parents:
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446 testData = glmLRT(fit, contrast=contrasts)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
447
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
448 # Select hairpins with FDR < 0.05 to highlight on plot
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
449 top <- topTags(testData, n=Inf)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
450 topIDs <- top$table[(top$table$FDR < fdrThresh) &
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
451 (abs(top$table$logFC) > lfcThresh), 1]
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
452 write.table(top, file=topOut[i], row.names=FALSE, sep="\t")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
453
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
454 linkName <- paste0("Top Tags Table(", contrastData[i], ") (.tsv)")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
455 linkAddr <- paste0("toptag(", contrastData[i], ").tsv")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
456 linkData <- rbind(linkData, c(linkName, linkAddr))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
457
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
458 # Make a plot of logFC versus logCPM
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
459 png(smearPng[i], height=600, width=600)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
460 plotTitle <- paste("Smear Plot:", gsub(".", " ", contrastData[i],
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
461 fixed=TRUE))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
462 plotSmear(testData, de.tags=topIDs, pch=20, cex=0.8, main=plotTitle)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
463 abline(h=c(-1, 0, 1), col=c("dodgerblue", "yellow", "dodgerblue"), lty=2)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
464
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
465 imgName <- paste0("Smear Plot(", contrastData[i], ")")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
466 imgAddr <- paste0("smear(", contrastData[i], ").png")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
467 imageData <- rbind(imageData, c(imgName, imgAddr))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
468 invisible(dev.off())
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
469
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
470 pdf(smearPdf[i])
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
471 plotTitle <- paste("Smear Plot:", gsub(".", " ", contrastData[i],
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
472 fixed=TRUE))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
473 plotSmear(testData, de.tags=topIDs, pch=20, cex=0.8, main=plotTitle)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
474 abline(h=c(-1, 0, 1), col=c("dodgerblue", "yellow", "dodgerblue"), lty=2)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
475
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
476 linkName <- paste0("Smear Plot(", contrastData[i], ") (.pdf)")
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shian_su <registertonysu@gmail.com>
parents:
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477 linkAddr <- paste0("smear(", contrastData[i], ").pdf")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
478 linkData <- rbind(linkData, c(linkName, linkAddr))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
479 invisible(dev.off())
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
480
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shian_su <registertonysu@gmail.com>
parents:
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481 genes <- as.character(data$genes$Gene)
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shian_su <registertonysu@gmail.com>
parents:
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482 unq <- unique(genes)
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shian_su <registertonysu@gmail.com>
parents:
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483 unq <- unq[!is.na(unq)]
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shian_su <registertonysu@gmail.com>
parents:
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484 geneList <- list()
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
485 for (gene in unq) {
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shian_su <registertonysu@gmail.com>
parents:
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486 if (length(which(genes==gene)) >= hairpinReq) {
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shian_su <registertonysu@gmail.com>
parents:
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487 geneList[[gene]] <- which(genes==gene)
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
488 }
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
489 }
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
490
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shian_su <registertonysu@gmail.com>
parents:
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491 if (wantRoast) {
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shian_su <registertonysu@gmail.com>
parents:
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492 # Input preparaton for roast
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shian_su <registertonysu@gmail.com>
parents:
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493 nrot = 9999
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
494 set.seed(602214129)
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shian_su <registertonysu@gmail.com>
parents:
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495 roastData <- mroast(data, index=geneList, design=design,
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shian_su <registertonysu@gmail.com>
parents:
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496 contrast=contrasts, nrot=nrot)
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shian_su <registertonysu@gmail.com>
parents:
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497 roastData <- cbind(GeneID=rownames(roastData), roastData)
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shian_su <registertonysu@gmail.com>
parents:
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498 write.table(roastData, file=roastOut[i], row.names=FALSE, sep="\t")
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shian_su <registertonysu@gmail.com>
parents:
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499 linkName <- paste0("Gene Level Analysis Table(", contrastData[i],
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shian_su <registertonysu@gmail.com>
parents:
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500 ") (.tsv)")
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shian_su <registertonysu@gmail.com>
parents:
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501 linkAddr <- paste0("roast(", contrastData[i], ").tsv")
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shian_su <registertonysu@gmail.com>
parents:
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502 linkData <- rbind(linkData, c(linkName, linkAddr))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
503 if (selectOpt=="rank") {
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shian_su <registertonysu@gmail.com>
parents:
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504 selectedGenes <- rownames(roastData)[selectVals]
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shian_su <registertonysu@gmail.com>
parents:
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505 } else {
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shian_su <registertonysu@gmail.com>
parents:
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506 selectedGenes <- selectVals
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
507 }
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
508
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shian_su <registertonysu@gmail.com>
parents:
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509 if (packageVersion("limma")<"3.19.19") {
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
510 png(barcodePng[i], width=600, height=length(selectedGenes)*150)
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shian_su <registertonysu@gmail.com>
parents:
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511 } else {
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shian_su <registertonysu@gmail.com>
parents:
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512 png(barcodePng[i], width=600, height=length(selectedGenes)*300)
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shian_su <registertonysu@gmail.com>
parents:
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513 }
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
514 par(mfrow=c(length(selectedGenes), 1))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
515 for (gene in selectedGenes) {
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
516 barcodeplot(testData$table$logFC, index=geneList[[gene]],
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
517 main=paste("Barcode Plot for", gene, "(logFCs)",
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
518 gsub(".", " ", contrastData[i])),
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
519 labels=c("Positive logFC", "Negative logFC"))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
520 }
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
521 imgName <- paste0("Barcode Plot(", contrastData[i], ")")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
522 imgAddr <- paste0("barcode(", contrastData[i], ").png")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
523 imageData <- rbind(imageData, c(imgName, imgAddr))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
524 dev.off()
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
525 if (packageVersion("limma")<"3.19.19") {
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
526 pdf(barcodePdf[i], width=8, height=2)
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
527 } else {
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
528 pdf(barcodePdf[i], width=8, height=4)
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
529 }
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
530 for (gene in selectedGenes) {
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
531 barcodeplot(testData$table$logFC, index=geneList[[gene]],
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
532 main=paste("Barcode Plot for", gene, "(logFCs)",
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
533 gsub(".", " ", contrastData[i])),
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
534 labels=c("Positive logFC", "Negative logFC"))
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
535 }
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
536 linkName <- paste0("Barcode Plot(", contrastData[i], ") (.pdf)")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
537 linkAddr <- paste0("barcode(", contrastData[i], ").pdf")
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
538 linkData <- rbind(linkData, c(linkName, linkAddr))
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
539 dev.off()
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
540 }
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
541 }
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
542 }
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
543
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
544 # Record ending time
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
545 timeEnd <- as.character(Sys.time())
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
546 ################################################################################
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
547 ### HTML Generation
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
548 ################################################################################
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
549 # Clear file
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shian_su <registertonysu@gmail.com>
parents:
diff changeset
550 cat("", file=htmlPath)
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
551
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
552 cata("<html>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
553 HtmlHead("EdgeR Output")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
554
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
555 cata("<body>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
556 cata("<h3>EdgeR Analysis Output:</h3>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
557 cata("<h4>Input Summary:</h4>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
558 if (inputType=="fastq") {
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
559 cata("<ul>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
560 ListItem(hpReadout[1])
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
561 ListItem(hpReadout[2])
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
562 cata("</ul>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
563 cata(hpReadout[3], "<br/>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
564 cata("<ul>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
565 ListItem(hpReadout[4])
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
566 ListItem(hpReadout[7])
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
567 cata("</ul>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
568 cata(hpReadout[8:11], sep="<br/>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
569 cata("<br />\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
570 cata("<b>Please check that read percentages are consistent with ")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
571 cata("expectations.</b><br >\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
572 } else if (inputType=="counts") {
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
573 cata("<ul>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
574 ListItem("Number of Samples: ", ncol(data$counts))
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
575 ListItem("Number of Hairpins: ", countsRows)
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
576 ListItem("Number of annotations provided: ", annoRows)
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
577 ListItem("Number of annotations matched to hairpin: ", annoMatched)
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
578 cata("</ul>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
579 }
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
580
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
581 cata("The estimated common biological coefficient of variation (BCV) is: ",
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
582 commonBCV, "<br />\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
583
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
584 cata("<h4>Output:</h4>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
585 cata("All images displayed have PDF copy at the bottom of the page, these can ")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
586 cata("exported in a pdf viewer to high resolution image format. <br/>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
587 for (i in 1:nrow(imageData)) {
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
588 if (grepl("barcode", imageData$Link[i])) {
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
589 if (packageVersion("limma")<"3.19.19") {
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
590 HtmlImage(imageData$Link[i], imageData$Label[i],
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
591 height=length(selectedGenes)*150)
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
592 } else {
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
593 HtmlImage(imageData$Link[i], imageData$Label[i],
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
594 height=length(selectedGenes)*300)
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
595 }
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
596 } else {
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
597 HtmlImage(imageData$Link[i], imageData$Label[i])
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
598 }
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
599 }
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
600 cata("<br/>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
601
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
602 cata("<h4>Plots:</h4>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
603 for (i in 1:nrow(linkData)) {
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
604 if (!grepl(".tsv", linkData$Link[i])) {
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
605 HtmlLink(linkData$Link[i], linkData$Label[i])
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
606 }
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
607 }
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
608
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
609 cata("<h4>Tables:</h4>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
610 for (i in 1:nrow(linkData)) {
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
611 if (grepl(".tsv", linkData$Link[i])) {
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
612 HtmlLink(linkData$Link[i], linkData$Label[i])
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
613 }
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
614 }
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
615
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
616 cata("<p>alt-click any of the links to download the file, or click the name ")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
617 cata("of this task in the galaxy history panel and click on the floppy ")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
618 cata("disk icon to download all files in a zip archive.</p>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
619 cata("<p>.tsv files are tab seperated files that can be viewed using Excel ")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
620 cata("or other spreadsheet programs</p>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
621 cata("<table border=\"0\">\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
622
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
623 cata("<tr>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
624 TableItem("Task started at:"); TableItem(timeStart)
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
625 cata("</tr>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
626 cata("<tr>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
627 TableItem("Task ended at:"); TableItem(timeEnd)
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
628 cata("</tr>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
629
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
630 cata("</body>\n")
076ca575208f First commit
shian_su <registertonysu@gmail.com>
parents:
diff changeset
631 cata("</html>")