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comparison template_script_DESeq2_CL.r @ 0:581d217c7337 draft

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author lgueguen
date Fri, 22 Jul 2016 05:39:13 -0400
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-1:000000000000 0:581d217c7337
1 #!/local/gensoft2/exe/R/3.1.2/bin/Rscript
2
3 # to run this script, use one of these commands:
4 # Rscript --no-save --no-restore --verbose template_script_DESeq2_CL.r -r raw -v group -c T0 > log.txt 2>&1
5 # Rscript template_script_DESeq2_CL.r -r raw -v group -c T0
6
7 # to get help:
8 # Rscript template_script_DESeq2_CL.r --help
9
10 ################################################################################
11 ### R script to compare several conditions with the SARTools and DESeq2 packages
12 ### Hugo Varet
13 ### April 20th, 2015
14 ### designed to be executed with SARTools 1.1.0
15 ################################################################################
16
17 rm(list=ls()) # remove all the objects from the R session
18 library(optparse) # to run the script in command lines
19
20 # options list with associated default value.
21 option_list <- list(
22 make_option(c("-P", "--projectName"),
23 default=basename(getwd()),
24 dest="projectName",
25 help="name of the project used for the report [default: name of the current directory]."),
26
27 make_option(c("-A", "--author"),
28 default=Sys.info()[7],
29 dest="author",
30 help="name of the report author [default: %default]."),
31
32 make_option(c("-t", "--targetFile"),
33 default="target.txt",
34 dest="targetFile",
35 help="path to the design/target file [default: %default]."),
36
37 make_option(c("-r", "--rawDir"),
38 default="raw",
39 dest="rawDir",
40 help="path to the directory containing the HTSeq files [default: %default]."),
41
42 make_option(c("-F", "--featuresToRemove"),
43 default="alignment_not_unique,ambiguous,no_feature,not_aligned,too_low_aQual",
44 dest="FTR",
45 help="names of the features to be removed, more than once can be specified [default: %default]"),
46
47 make_option(c("-v", "--varInt"),
48 default="group",
49 dest="varInt",
50 help="factor of interest [default: %default]"),
51
52 make_option(c("-c", "--condRef"),
53 default="WT",
54 dest="condRef",
55 help="reference biological condition [default: %default]"),
56
57 make_option(c("-b", "--batch"),
58 default=NULL,
59 dest="batch",
60 help="blocking factor [default: %default] or \"batch\" for example"),
61
62 make_option(c("-f", "--fitType"),
63 default="parametric",
64 dest="fitType",
65 help="mean-variance relationship: [default: %default] or local"),
66
67 make_option(c("-o", "--cooksCutoff"),
68 default=TRUE,
69 dest="cooksCutoff",
70 help="perform the outliers detection (default is TRUE)"),
71
72 make_option(c("-i", "--independentFiltering"),
73 default=TRUE,
74 dest="independentFiltering",
75 help="perform independent filtering (default is TRUE)"),
76
77 make_option(c("-a", "--alpha"),
78 default=0.05,
79 dest="alpha",
80 help="threshold of statistical significance [default: %default]"),
81
82 make_option(c("-p", "--pAdjustMethod"),
83 default="BH",
84 dest="pAdjustMethod",
85 help="p-value adjustment method: \"BH\" or \"BY\" [default: %default]"),
86
87 make_option(c("-T", "--typeTrans"),
88 default="VST",
89 dest="typeTrans",
90 help="transformation for PCA/clustering: \"VST\" ou \"rlog\" [default: %default]"),
91
92 make_option(c("-l", "--locfunc"),
93 default="median",
94 dest="locfunc",
95 help="median or shorth to estimate the size factors [default: %default]"),
96
97 make_option(c("-C", "--colors"),
98 default="dodgerblue,firebrick1,MediumVioletRed,SpringGreen,chartreuse,cyan,darkorchid,darkorange",
99 dest="cols",
100 help="colors of each biological condition on the plots\n\t\t\"col1,col2,col3,col4\"\n\t\t[default: %default]")
101 )
102
103 # now parse the command line to check which option is given and get associated values
104 parser <- OptionParser(usage="usage: %prog [options]",
105 option_list=option_list,
106 description="Compare two or more biological conditions in a RNA-Seq framework with DESeq2.",
107 epilogue="For comments, bug reports etc... please contact Hugo Varet <hugo.varet@pasteur.fr>")
108 opt <- parse_args(parser, args=commandArgs(trailingOnly=TRUE), positional_arguments=0)$options
109
110 # get options and arguments
111 workDir <- getwd()
112 projectName <- opt$projectName # name of the project
113 author <- opt$author # author of the statistical analysis/report
114 targetFile <- opt$targetFile # path to the design/target file
115 rawDir <- opt$rawDir # path to the directory containing raw counts files
116 featuresToRemove <- unlist(strsplit(opt$FTR, ",")) # names of the features to be removed (specific HTSeq-count information and rRNA for example)
117 varInt <- opt$varInt # factor of interest
118 condRef <- opt$condRef # reference biological condition
119 batch <- opt$batch # blocking factor: NULL (default) or "batch" for example
120 fitType <- opt$fitType # mean-variance relationship: "parametric" (default) or "local"
121 cooksCutoff <- opt$cooksCutoff # outliers detection threshold (NULL to let DESeq2 choosing it)
122 independentFiltering <- opt$independentFiltering # TRUE/FALSE to perform independent filtering (default is TRUE)
123 alpha <- as.numeric(opt$alpha) # threshold of statistical significance
124 pAdjustMethod <- opt$pAdjustMethod # p-value adjustment method: "BH" (default) or "BY"
125 typeTrans <- opt$typeTrans # transformation for PCA/clustering: "VST" ou "rlog"
126 locfunc <- opt$locfunc # "median" (default) or "shorth" to estimate the size factors
127 colors <- unlist(strsplit(opt$cols, ",")) # vector of colors of each biologicial condition on the plots
128
129 # print(paste("workDir", workDir))
130 # print(paste("projectName", projectName))
131 # print(paste("author", author))
132 # print(paste("targetFile", targetFile))
133 # print(paste("rawDir", rawDir))
134 # print(paste("varInt", varInt))
135 # print(paste("condRef", condRef))
136 # print(paste("batch", batch))
137 # print(paste("fitType", fitType))
138 # print(paste("cooksCutoff", cooksCutoff))
139 # print(paste("independentFiltering", independentFiltering))
140 # print(paste("alpha", alpha))
141 # print(paste("pAdjustMethod", pAdjustMethod))
142 # print(paste("typeTrans", typeTrans))
143 # print(paste("locfunc", locfunc))
144 # print(paste("featuresToRemove", featuresToRemove))
145 # print(paste("colors", colors))
146
147 ################################################################################
148 ### running script ###
149 ################################################################################
150 # setwd(workDir)
151 library(SARTools)
152
153 # checking parameters
154 problem <- checkParameters.DESeq2(projectName=projectName,author=author,targetFile=targetFile,
155 rawDir=rawDir,featuresToRemove=featuresToRemove,varInt=varInt,
156 condRef=condRef,batch=batch,fitType=fitType,cooksCutoff=cooksCutoff,
157 independentFiltering=independentFiltering,alpha=alpha,pAdjustMethod=pAdjustMethod,
158 typeTrans=typeTrans,locfunc=locfunc,colors=colors)
159 if (problem) quit(save="yes")
160
161 # loading target file
162 target <- loadTargetFile(targetFile=targetFile, varInt=varInt, condRef=condRef, batch=batch)
163
164 # loading counts
165 counts <- loadCountData(target=target, rawDir=rawDir, featuresToRemove=featuresToRemove)
166
167 # description plots
168 majSequences <- descriptionPlots(counts=counts, group=target[,varInt], col=colors)
169
170 # analysis with DESeq2
171 out.DESeq2 <- run.DESeq2(counts=counts, target=target, varInt=varInt, batch=batch,
172 locfunc=locfunc, fitType=fitType, pAdjustMethod=pAdjustMethod,
173 cooksCutoff=cooksCutoff, independentFiltering=independentFiltering, alpha=alpha)
174
175 # PCA + clustering
176 exploreCounts(object=out.DESeq2$dds, group=target[,varInt], typeTrans=typeTrans, col=colors)
177
178 # summary of the analysis (boxplots, dispersions, diag size factors, export table, nDiffTotal, histograms, MA plot)
179 summaryResults <- summarizeResults.DESeq2(out.DESeq2, group=target[,varInt], col=colors,
180 independentFiltering=independentFiltering,
181 cooksCutoff=cooksCutoff, alpha=alpha)
182
183 # save image of the R session
184 save.image(file=paste0(projectName, ".RData"))
185
186 # generating HTML report
187 writeReport.DESeq2(target=target, counts=counts, out.DESeq2=out.DESeq2, summaryResults=summaryResults,
188 majSequences=majSequences, workDir=workDir, projectName=projectName, author=author,
189 targetFile=targetFile, rawDir=rawDir, featuresToRemove=featuresToRemove, varInt=varInt,
190 condRef=condRef, batch=batch, fitType=fitType, cooksCutoff=cooksCutoff,
191 independentFiltering=independentFiltering, alpha=alpha, pAdjustMethod=pAdjustMethod,
192 typeTrans=typeTrans, locfunc=locfunc, colors=colors)