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1 ################################################################################
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2 ### R script to compare several conditions with the SARTools and DESeq2 packages
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3 ### Hugo Varet
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4 ### March 20th, 2018
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5 ### designed to be executed with SARTools 1.6.3
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6 ### run "Rscript template_script_DESeq2_CL.r --help" to get some help
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7 ################################################################################
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8
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9 rm(list=ls()) # remove all the objects from the R session
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10 library(optparse) # to run the script in command lines
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11
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12 # options list with associated default value.
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13 option_list <- list(
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14 make_option(c("-P", "--projectName"),
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15 default=basename(getwd()),
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16 dest="projectName",
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17 help="name of the project used for the report [default: name of the current directory]."),
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18
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19 make_option(c("-A", "--author"),
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20 default=Sys.info()[7],
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21 dest="author",
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22 help="name of the report author [default: %default]."),
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23
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24 make_option(c("-t", "--targetFile"),
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25 default="target.txt",
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26 dest="targetFile",
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27 help="path to the design/target file [default: %default]."),
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28
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29 make_option(c("-r", "--rawDir"),
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30 default="raw",
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31 dest="rawDir",
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32 help="path to the directory containing the HTSeq files [default: %default]."),
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33
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34 make_option(c("-F", "--featuresToRemove"),
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35 default="alignment_not_unique,ambiguous,no_feature,not_aligned,too_low_aQual",
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36 dest="FTR",
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37 help="names of the features to be removed, more than once can be specified [default: %default]"),
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38
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39 make_option(c("-v", "--varInt"),
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40 default="group",
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41 dest="varInt",
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42 help="factor of interest [default: %default]"),
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43
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44 make_option(c("-c", "--condRef"),
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45 default="WT",
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46 dest="condRef",
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47 help="reference biological condition [default: %default]"),
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48
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49 make_option(c("-b", "--batch"),
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50 default=NULL,
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51 dest="batch",
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52 help="blocking factor [default: %default] or \"batch\" for example"),
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53
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54 make_option(c("-f", "--fitType"),
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55 default="parametric",
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56 dest="fitType",
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57 help="mean-variance relationship: [default: %default], local or mean"),
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58
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59 make_option(c("-o", "--cooksCutoff"),
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60 default=TRUE,
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61 dest="cooksCutoff",
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62 help="perform the outliers detection (default is TRUE)"),
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63
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64 make_option(c("-i", "--independentFiltering"),
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65 default=TRUE,
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66 dest="independentFiltering",
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67 help="perform independent filtering (default is TRUE)"),
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68
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69 make_option(c("-a", "--alpha"),
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70 default=0.05,
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71 dest="alpha",
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72 help="threshold of statistical significance [default: %default]"),
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73
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74 make_option(c("-p", "--pAdjustMethod"),
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75 default="BH",
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76 dest="pAdjustMethod",
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77 help="p-value adjustment method: \"BH\" or \"BY\" [default: %default]"),
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78
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79 make_option(c("-T", "--typeTrans"),
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80 default="VST",
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81 dest="typeTrans",
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82 help="transformation for PCA/clustering: \"VST\" ou \"rlog\" [default: %default]"),
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83
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84 make_option(c("-l", "--locfunc"),
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85 default="median",
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86 dest="locfunc",
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87 help="median or shorth to estimate the size factors [default: %default]"),
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88
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89 make_option(c("-C", "--colors"),
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90 default="dodgerblue,firebrick1,MediumVioletRed,SpringGreen,chartreuse,cyan,darkorchid,darkorange",
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91 dest="cols",
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92 help="colors of each biological condition on the plots\n\t\t\"col1,col2,col3,col4\"\n\t\t[default: %default]"),
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93
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94 make_option(c("-g", "--forceCairoGraph"),
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95 action="store_true",
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96 default=FALSE,
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97 dest="forceCairoGraph",
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98 help="activate cairo type")
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99
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100 )
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101
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102 # now parse the command line to check which option is given and get associated values
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103 parser <- OptionParser(usage="usage: %prog [options]",
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104 option_list=option_list,
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105 description="Compare two or more biological conditions in a RNA-Seq framework with DESeq2.",
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106 epilogue="For comments, bug reports etc... please contact Hugo Varet <hugo.varet@pasteur.fr>")
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107 opt <- parse_args(parser, args=commandArgs(trailingOnly=TRUE), positional_arguments=0)$options
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108
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109 # get options and arguments
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110 workDir <- getwd()
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111 projectName <- opt$projectName # name of the project
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112 author <- opt$author # author of the statistical analysis/report
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113 targetFile <- opt$targetFile # path to the design/target file
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114 rawDir <- opt$rawDir # path to the directory containing raw counts files
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115 featuresToRemove <- unlist(strsplit(opt$FTR, ",")) # names of the features to be removed (specific HTSeq-count information and rRNA for example)
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116 varInt <- opt$varInt # factor of interest
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117 condRef <- opt$condRef # reference biological condition
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118 batch <- opt$batch # blocking factor: NULL (default) or "batch" for example
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119 fitType <- opt$fitType # mean-variance relationship: "parametric" (default), "local" or "mean"
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120 cooksCutoff <- opt$cooksCutoff # outliers detection threshold (NULL to let DESeq2 choosing it)
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121 independentFiltering <- opt$independentFiltering # TRUE/FALSE to perform independent filtering (default is TRUE)
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122 alpha <- as.numeric(opt$alpha) # threshold of statistical significance
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123 pAdjustMethod <- opt$pAdjustMethod # p-value adjustment method: "BH" (default) or "BY"
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124 typeTrans <- opt$typeTrans # transformation for PCA/clustering: "VST" ou "rlog"
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125 locfunc <- opt$locfunc # "median" (default) or "shorth" to estimate the size factors
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126 colors <- unlist(strsplit(opt$cols, ",")) # vector of colors of each biologicial condition on the plots
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127 forceCairoGraph <- opt$forceCairoGraph # force cairo as plotting device if enabled
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128 # print(paste("workDir", workDir))
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129 # print(paste("projectName", projectName))
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130 # print(paste("author", author))
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131 # print(paste("targetFile", targetFile))
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132 # print(paste("rawDir", rawDir))
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133 # print(paste("varInt", varInt))
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134 # print(paste("condRef", condRef))
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135 # print(paste("batch", batch))
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136 # print(paste("fitType", fitType))
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137 # print(paste("cooksCutoff", cooksCutoff))
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138 # print(paste("independentFiltering", independentFiltering))
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139 # print(paste("alpha", alpha))
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140 # print(paste("pAdjustMethod", pAdjustMethod))
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141 # print(paste("typeTrans", typeTrans))
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142 # print(paste("locfunc", locfunc))
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143 # print(paste("featuresToRemove", featuresToRemove))
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144 # print(paste("colors", colors))
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145
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146 ################################################################################
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147 ### running script ###
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148 ################################################################################
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149 # setwd(workDir)
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150 library(SARTools)
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151 if (forceCairoGraph) options(bitmapType="cairo")
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152
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153 # checking parameters
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154 problem <- checkParameters.DESeq2(projectName=projectName,author=author,targetFile=targetFile,
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155 rawDir=rawDir,featuresToRemove=featuresToRemove,varInt=varInt,
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156 condRef=condRef,batch=batch,fitType=fitType,cooksCutoff=cooksCutoff,
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157 independentFiltering=independentFiltering,alpha=alpha,pAdjustMethod=pAdjustMethod,
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158 typeTrans=typeTrans,locfunc=locfunc,colors=colors)
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159 if (problem) quit(save="yes")
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160
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161 # loading target file
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162 target <- loadTargetFile(targetFile=targetFile, varInt=varInt, condRef=condRef, batch=batch)
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163
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164 # loading counts
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165 counts <- loadCountData(target=target, rawDir=rawDir, featuresToRemove=featuresToRemove)
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166
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167 # description plots
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168 majSequences <- descriptionPlots(counts=counts, group=target[,varInt], col=colors)
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169
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170 # analysis with DESeq2
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171 out.DESeq2 <- run.DESeq2(counts=counts, target=target, varInt=varInt, batch=batch,
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172 locfunc=locfunc, fitType=fitType, pAdjustMethod=pAdjustMethod,
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173 cooksCutoff=cooksCutoff, independentFiltering=independentFiltering, alpha=alpha)
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174
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175 # PCA + clustering
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176 exploreCounts(object=out.DESeq2$dds, group=target[,varInt], typeTrans=typeTrans, col=colors)
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177
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178 # summary of the analysis (boxplots, dispersions, diag size factors, export table, nDiffTotal, histograms, MA plot)
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179 summaryResults <- summarizeResults.DESeq2(out.DESeq2, group=target[,varInt], col=colors,
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180 independentFiltering=independentFiltering,
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181 cooksCutoff=cooksCutoff, alpha=alpha)
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182
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183 # save image of the R session
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184 save.image(file=paste0(projectName, ".RData"))
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185
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186 # generating HTML report
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4
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187 Sys.setenv(HOME = getwd())
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3
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188 writeReport.DESeq2(target=target, counts=counts, out.DESeq2=out.DESeq2, summaryResults=summaryResults,
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189 majSequences=majSequences, workDir=workDir, projectName=projectName, author=author,
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190 targetFile=targetFile, rawDir=rawDir, featuresToRemove=featuresToRemove, varInt=varInt,
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191 condRef=condRef, batch=batch, fitType=fitType, cooksCutoff=cooksCutoff,
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192 independentFiltering=independentFiltering, alpha=alpha, pAdjustMethod=pAdjustMethod,
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193 typeTrans=typeTrans, locfunc=locfunc, colors=colors)
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