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12
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1 # ---------------------- load/install packages ----------------------
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2
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3 if (!("gridExtra" %in% rownames(installed.packages()))) {
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4 install.packages("gridExtra", repos="http://cran.xl-mirror.nl/")
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5 }
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6 library(gridExtra)
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7 if (!("ggplot2" %in% rownames(installed.packages()))) {
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8 install.packages("ggplot2", repos="http://cran.xl-mirror.nl/")
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9 }
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10 library(ggplot2)
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11 if (!("plyr" %in% rownames(installed.packages()))) {
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12 install.packages("plyr", repos="http://cran.xl-mirror.nl/")
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13 }
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14 library(plyr)
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15
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16 if (!("data.table" %in% rownames(installed.packages()))) {
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17 install.packages("data.table", repos="http://cran.xl-mirror.nl/")
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18 }
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19 library(data.table)
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20
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21 if (!("reshape2" %in% rownames(installed.packages()))) {
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22 install.packages("reshape2", repos="http://cran.xl-mirror.nl/")
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23 }
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24 library(reshape2)
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25
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26 if (!("lymphclon" %in% rownames(installed.packages()))) {
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27 install.packages("lymphclon", repos="http://cran.xl-mirror.nl/")
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28 }
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29 library(lymphclon)
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30
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31 # ---------------------- parameters ----------------------
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32
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33 args <- commandArgs(trailingOnly = TRUE)
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34
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35 infile = args[1] #path to input file
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36 outfile = args[2] #path to output file
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37 outdir = args[3] #path to output folder (html/images/data)
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38 clonaltype = args[4] #clonaltype definition, or 'none' for no unique filtering
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39 ct = unlist(strsplit(clonaltype, ","))
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40 species = args[5] #human or mouse
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41 locus = args[6] # IGH, IGK, IGL, TRB, TRA, TRG or TRD
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42 filterproductive = ifelse(args[7] == "yes", T, F) #should unproductive sequences be filtered out? (yes/no)
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43 clonality_method = args[8]
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47
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44 filter_uniques = args[9]
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12
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45
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50
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46 print(paste("filter_uniques", filter_uniques))
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47
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12
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48 # ---------------------- Data preperation ----------------------
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49
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18
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50 print("Report Clonality - Data preperation")
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51
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12
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52 inputdata = read.table(infile, sep="\t", header=TRUE, fill=T, comment.char="")
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53
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54 setwd(outdir)
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55
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56 # remove weird rows
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57 inputdata = inputdata[inputdata$Sample != "",]
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58
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59 #remove the allele from the V,D and J genes
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60 inputdata$Top.V.Gene = gsub("[*]([0-9]+)", "", inputdata$Top.V.Gene)
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61 inputdata$Top.D.Gene = gsub("[*]([0-9]+)", "", inputdata$Top.D.Gene)
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62 inputdata$Top.J.Gene = gsub("[*]([0-9]+)", "", inputdata$Top.J.Gene)
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63
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47
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64 #filter uniques
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65 inputdata.removed = inputdata[NULL,]
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66
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48
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67 filter_uniques = filter_uniques == "yes" && c("CDR1.Seq", "CDR2.Seq", "CDR3.Seq", "FR1.IMGT", "FR2.IMGT", "FR3.IMGT") %in% names(inputdata)
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68
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50
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69 print(paste("filter_uniques", filter_uniques))
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70
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48
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71 if(filter_uniques){
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47
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72
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73 clmns = names(inputdata)
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74
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75 inputdata$unique.def = paste(inputdata$CDR1.Seq, inputdata$CDR2.Seq, inputdata$CDR3.Seq, inputdata$FR1.IMGT, inputdata$FR2.IMGT, inputdata$FR3.IMGT)
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76 inputdata.filtered = inputdata[duplicated(inputdata$unique.def),]
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77 fltr = inputdata$unique.def %in% inputdata.filtered$unique.def
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78
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79 inputdata.removed = inputdata[!fltr,]
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80 inputdata.removed$samples_replicates = paste(inputdata.removed$Sample, inputdata.removed$Replicate, sep="_")
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81
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82 inputdata = inputdata[fltr,]
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83
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84 inputdata = inputdata[,clmns]
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85
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86 write.table(inputdata.removed, "unique_removed.csv", sep=",",quote=F,row.names=F,col.names=T)
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87 }
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88
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89
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12
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90 inputdata$clonaltype = 1:nrow(inputdata)
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91
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92 PRODF = inputdata
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93 UNPROD = inputdata
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94 if(filterproductive){
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95 if("Functionality" %in% colnames(inputdata)) { # "Functionality" is an IMGT column
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96 PRODF = inputdata[inputdata$Functionality == "productive" | inputdata$Functionality == "productive (see comment)", ]
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97 UNPROD = inputdata[!(inputdata$Functionality == "productive" | inputdata$Functionality == "productive (see comment)"), ]
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98 } else {
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99 PRODF = inputdata[inputdata$VDJ.Frame != "In-frame with stop codon" & inputdata$VDJ.Frame != "Out-of-frame" & inputdata$CDR3.Found.How != "NOT_FOUND" , ]
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100 UNPROD = inputdata[!(inputdata$VDJ.Frame != "In-frame with stop codon" & inputdata$VDJ.Frame != "Out-of-frame" & inputdata$CDR3.Found.How != "NOT_FOUND" ), ]
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101 }
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102 }
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103
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104 clonalityFrame = PRODF
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105
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106 #remove duplicates based on the clonaltype
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107 if(clonaltype != "none"){
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108 clonaltype = paste(clonaltype, ",Sample", sep="") #add sample column to clonaltype, unique within samples
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109 PRODF$clonaltype = do.call(paste, c(PRODF[unlist(strsplit(clonaltype, ","))], sep = ":"))
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110 PRODF = PRODF[!duplicated(PRODF$clonaltype), ]
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111
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112 UNPROD$clonaltype = do.call(paste, c(UNPROD[unlist(strsplit(clonaltype, ","))], sep = ":"))
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113 UNPROD = UNPROD[!duplicated(UNPROD$clonaltype), ]
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114
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115 #again for clonalityFrame but with sample+replicate
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116 clonalityFrame$clonaltype = do.call(paste, c(clonalityFrame[unlist(strsplit(clonaltype, ","))], sep = ":"))
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117 clonalityFrame$clonality_clonaltype = do.call(paste, c(clonalityFrame[unlist(strsplit(paste(clonaltype, ",Replicate", sep=""), ","))], sep = ":"))
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118 clonalityFrame = clonalityFrame[!duplicated(clonalityFrame$clonality_clonaltype), ]
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119 }
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120
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121 PRODF$freq = 1
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122
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123 if(any(grepl(pattern="_", x=PRODF$ID))){ #the frequency can be stored in the ID with the pattern ".*_freq_.*"
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124 PRODF$freq = gsub("^[0-9]+_", "", PRODF$ID)
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125 PRODF$freq = gsub("_.*", "", PRODF$freq)
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126 PRODF$freq = as.numeric(PRODF$freq)
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127 if(any(is.na(PRODF$freq))){ #if there was an "_" in the ID, but not the frequency, go back to frequency of 1 for every sequence
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128 PRODF$freq = 1
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129 }
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130 }
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131
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132
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133
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134 #write the complete dataset that is left over, will be the input if 'none' for clonaltype and 'no' for filterproductive
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135 write.table(PRODF, "allUnique.txt", sep=",",quote=F,row.names=F,col.names=T)
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136 write.table(PRODF, "allUnique.csv", sep="\t",quote=F,row.names=F,col.names=T)
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137 write.table(UNPROD, "allUnproductive.csv", sep=",",quote=F,row.names=F,col.names=T)
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138
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139 #write the samples to a file
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140 sampleFile <- file("samples.txt")
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141 un = unique(inputdata$Sample)
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142 un = paste(un, sep="\n")
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143 writeLines(un, sampleFile)
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144 close(sampleFile)
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145
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146 # ---------------------- Counting the productive/unproductive and unique sequences ----------------------
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147
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18
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148 print("Report Clonality - counting productive/unproductive/unique")
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149
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13
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150 if(!("Functionality" %in% inputdata)){ #add a functionality column to the igblast data
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151 inputdata$Functionality = "unproductive"
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152 search = (inputdata$VDJ.Frame != "In-frame with stop codon" & inputdata$VDJ.Frame != "Out-of-frame" & inputdata$CDR3.Found.How != "NOT_FOUND")
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153 if(sum(search) > 0){
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154 inputdata[search,]$Functionality = "productive"
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155 }
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156 }
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157
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12
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158 inputdata.dt = data.table(inputdata) #for speed
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159
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160 if(clonaltype == "none"){
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161 ct = c("clonaltype")
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162 }
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163
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164 inputdata.dt$samples_replicates = paste(inputdata.dt$Sample, inputdata.dt$Replicate, sep="_")
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165 samples_replicates = c(unique(inputdata.dt$samples_replicates), unique(as.character(inputdata.dt$Sample)))
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166 frequency_table = data.frame(ID = samples_replicates[order(samples_replicates)])
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167
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168
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169 sample_productive_count = inputdata.dt[, list(All=.N,
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170 Productive = nrow(.SD[.SD$Functionality == "productive" | .SD$Functionality == "productive (see comment)",]),
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171 perc_prod = 1,
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172 Productive_unique = nrow(.SD[.SD$Functionality == "productive" | .SD$Functionality == "productive (see comment)",list(count=.N),by=ct]),
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173 perc_prod_un = 1,
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174 Unproductive= nrow(.SD[.SD$Functionality != "productive" & .SD$Functionality != "productive (see comment)",]),
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175 perc_unprod = 1,
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176 Unproductive_unique =nrow(.SD[.SD$Functionality != "productive" & .SD$Functionality != "productive (see comment)",list(count=.N),by=ct]),
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177 perc_unprod_un = 1),
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178 by=c("Sample")]
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179
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180 sample_productive_count$perc_prod = round(sample_productive_count$Productive / sample_productive_count$All * 100)
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181 sample_productive_count$perc_prod_un = round(sample_productive_count$Productive_unique / sample_productive_count$All * 100)
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182
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183 sample_productive_count$perc_unprod = round(sample_productive_count$Unproductive / sample_productive_count$All * 100)
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184 sample_productive_count$perc_unprod_un = round(sample_productive_count$Unproductive_unique / sample_productive_count$All * 100)
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185
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48
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186
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187 if(filter_uniques){
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188 inputdata.removed.s = data.table(inputdata.removed)[, list(UniqueRemoved=.N), by=c("Sample")]
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189
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190 sample_productive_count = merge(sample_productive_count, inputdata.removed.s, by="Sample")
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47
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191
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48
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192 sample_productive_count$perc_rem = round(sample_productive_count$UniqueRemoved / sample_productive_count$All * 100)
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193 } else {
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194 sample_productive_count$UniqueRemoved = 0
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195 sample_productive_count$perc_rem = 0
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196 }
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12
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197
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198 sample_replicate_productive_count = inputdata.dt[, list(All=.N,
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199 Productive = nrow(.SD[.SD$Functionality == "productive" | .SD$Functionality == "productive (see comment)",]),
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200 perc_prod = 1,
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201 Productive_unique = nrow(.SD[.SD$Functionality == "productive" | .SD$Functionality == "productive (see comment)",list(count=.N),by=ct]),
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202 perc_prod_un = 1,
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203 Unproductive= nrow(.SD[.SD$Functionality != "productive" & .SD$Functionality != "productive (see comment)",]),
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204 perc_unprod = 1,
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205 Unproductive_unique =nrow(.SD[.SD$Functionality != "productive" & .SD$Functionality != "productive (see comment)",list(count=.N),by=ct]),
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206 perc_unprod_un = 1),
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207 by=c("samples_replicates")]
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208
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209 sample_replicate_productive_count$perc_prod = round(sample_replicate_productive_count$Productive / sample_replicate_productive_count$All * 100)
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210 sample_replicate_productive_count$perc_prod_un = round(sample_replicate_productive_count$Productive_unique / sample_replicate_productive_count$All * 100)
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211
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212 sample_replicate_productive_count$perc_unprod = round(sample_replicate_productive_count$Unproductive / sample_replicate_productive_count$All * 100)
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213 sample_replicate_productive_count$perc_unprod_un = round(sample_replicate_productive_count$Unproductive_unique / sample_replicate_productive_count$All * 100)
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214
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48
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215
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216 if(filter_uniques){
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217 inputdata.removed.sr = data.table(inputdata.removed)[, list(UniqueRemoved=.N), by=c("samples_replicates")]
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218
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219 sample_replicate_productive_count = merge(sample_replicate_productive_count, inputdata.removed.sr, by="samples_replicates")
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47
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220
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48
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221 sample_replicate_productive_count$perc_rem = round(sample_replicate_productive_count$UniqueRemoved / sample_productive_count$All * 100)
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222 } else {
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223 sample_replicate_productive_count$UniqueRemoved = 0
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224 sample_replicate_productive_count$perc_rem = 0
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225 }
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47
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226
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12
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227 setnames(sample_replicate_productive_count, colnames(sample_productive_count))
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228
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229 counts = rbind(sample_replicate_productive_count, sample_productive_count)
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230 counts = counts[order(counts$Sample),]
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231
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232 write.table(x=counts, file="productive_counting.txt", sep=",",quote=F,row.names=F,col.names=F)
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233
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234 # ---------------------- Frequency calculation for V, D and J ----------------------
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235
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18
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236 print("Report Clonality - frequency calculation V, D and J")
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237
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12
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238 PRODFV = data.frame(data.table(PRODF)[, list(Length=sum(freq)), by=c("Sample", "Top.V.Gene")])
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239 Total = ddply(PRODFV, .(Sample), function(x) data.frame(Total = sum(x$Length)))
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240 PRODFV = merge(PRODFV, Total, by.x='Sample', by.y='Sample', all.x=TRUE)
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241 PRODFV = ddply(PRODFV, c("Sample", "Top.V.Gene"), summarise, relFreq= (Length*100 / Total))
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242
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243 PRODFD = data.frame(data.table(PRODF)[, list(Length=sum(freq)), by=c("Sample", "Top.D.Gene")])
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244 Total = ddply(PRODFD, .(Sample), function(x) data.frame(Total = sum(x$Length)))
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245 PRODFD = merge(PRODFD, Total, by.x='Sample', by.y='Sample', all.x=TRUE)
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246 PRODFD = ddply(PRODFD, c("Sample", "Top.D.Gene"), summarise, relFreq= (Length*100 / Total))
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247
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248 PRODFJ = data.frame(data.table(PRODF)[, list(Length=sum(freq)), by=c("Sample", "Top.J.Gene")])
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249 Total = ddply(PRODFJ, .(Sample), function(x) data.frame(Total = sum(x$Length)))
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250 PRODFJ = merge(PRODFJ, Total, by.x='Sample', by.y='Sample', all.x=TRUE)
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251 PRODFJ = ddply(PRODFJ, c("Sample", "Top.J.Gene"), summarise, relFreq= (Length*100 / Total))
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252
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253 # ---------------------- Setting up the gene names for the different species/loci ----------------------
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254
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18
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255 print("Report Clonality - getting genes for species/loci")
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256
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12
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257 Vchain = ""
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258 Dchain = ""
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259 Jchain = ""
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260
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261 if(species == "custom"){
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262 print("Custom genes: ")
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263 splt = unlist(strsplit(locus, ";"))
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264 print(paste("V:", splt[1]))
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265 print(paste("D:", splt[2]))
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266 print(paste("J:", splt[3]))
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267
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268 Vchain = unlist(strsplit(splt[1], ","))
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269 Vchain = data.frame(v.name = Vchain, chr.orderV = 1:length(Vchain))
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270
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271 Dchain = unlist(strsplit(splt[2], ","))
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272 if(length(Dchain) > 0){
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273 Dchain = data.frame(v.name = Dchain, chr.orderD = 1:length(Dchain))
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274 } else {
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275 Dchain = data.frame(v.name = character(0), chr.orderD = numeric(0))
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276 }
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277
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278 Jchain = unlist(strsplit(splt[3], ","))
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279 Jchain = data.frame(v.name = Jchain, chr.orderJ = 1:length(Jchain))
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280
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281 } else {
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282 genes = read.table("genes.txt", sep="\t", header=TRUE, fill=T, comment.char="")
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283
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284 Vchain = genes[grepl(species, genes$Species) & genes$locus == locus & genes$region == "V",c("IMGT.GENE.DB", "chr.order")]
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285 colnames(Vchain) = c("v.name", "chr.orderV")
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286 Dchain = genes[grepl(species, genes$Species) & genes$locus == locus & genes$region == "D",c("IMGT.GENE.DB", "chr.order")]
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287 colnames(Dchain) = c("v.name", "chr.orderD")
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288 Jchain = genes[grepl(species, genes$Species) & genes$locus == locus & genes$region == "J",c("IMGT.GENE.DB", "chr.order")]
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289 colnames(Jchain) = c("v.name", "chr.orderJ")
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290 }
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291 useD = TRUE
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292 if(nrow(Dchain) == 0){
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293 useD = FALSE
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294 cat("No D Genes in this species/locus")
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295 }
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18
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296 print(paste(nrow(Vchain), "genes in V"))
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297 print(paste(nrow(Dchain), "genes in D"))
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298 print(paste(nrow(Jchain), "genes in J"))
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12
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299
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300 # ---------------------- merge with the frequency count ----------------------
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301
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302 PRODFV = merge(PRODFV, Vchain, by.x='Top.V.Gene', by.y='v.name', all.x=TRUE)
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303
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304 PRODFD = merge(PRODFD, Dchain, by.x='Top.D.Gene', by.y='v.name', all.x=TRUE)
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305
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306 PRODFJ = merge(PRODFJ, Jchain, by.x='Top.J.Gene', by.y='v.name', all.x=TRUE)
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307
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308 # ---------------------- Create the V, D and J frequency plots and write the data.frame for every plot to a file ----------------------
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309
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18
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310 print("Report Clonality - V, D and J frequency plots")
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311
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12
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312 pV = ggplot(PRODFV)
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313 pV = pV + geom_bar( aes( x=factor(reorder(Top.V.Gene, chr.orderV)), y=relFreq, fill=Sample), stat='identity', position="dodge") + theme(axis.text.x = element_text(angle = 90, hjust = 1))
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314 pV = pV + xlab("Summary of V gene") + ylab("Frequency") + ggtitle("Relative frequency of V gene usage")
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315 write.table(x=PRODFV, file="VFrequency.csv", sep=",",quote=F,row.names=F,col.names=T)
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316
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317 png("VPlot.png",width = 1280, height = 720)
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318 pV
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319 dev.off();
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320
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|
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321 if(useD){
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322 pD = ggplot(PRODFD)
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323 pD = pD + geom_bar( aes( x=factor(reorder(Top.D.Gene, chr.orderD)), y=relFreq, fill=Sample), stat='identity', position="dodge") + theme(axis.text.x = element_text(angle = 90, hjust = 1))
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324 pD = pD + xlab("Summary of D gene") + ylab("Frequency") + ggtitle("Relative frequency of D gene usage")
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325 write.table(x=PRODFD, file="DFrequency.csv", sep=",",quote=F,row.names=F,col.names=T)
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326
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327 png("DPlot.png",width = 800, height = 600)
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328 print(pD)
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329 dev.off();
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330 }
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331
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332 pJ = ggplot(PRODFJ)
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333 pJ = pJ + geom_bar( aes( x=factor(reorder(Top.J.Gene, chr.orderJ)), y=relFreq, fill=Sample), stat='identity', position="dodge") + theme(axis.text.x = element_text(angle = 90, hjust = 1))
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334 pJ = pJ + xlab("Summary of J gene") + ylab("Frequency") + ggtitle("Relative frequency of J gene usage")
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335 write.table(x=PRODFJ, file="JFrequency.csv", sep=",",quote=F,row.names=F,col.names=T)
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336
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337 png("JPlot.png",width = 800, height = 600)
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338 pJ
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339 dev.off();
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340
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341 pJ = ggplot(PRODFJ)
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342 pJ = pJ + geom_bar( aes( x=factor(reorder(Top.J.Gene, chr.orderJ)), y=relFreq, fill=Sample), stat='identity', position="dodge") + theme(axis.text.x = element_text(angle = 90, hjust = 1))
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343 pJ = pJ + xlab("Summary of J gene") + ylab("Frequency") + ggtitle("Relative frequency of J gene usage")
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344 write.table(x=PRODFJ, file="JFrequency.csv", sep=",",quote=F,row.names=F,col.names=T)
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345
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346 png("JPlot.png",width = 800, height = 600)
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347 pJ
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|
348 dev.off();
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349
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350 # ---------------------- Now the frequency plots of the V, D and J families ----------------------
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351
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18
|
352 print("Report Clonality - V, D and J family plots")
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|
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353
|
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12
|
354 VGenes = PRODF[,c("Sample", "Top.V.Gene")]
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355 VGenes$Top.V.Gene = gsub("-.*", "", VGenes$Top.V.Gene)
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356 VGenes = data.frame(data.table(VGenes)[, list(Count=.N), by=c("Sample", "Top.V.Gene")])
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|
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357 TotalPerSample = data.frame(data.table(VGenes)[, list(total=sum(.SD$Count)), by=Sample])
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358 VGenes = merge(VGenes, TotalPerSample, by="Sample")
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359 VGenes$Frequency = VGenes$Count * 100 / VGenes$total
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360 VPlot = ggplot(VGenes)
|
|
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361 VPlot = VPlot + geom_bar(aes( x = Top.V.Gene, y = Frequency, fill = Sample), stat='identity', position='dodge' ) + theme(axis.text.x = element_text(angle = 90, hjust = 1)) +
|
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362 ggtitle("Distribution of V gene families") +
|
|
|
363 ylab("Percentage of sequences")
|
|
|
364 png("VFPlot.png")
|
|
|
365 VPlot
|
|
|
366 dev.off();
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|
|
367 write.table(x=VGenes, file="VFFrequency.csv", sep=",",quote=F,row.names=F,col.names=T)
|
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|
368
|
|
|
369 if(useD){
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|
370 DGenes = PRODF[,c("Sample", "Top.D.Gene")]
|
|
|
371 DGenes$Top.D.Gene = gsub("-.*", "", DGenes$Top.D.Gene)
|
|
|
372 DGenes = data.frame(data.table(DGenes)[, list(Count=.N), by=c("Sample", "Top.D.Gene")])
|
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|
373 TotalPerSample = data.frame(data.table(DGenes)[, list(total=sum(.SD$Count)), by=Sample])
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|
|
374 DGenes = merge(DGenes, TotalPerSample, by="Sample")
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|
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375 DGenes$Frequency = DGenes$Count * 100 / DGenes$total
|
|
|
376 DPlot = ggplot(DGenes)
|
|
|
377 DPlot = DPlot + geom_bar(aes( x = Top.D.Gene, y = Frequency, fill = Sample), stat='identity', position='dodge' ) + theme(axis.text.x = element_text(angle = 90, hjust = 1)) +
|
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378 ggtitle("Distribution of D gene families") +
|
|
|
379 ylab("Percentage of sequences")
|
|
|
380 png("DFPlot.png")
|
|
|
381 print(DPlot)
|
|
|
382 dev.off();
|
|
|
383 write.table(x=DGenes, file="DFFrequency.csv", sep=",",quote=F,row.names=F,col.names=T)
|
|
|
384 }
|
|
|
385
|
|
|
386 JGenes = PRODF[,c("Sample", "Top.J.Gene")]
|
|
|
387 JGenes$Top.J.Gene = gsub("-.*", "", JGenes$Top.J.Gene)
|
|
|
388 JGenes = data.frame(data.table(JGenes)[, list(Count=.N), by=c("Sample", "Top.J.Gene")])
|
|
|
389 TotalPerSample = data.frame(data.table(JGenes)[, list(total=sum(.SD$Count)), by=Sample])
|
|
|
390 JGenes = merge(JGenes, TotalPerSample, by="Sample")
|
|
|
391 JGenes$Frequency = JGenes$Count * 100 / JGenes$total
|
|
|
392 JPlot = ggplot(JGenes)
|
|
|
393 JPlot = JPlot + geom_bar(aes( x = Top.J.Gene, y = Frequency, fill = Sample), stat='identity', position='dodge' ) + theme(axis.text.x = element_text(angle = 90, hjust = 1)) +
|
|
|
394 ggtitle("Distribution of J gene families") +
|
|
|
395 ylab("Percentage of sequences")
|
|
|
396 png("JFPlot.png")
|
|
|
397 JPlot
|
|
|
398 dev.off();
|
|
|
399 write.table(x=JGenes, file="JFFrequency.csv", sep=",",quote=F,row.names=F,col.names=T)
|
|
|
400
|
|
|
401 # ---------------------- Plotting the cdr3 length ----------------------
|
|
|
402
|
|
18
|
403 print("Report Clonality - CDR3 length plot")
|
|
|
404
|
|
12
|
405 CDR3Length = data.frame(data.table(PRODF)[, list(Count=.N), by=c("Sample", "CDR3.Length.DNA")])
|
|
|
406 TotalPerSample = data.frame(data.table(CDR3Length)[, list(total=sum(.SD$Count)), by=Sample])
|
|
|
407 CDR3Length = merge(CDR3Length, TotalPerSample, by="Sample")
|
|
|
408 CDR3Length$Frequency = CDR3Length$Count * 100 / CDR3Length$total
|
|
|
409 CDR3LengthPlot = ggplot(CDR3Length)
|
|
|
410 CDR3LengthPlot = CDR3LengthPlot + geom_bar(aes( x = CDR3.Length.DNA, y = Frequency, fill = Sample), stat='identity', position='dodge' ) + theme(axis.text.x = element_text(angle = 90, hjust = 1)) +
|
|
|
411 ggtitle("Length distribution of CDR3") +
|
|
|
412 xlab("CDR3 Length") +
|
|
|
413 ylab("Percentage of sequences")
|
|
|
414 png("CDR3LengthPlot.png",width = 1280, height = 720)
|
|
|
415 CDR3LengthPlot
|
|
|
416 dev.off()
|
|
|
417 write.table(x=CDR3Length, file="CDR3LengthPlot.csv", sep=",",quote=F,row.names=F,col.names=T)
|
|
|
418
|
|
|
419 # ---------------------- Plot the heatmaps ----------------------
|
|
|
420
|
|
|
421 #get the reverse order for the V and D genes
|
|
|
422 revVchain = Vchain
|
|
|
423 revDchain = Dchain
|
|
|
424 revVchain$chr.orderV = rev(revVchain$chr.orderV)
|
|
|
425 revDchain$chr.orderD = rev(revDchain$chr.orderD)
|
|
|
426
|
|
|
427 if(useD){
|
|
18
|
428 print("Report Clonality - Heatmaps VD")
|
|
12
|
429 plotVD <- function(dat){
|
|
|
430 if(length(dat[,1]) == 0){
|
|
|
431 return()
|
|
|
432 }
|
|
|
433 img = ggplot() +
|
|
|
434 geom_tile(data=dat, aes(x=factor(reorder(Top.D.Gene, chr.orderD)), y=factor(reorder(Top.V.Gene, chr.orderV)), fill=relLength)) +
|
|
|
435 theme(axis.text.x = element_text(angle = 90, hjust = 1)) +
|
|
|
436 scale_fill_gradient(low="gold", high="blue", na.value="white") +
|
|
|
437 ggtitle(paste(unique(dat$Sample), " (N=" , sum(dat$Length, na.rm=T) ,")", sep="")) +
|
|
|
438 xlab("D genes") +
|
|
|
439 ylab("V Genes")
|
|
|
440
|
|
|
441 png(paste("HeatmapVD_", unique(dat[3])[1,1] , ".png", sep=""), width=150+(15*length(Dchain$v.name)), height=100+(15*length(Vchain$v.name)))
|
|
|
442 print(img)
|
|
|
443 dev.off()
|
|
|
444 write.table(x=acast(dat, Top.V.Gene~Top.D.Gene, value.var="Length"), file=paste("HeatmapVD_", unique(dat[3])[1,1], ".csv", sep=""), sep=",",quote=F,row.names=T,col.names=NA)
|
|
|
445 }
|
|
|
446
|
|
|
447 VandDCount = data.frame(data.table(PRODF)[, list(Length=.N), by=c("Top.V.Gene", "Top.D.Gene", "Sample")])
|
|
|
448
|
|
|
449 VandDCount$l = log(VandDCount$Length)
|
|
|
450 maxVD = data.frame(data.table(VandDCount)[, list(max=max(l)), by=c("Sample")])
|
|
|
451 VandDCount = merge(VandDCount, maxVD, by.x="Sample", by.y="Sample", all.x=T)
|
|
|
452 VandDCount$relLength = VandDCount$l / VandDCount$max
|
|
|
453
|
|
|
454 cartegianProductVD = expand.grid(Top.V.Gene = Vchain$v.name, Top.D.Gene = Dchain$v.name, Sample = unique(inputdata$Sample))
|
|
|
455
|
|
|
456 completeVD = merge(VandDCount, cartegianProductVD, all.y=TRUE)
|
|
|
457 completeVD = merge(completeVD, revVchain, by.x="Top.V.Gene", by.y="v.name", all.x=TRUE)
|
|
|
458 completeVD = merge(completeVD, Dchain, by.x="Top.D.Gene", by.y="v.name", all.x=TRUE)
|
|
|
459 VDList = split(completeVD, f=completeVD[,"Sample"])
|
|
|
460 lapply(VDList, FUN=plotVD)
|
|
|
461 }
|
|
|
462
|
|
18
|
463 print("Report Clonality - Heatmaps VJ")
|
|
|
464
|
|
12
|
465 plotVJ <- function(dat){
|
|
|
466 if(length(dat[,1]) == 0){
|
|
|
467 return()
|
|
|
468 }
|
|
|
469 cat(paste(unique(dat[3])[1,1]))
|
|
|
470 img = ggplot() +
|
|
|
471 geom_tile(data=dat, aes(x=factor(reorder(Top.J.Gene, chr.orderJ)), y=factor(reorder(Top.V.Gene, chr.orderV)), fill=relLength)) +
|
|
|
472 theme(axis.text.x = element_text(angle = 90, hjust = 1)) +
|
|
|
473 scale_fill_gradient(low="gold", high="blue", na.value="white") +
|
|
|
474 ggtitle(paste(unique(dat$Sample), " (N=" , sum(dat$Length, na.rm=T) ,")", sep="")) +
|
|
|
475 xlab("J genes") +
|
|
|
476 ylab("V Genes")
|
|
|
477
|
|
|
478 png(paste("HeatmapVJ_", unique(dat[3])[1,1] , ".png", sep=""), width=150+(15*length(Jchain$v.name)), height=100+(15*length(Vchain$v.name)))
|
|
|
479 print(img)
|
|
|
480 dev.off()
|
|
|
481 write.table(x=acast(dat, Top.V.Gene~Top.J.Gene, value.var="Length"), file=paste("HeatmapVJ_", unique(dat[3])[1,1], ".csv", sep=""), sep=",",quote=F,row.names=T,col.names=NA)
|
|
|
482 }
|
|
|
483
|
|
|
484 VandJCount = data.frame(data.table(PRODF)[, list(Length=.N), by=c("Top.V.Gene", "Top.J.Gene", "Sample")])
|
|
|
485
|
|
|
486 VandJCount$l = log(VandJCount$Length)
|
|
|
487 maxVJ = data.frame(data.table(VandJCount)[, list(max=max(l)), by=c("Sample")])
|
|
|
488 VandJCount = merge(VandJCount, maxVJ, by.x="Sample", by.y="Sample", all.x=T)
|
|
|
489 VandJCount$relLength = VandJCount$l / VandJCount$max
|
|
|
490
|
|
|
491 cartegianProductVJ = expand.grid(Top.V.Gene = Vchain$v.name, Top.J.Gene = Jchain$v.name, Sample = unique(inputdata$Sample))
|
|
|
492
|
|
|
493 completeVJ = merge(VandJCount, cartegianProductVJ, all.y=TRUE)
|
|
|
494 completeVJ = merge(completeVJ, revVchain, by.x="Top.V.Gene", by.y="v.name", all.x=TRUE)
|
|
|
495 completeVJ = merge(completeVJ, Jchain, by.x="Top.J.Gene", by.y="v.name", all.x=TRUE)
|
|
|
496 VJList = split(completeVJ, f=completeVJ[,"Sample"])
|
|
|
497 lapply(VJList, FUN=plotVJ)
|
|
|
498
|
|
18
|
499
|
|
|
500
|
|
12
|
501 if(useD){
|
|
18
|
502 print("Report Clonality - Heatmaps DJ")
|
|
12
|
503 plotDJ <- function(dat){
|
|
|
504 if(length(dat[,1]) == 0){
|
|
|
505 return()
|
|
|
506 }
|
|
|
507 img = ggplot() +
|
|
|
508 geom_tile(data=dat, aes(x=factor(reorder(Top.J.Gene, chr.orderJ)), y=factor(reorder(Top.D.Gene, chr.orderD)), fill=relLength)) +
|
|
|
509 theme(axis.text.x = element_text(angle = 90, hjust = 1)) +
|
|
|
510 scale_fill_gradient(low="gold", high="blue", na.value="white") +
|
|
|
511 ggtitle(paste(unique(dat$Sample), " (N=" , sum(dat$Length, na.rm=T) ,")", sep="")) +
|
|
|
512 xlab("J genes") +
|
|
|
513 ylab("D Genes")
|
|
|
514
|
|
|
515 png(paste("HeatmapDJ_", unique(dat[3])[1,1] , ".png", sep=""), width=150+(15*length(Jchain$v.name)), height=100+(15*length(Dchain$v.name)))
|
|
|
516 print(img)
|
|
|
517 dev.off()
|
|
|
518 write.table(x=acast(dat, Top.D.Gene~Top.J.Gene, value.var="Length"), file=paste("HeatmapDJ_", unique(dat[3])[1,1], ".csv", sep=""), sep=",",quote=F,row.names=T,col.names=NA)
|
|
|
519 }
|
|
|
520
|
|
|
521
|
|
|
522 DandJCount = data.frame(data.table(PRODF)[, list(Length=.N), by=c("Top.D.Gene", "Top.J.Gene", "Sample")])
|
|
|
523
|
|
|
524 DandJCount$l = log(DandJCount$Length)
|
|
|
525 maxDJ = data.frame(data.table(DandJCount)[, list(max=max(l)), by=c("Sample")])
|
|
|
526 DandJCount = merge(DandJCount, maxDJ, by.x="Sample", by.y="Sample", all.x=T)
|
|
|
527 DandJCount$relLength = DandJCount$l / DandJCount$max
|
|
|
528
|
|
|
529 cartegianProductDJ = expand.grid(Top.D.Gene = Dchain$v.name, Top.J.Gene = Jchain$v.name, Sample = unique(inputdata$Sample))
|
|
|
530
|
|
|
531 completeDJ = merge(DandJCount, cartegianProductDJ, all.y=TRUE)
|
|
|
532 completeDJ = merge(completeDJ, revDchain, by.x="Top.D.Gene", by.y="v.name", all.x=TRUE)
|
|
|
533 completeDJ = merge(completeDJ, Jchain, by.x="Top.J.Gene", by.y="v.name", all.x=TRUE)
|
|
|
534 DJList = split(completeDJ, f=completeDJ[,"Sample"])
|
|
|
535 lapply(DJList, FUN=plotDJ)
|
|
|
536 }
|
|
|
537
|
|
|
538
|
|
34
|
539 # ---------------------- output tables for the circos plots ----------------------
|
|
|
540
|
|
|
541 print("Report Clonality - Circos data")
|
|
|
542
|
|
|
543 for(smpl in unique(PRODF$Sample)){
|
|
|
544 PRODF.sample = PRODF[PRODF$Sample == smpl,]
|
|
|
545
|
|
|
546 fltr = PRODF.sample$Top.V.Gene == ""
|
|
36
|
547 if(any(fltr, na.rm=T)){
|
|
34
|
548 PRODF.sample[fltr, "Top.V.Gene"] = "NA"
|
|
|
549 }
|
|
36
|
550
|
|
34
|
551 fltr = PRODF.sample$Top.D.Gene == ""
|
|
36
|
552 if(any(fltr, na.rm=T)){
|
|
34
|
553 PRODF.sample[fltr, "Top.D.Gene"] = "NA"
|
|
|
554 }
|
|
|
555
|
|
|
556 fltr = PRODF.sample$Top.J.Gene == ""
|
|
36
|
557 if(any(fltr, na.rm=T)){
|
|
34
|
558 PRODF.sample[fltr, "Top.J.Gene"] = "NA"
|
|
|
559 }
|
|
|
560
|
|
|
561 v.d = table(PRODF.sample$Top.V.Gene, PRODF.sample$Top.D.Gene)
|
|
|
562 v.j = table(PRODF.sample$Top.V.Gene, PRODF.sample$Top.J.Gene)
|
|
|
563 d.j = table(PRODF.sample$Top.D.Gene, PRODF.sample$Top.J.Gene)
|
|
|
564
|
|
|
565 write.table(v.d, file=paste(smpl, "_VD_circos.txt", sep=""), sep="\t", quote=F, row.names=T, col.names=NA)
|
|
|
566 write.table(v.j, file=paste(smpl, "_VJ_circos.txt", sep=""), sep="\t", quote=F, row.names=T, col.names=NA)
|
|
|
567 write.table(d.j, file=paste(smpl, "_DJ_circos.txt", sep=""), sep="\t", quote=F, row.names=T, col.names=NA)
|
|
|
568 }
|
|
|
569
|
|
12
|
570 # ---------------------- calculating the clonality score ----------------------
|
|
|
571
|
|
|
572 if("Replicate" %in% colnames(inputdata)) #can only calculate clonality score when replicate information is available
|
|
|
573 {
|
|
34
|
574 print("Report Clonality - Clonality")
|
|
12
|
575 if(clonality_method == "boyd"){
|
|
|
576 samples = split(clonalityFrame, clonalityFrame$Sample, drop=T)
|
|
|
577
|
|
|
578 for (sample in samples){
|
|
|
579 res = data.frame(paste=character(0))
|
|
|
580 sample_id = unique(sample$Sample)[[1]]
|
|
|
581 for(replicate in unique(sample$Replicate)){
|
|
|
582 tmp = sample[sample$Replicate == replicate,]
|
|
|
583 clone_table = data.frame(table(tmp$clonaltype))
|
|
|
584 clone_col_name = paste("V", replicate, sep="")
|
|
|
585 colnames(clone_table) = c("paste", clone_col_name)
|
|
|
586 res = merge(res, clone_table, by="paste", all=T)
|
|
|
587 }
|
|
|
588
|
|
|
589 res[is.na(res)] = 0
|
|
|
590 infer.result = infer.clonality(as.matrix(res[,2:ncol(res)]))
|
|
|
591
|
|
|
592 write.table(data.table(infer.result[[12]]), file=paste("lymphclon_clonality_", sample_id, ".csv", sep=""), sep=",",quote=F,row.names=F,col.names=F)
|
|
|
593
|
|
|
594 res$type = rowSums(res[,2:ncol(res)])
|
|
|
595
|
|
|
596 coincidence.table = data.frame(table(res$type))
|
|
|
597 colnames(coincidence.table) = c("Coincidence Type", "Raw Coincidence Freq")
|
|
|
598 write.table(coincidence.table, file=paste("lymphclon_coincidences_", sample_id, ".csv", sep=""), sep=",",quote=F,row.names=F,col.names=T)
|
|
|
599 }
|
|
|
600 } else {
|
|
|
601 write.table(clonalityFrame, "clonalityComplete.csv", sep=",",quote=F,row.names=F,col.names=T)
|
|
|
602
|
|
|
603 clonalFreq = data.frame(data.table(clonalityFrame)[, list(Type=.N), by=c("Sample", "clonaltype")])
|
|
|
604 clonalFreqCount = data.frame(data.table(clonalFreq)[, list(Count=.N), by=c("Sample", "Type")])
|
|
|
605 clonalFreqCount$realCount = clonalFreqCount$Type * clonalFreqCount$Count
|
|
|
606 clonalSum = data.frame(data.table(clonalFreqCount)[, list(Reads=sum(realCount)), by=c("Sample")])
|
|
|
607 clonalFreqCount = merge(clonalFreqCount, clonalSum, by.x="Sample", by.y="Sample")
|
|
|
608
|
|
|
609 ct = c('Type\tWeight\n2\t1\n3\t3\n4\t6\n5\t10\n6\t15')
|
|
|
610 tcct = textConnection(ct)
|
|
|
611 CT = read.table(tcct, sep="\t", header=TRUE)
|
|
|
612 close(tcct)
|
|
|
613 clonalFreqCount = merge(clonalFreqCount, CT, by.x="Type", by.y="Type", all.x=T)
|
|
|
614 clonalFreqCount$WeightedCount = clonalFreqCount$Count * clonalFreqCount$Weight
|
|
|
615
|
|
|
616 ReplicateReads = data.frame(data.table(clonalityFrame)[, list(Type=.N), by=c("Sample", "Replicate", "clonaltype")])
|
|
|
617 ReplicateReads = data.frame(data.table(ReplicateReads)[, list(Reads=.N), by=c("Sample", "Replicate")])
|
|
|
618 clonalFreqCount$Reads = as.numeric(clonalFreqCount$Reads)
|
|
39
|
619 ReplicateReads$Reads = as.numeric(ReplicateReads$Reads)
|
|
|
620 ReplicateReads$squared = as.numeric(ReplicateReads$Reads * ReplicateReads$Reads)
|
|
12
|
621
|
|
|
622 ReplicatePrint <- function(dat){
|
|
|
623 write.table(dat[-1], paste("ReplicateReads_", unique(dat[1])[1,1] , ".csv", sep=""), sep=",",quote=F,na="-",row.names=F,col.names=F)
|
|
|
624 }
|
|
|
625
|
|
|
626 ReplicateSplit = split(ReplicateReads, f=ReplicateReads[,"Sample"])
|
|
|
627 lapply(ReplicateSplit, FUN=ReplicatePrint)
|
|
|
628
|
|
|
629 ReplicateReads = data.frame(data.table(ReplicateReads)[, list(ReadsSum=sum(as.numeric(Reads)), ReadsSquaredSum=sum(as.numeric(squared))), by=c("Sample")])
|
|
|
630 clonalFreqCount = merge(clonalFreqCount, ReplicateReads, by.x="Sample", by.y="Sample", all.x=T)
|
|
|
631
|
|
|
632 ReplicateSumPrint <- function(dat){
|
|
|
633 write.table(dat[-1], paste("ReplicateSumReads_", unique(dat[1])[1,1] , ".csv", sep=""), sep=",",quote=F,na="-",row.names=F,col.names=F)
|
|
|
634 }
|
|
|
635
|
|
|
636 ReplicateSumSplit = split(ReplicateReads, f=ReplicateReads[,"Sample"])
|
|
|
637 lapply(ReplicateSumSplit, FUN=ReplicateSumPrint)
|
|
|
638
|
|
|
639 clonalFreqCountSum = data.frame(data.table(clonalFreqCount)[, list(Numerator=sum(WeightedCount, na.rm=T)), by=c("Sample")])
|
|
|
640 clonalFreqCount = merge(clonalFreqCount, clonalFreqCountSum, by.x="Sample", by.y="Sample", all.x=T)
|
|
|
641 clonalFreqCount$ReadsSum = as.numeric(clonalFreqCount$ReadsSum) #prevent integer overflow
|
|
|
642 clonalFreqCount$Denominator = (((clonalFreqCount$ReadsSum * clonalFreqCount$ReadsSum) - clonalFreqCount$ReadsSquaredSum) / 2)
|
|
|
643 clonalFreqCount$Result = (clonalFreqCount$Numerator + 1) / (clonalFreqCount$Denominator + 1)
|
|
|
644
|
|
|
645 ClonalityScorePrint <- function(dat){
|
|
|
646 write.table(dat$Result, paste("ClonalityScore_", unique(dat[1])[1,1] , ".csv", sep=""), sep=",",quote=F,na="-",row.names=F,col.names=F)
|
|
|
647 }
|
|
|
648
|
|
|
649 clonalityScore = clonalFreqCount[c("Sample", "Result")]
|
|
|
650 clonalityScore = unique(clonalityScore)
|
|
|
651
|
|
|
652 clonalityScoreSplit = split(clonalityScore, f=clonalityScore[,"Sample"])
|
|
|
653 lapply(clonalityScoreSplit, FUN=ClonalityScorePrint)
|
|
|
654
|
|
|
655 clonalityOverview = clonalFreqCount[c("Sample", "Type", "Count", "Weight", "WeightedCount")]
|
|
|
656
|
|
|
657
|
|
|
658
|
|
|
659 ClonalityOverviewPrint <- function(dat){
|
|
|
660 write.table(dat[-1], paste("ClonalityOverView_", unique(dat[1])[1,1] , ".csv", sep=""), sep=",",quote=F,na="-",row.names=F,col.names=F)
|
|
|
661 }
|
|
|
662
|
|
|
663 clonalityOverviewSplit = split(clonalityOverview, f=clonalityOverview$Sample)
|
|
|
664 lapply(clonalityOverviewSplit, FUN=ClonalityOverviewPrint)
|
|
|
665 }
|
|
|
666 }
|
|
|
667
|
|
|
668 imgtcolumns = c("X3V.REGION.trimmed.nt.nb","P3V.nt.nb", "N1.REGION.nt.nb", "P5D.nt.nb", "X5D.REGION.trimmed.nt.nb", "X3D.REGION.trimmed.nt.nb", "P3D.nt.nb", "N2.REGION.nt.nb", "P5J.nt.nb", "X5J.REGION.trimmed.nt.nb", "X3V.REGION.trimmed.nt.nb", "X5D.REGION.trimmed.nt.nb", "X3D.REGION.trimmed.nt.nb", "X5J.REGION.trimmed.nt.nb", "N1.REGION.nt.nb", "N2.REGION.nt.nb", "P3V.nt.nb", "P5D.nt.nb", "P3D.nt.nb", "P5J.nt.nb")
|
|
|
669 if(all(imgtcolumns %in% colnames(inputdata)))
|
|
|
670 {
|
|
17
|
671 print("found IMGT columns, running junction analysis")
|
|
19
|
672
|
|
|
673 if(locus %in% c("IGK","IGL", "TRA", "TRG")){
|
|
|
674 print("VJ recombination, using N column for junction analysis")
|
|
|
675 print(names(PRODF))
|
|
|
676 print(head(PRODF$N.REGION.nt.nb, 30))
|
|
|
677 PRODF$N1.REGION.nt.nb = PRODF$N.REGION.nt.nb
|
|
|
678 }
|
|
|
679
|
|
12
|
680 newData = data.frame(data.table(PRODF)[,list(unique=.N,
|
|
|
681 VH.DEL=mean(.SD$X3V.REGION.trimmed.nt.nb, na.rm=T),
|
|
|
682 P1=mean(.SD$P3V.nt.nb, na.rm=T),
|
|
|
683 N1=mean(.SD$N1.REGION.nt.nb, na.rm=T),
|
|
|
684 P2=mean(.SD$P5D.nt.nb, na.rm=T),
|
|
|
685 DEL.DH=mean(.SD$X5D.REGION.trimmed.nt.nb, na.rm=T),
|
|
|
686 DH.DEL=mean(.SD$X3D.REGION.trimmed.nt.nb, na.rm=T),
|
|
|
687 P3=mean(.SD$P3D.nt.nb, na.rm=T),
|
|
|
688 N2=mean(.SD$N2.REGION.nt.nb, na.rm=T),
|
|
|
689 P4=mean(.SD$P5J.nt.nb, na.rm=T),
|
|
|
690 DEL.JH=mean(.SD$X5J.REGION.trimmed.nt.nb, na.rm=T),
|
|
|
691 Total.Del=( mean(.SD$X3V.REGION.trimmed.nt.nb, na.rm=T) +
|
|
|
692 mean(.SD$X5D.REGION.trimmed.nt.nb, na.rm=T) +
|
|
|
693 mean(.SD$X3D.REGION.trimmed.nt.nb, na.rm=T) +
|
|
|
694 mean(.SD$X5J.REGION.trimmed.nt.nb, na.rm=T)),
|
|
|
695
|
|
|
696 Total.N=( mean(.SD$N1.REGION.nt.nb, na.rm=T) +
|
|
|
697 mean(.SD$N2.REGION.nt.nb, na.rm=T)),
|
|
|
698
|
|
|
699 Total.P=( mean(.SD$P3V.nt.nb, na.rm=T) +
|
|
|
700 mean(.SD$P5D.nt.nb, na.rm=T) +
|
|
|
701 mean(.SD$P3D.nt.nb, na.rm=T) +
|
|
|
702 mean(.SD$P5J.nt.nb, na.rm=T))),
|
|
|
703 by=c("Sample")])
|
|
16
|
704 newData[,sapply(newData, is.numeric)] = round(newData[,sapply(newData, is.numeric)],1)
|
|
12
|
705 write.table(newData, "junctionAnalysisProd.csv" , sep=",",quote=F,na="-",row.names=F,col.names=F)
|
|
|
706
|
|
|
707 newData = data.frame(data.table(UNPROD)[,list(unique=.N,
|
|
|
708 VH.DEL=mean(.SD$X3V.REGION.trimmed.nt.nb, na.rm=T),
|
|
|
709 P1=mean(.SD$P3V.nt.nb, na.rm=T),
|
|
|
710 N1=mean(.SD$N1.REGION.nt.nb, na.rm=T),
|
|
|
711 P2=mean(.SD$P5D.nt.nb, na.rm=T),
|
|
|
712 DEL.DH=mean(.SD$X5D.REGION.trimmed.nt.nb, na.rm=T),
|
|
|
713 DH.DEL=mean(.SD$X3D.REGION.trimmed.nt.nb, na.rm=T),
|
|
|
714 P3=mean(.SD$P3D.nt.nb, na.rm=T),
|
|
|
715 N2=mean(.SD$N2.REGION.nt.nb, na.rm=T),
|
|
|
716 P4=mean(.SD$P5J.nt.nb, na.rm=T),
|
|
|
717 DEL.JH=mean(.SD$X5J.REGION.trimmed.nt.nb, na.rm=T),
|
|
17
|
718 Total.Del=(mean(.SD$X3V.REGION.trimmed.nt.nb, na.rm=T) +
|
|
|
719 mean(.SD$X5D.REGION.trimmed.nt.nb, na.rm=T) +
|
|
|
720 mean(.SD$X3D.REGION.trimmed.nt.nb, na.rm=T) +
|
|
|
721 mean(.SD$X5J.REGION.trimmed.nt.nb, na.rm=T)),
|
|
|
722 Total.N=( mean(.SD$N1.REGION.nt.nb, na.rm=T) +
|
|
|
723 mean(.SD$N2.REGION.nt.nb, na.rm=T)),
|
|
|
724 Total.P=( mean(.SD$P3V.nt.nb, na.rm=T) +
|
|
|
725 mean(.SD$P5D.nt.nb, na.rm=T) +
|
|
|
726 mean(.SD$P3D.nt.nb, na.rm=T) +
|
|
|
727 mean(.SD$P5J.nt.nb, na.rm=T))),
|
|
12
|
728 by=c("Sample")])
|
|
16
|
729 newData[,sapply(newData, is.numeric)] = round(newData[,sapply(newData, is.numeric)],1)
|
|
12
|
730 write.table(newData, "junctionAnalysisUnProd.csv" , sep=",",quote=F,na="-",row.names=F,col.names=F)
|
|
|
731 }
|
|
|
732
|
|
|
733 # ---------------------- AA composition in CDR3 ----------------------
|
|
|
734
|
|
|
735 AACDR3 = PRODF[,c("Sample", "CDR3.Seq")]
|
|
|
736
|
|
|
737 TotalPerSample = data.frame(data.table(AACDR3)[, list(total=sum(nchar(as.character(.SD$CDR3.Seq)))), by=Sample])
|
|
|
738
|
|
|
739 AAfreq = list()
|
|
|
740
|
|
|
741 for(i in 1:nrow(TotalPerSample)){
|
|
|
742 sample = TotalPerSample$Sample[i]
|
|
|
743 AAfreq[[i]] = data.frame(table(unlist(strsplit(as.character(AACDR3[AACDR3$Sample == sample,c("CDR3.Seq")]), ""))))
|
|
|
744 AAfreq[[i]]$Sample = sample
|
|
|
745 }
|
|
|
746
|
|
|
747 AAfreq = ldply(AAfreq, data.frame)
|
|
|
748 AAfreq = merge(AAfreq, TotalPerSample, by="Sample", all.x = T)
|
|
|
749 AAfreq$freq_perc = as.numeric(AAfreq$Freq / AAfreq$total * 100)
|
|
|
750
|
|
|
751
|
|
|
752 AAorder = read.table(sep="\t", header=TRUE, text="order.aa\tAA\n1\tR\n2\tK\n3\tN\n4\tD\n5\tQ\n6\tE\n7\tH\n8\tP\n9\tY\n10\tW\n11\tS\n12\tT\n13\tG\n14\tA\n15\tM\n16\tC\n17\tF\n18\tL\n19\tV\n20\tI")
|
|
|
753 AAfreq = merge(AAfreq, AAorder, by.x='Var1', by.y='AA', all.x=TRUE)
|
|
|
754
|
|
|
755 AAfreq = AAfreq[!is.na(AAfreq$order.aa),]
|
|
|
756
|
|
|
757 AAfreqplot = ggplot(AAfreq)
|
|
|
758 AAfreqplot = AAfreqplot + geom_bar(aes( x=factor(reorder(Var1, order.aa)), y = freq_perc, fill = Sample), stat='identity', position='dodge' )
|
|
|
759 AAfreqplot = AAfreqplot + annotate("rect", xmin = 0.5, xmax = 2.5, ymin = 0, ymax = Inf, fill = "red", alpha = 0.2)
|
|
|
760 AAfreqplot = AAfreqplot + annotate("rect", xmin = 3.5, xmax = 4.5, ymin = 0, ymax = Inf, fill = "blue", alpha = 0.2)
|
|
|
761 AAfreqplot = AAfreqplot + annotate("rect", xmin = 5.5, xmax = 6.5, ymin = 0, ymax = Inf, fill = "blue", alpha = 0.2)
|
|
|
762 AAfreqplot = AAfreqplot + annotate("rect", xmin = 6.5, xmax = 7.5, ymin = 0, ymax = Inf, fill = "red", alpha = 0.2)
|
|
|
763 AAfreqplot = AAfreqplot + ggtitle("Amino Acid Composition in the CDR3") + xlab("Amino Acid, from Hydrophilic (left) to Hydrophobic (right)") + ylab("Percentage")
|
|
|
764
|
|
|
765 png("AAComposition.png",width = 1280, height = 720)
|
|
|
766 AAfreqplot
|
|
|
767 dev.off()
|
|
|
768 write.table(AAfreq, "AAComposition.csv" , sep=",",quote=F,na="-",row.names=F,col.names=T)
|
|
|
769
|
|
|
770
|
