Mercurial > repos > davidvanzessen > argalaxy_tools
annotate report_clonality/RScript.r @ 58:4c6df851e262 draft default tip
"planemo upload commit 1a7731bb4fe2e80fb1fdc88121783068af3edb08"
author | rhpvorderman |
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date | Fri, 21 Apr 2023 10:04:39 +0000 |
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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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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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44 |
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46 # ---------------------- Data preperation ---------------------- |
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48 print("Report Clonality - Data preperation") |
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49 |
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50 inputdata = read.table(infile, sep="\t", header=TRUE, fill=T, comment.char="", stringsAsFactors=F) |
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51 |
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52 inputdata$Sample = as.character(inputdata$Sample) |
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53 |
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54 |
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55 print(paste("nrows: ", nrow(inputdata))) |
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56 |
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57 setwd(outdir) |
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58 |
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59 # remove weird rows |
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60 inputdata = inputdata[inputdata$Sample != "",] |
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61 |
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62 print(paste("nrows: ", nrow(inputdata))) |
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63 |
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64 #remove the allele from the V,D and J genes |
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65 inputdata$Top.V.Gene = gsub("[*]([0-9]+)", "", inputdata$Top.V.Gene) |
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66 inputdata$Top.D.Gene = gsub("[*]([0-9]+)", "", inputdata$Top.D.Gene) |
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67 inputdata$Top.J.Gene = gsub("[*]([0-9]+)", "", inputdata$Top.J.Gene) |
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68 |
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69 print(paste("nrows: ", nrow(inputdata))) |
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70 |
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71 #filter uniques |
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72 inputdata.removed = inputdata[NULL,] |
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73 |
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74 print(paste("nrows: ", nrow(inputdata))) |
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75 |
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76 inputdata$clonaltype = 1:nrow(inputdata) |
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77 |
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78 #keep track of the count of sequences in samples or samples/replicates for the front page overview |
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79 input.sample.count = data.frame(data.table(inputdata)[, list(All=.N), by=c("Sample")]) |
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80 input.rep.count = data.frame(data.table(inputdata)[, list(All=.N), by=c("Sample", "Replicate")]) |
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81 |
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82 PRODF = inputdata |
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83 UNPROD = inputdata |
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84 if(filterproductive){ |
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85 if("Functionality" %in% colnames(inputdata)) { # "Functionality" is an IMGT column |
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86 #PRODF = inputdata[inputdata$Functionality == "productive" | inputdata$Functionality == "productive (see comment)", ] |
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87 PRODF = inputdata[inputdata$Functionality %in% c("productive (see comment)","productive"),] |
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88 |
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89 PRODF.count = data.frame(data.table(PRODF)[, list(count=.N), by=c("Sample")]) |
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90 |
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91 UNPROD = inputdata[inputdata$Functionality %in% c("unproductive (see comment)","unproductive"), ] |
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92 } else { |
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93 PRODF = inputdata[inputdata$VDJ.Frame != "In-frame with stop codon" & inputdata$VDJ.Frame != "Out-of-frame" & inputdata$CDR3.Found.How != "NOT_FOUND" , ] |
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94 UNPROD = inputdata[!(inputdata$VDJ.Frame != "In-frame with stop codon" & inputdata$VDJ.Frame != "Out-of-frame" & inputdata$CDR3.Found.How != "NOT_FOUND" ), ] |
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95 } |
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96 } |
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97 |
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98 for(i in 1:nrow(UNPROD)){ |
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99 if(!is.numeric(UNPROD[i,"CDR3.Length"])){ |
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100 UNPROD[i,"CDR3.Length"] = 0 |
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101 } |
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102 } |
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103 |
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104 prod.sample.count = data.frame(data.table(PRODF)[, list(Productive=.N), by=c("Sample")]) |
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105 prod.rep.count = data.frame(data.table(PRODF)[, list(Productive=.N), by=c("Sample", "Replicate")]) |
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106 |
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107 unprod.sample.count = data.frame(data.table(UNPROD)[, list(Unproductive=.N), by=c("Sample")]) |
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108 unprod.rep.count = data.frame(data.table(UNPROD)[, list(Unproductive=.N), by=c("Sample", "Replicate")]) |
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109 |
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110 clonalityFrame = PRODF |
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111 |
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112 #remove duplicates based on the clonaltype |
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113 if(clonaltype != "none"){ |
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114 clonaltype = paste(clonaltype, ",Sample", sep="") #add sample column to clonaltype, unique within samples |
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115 PRODF$clonaltype = do.call(paste, c(PRODF[unlist(strsplit(clonaltype, ","))], sep = ":")) |
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116 PRODF = PRODF[!duplicated(PRODF$clonaltype), ] |
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117 |
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118 UNPROD$clonaltype = do.call(paste, c(UNPROD[unlist(strsplit(clonaltype, ","))], sep = ":")) |
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119 UNPROD = UNPROD[!duplicated(UNPROD$clonaltype), ] |
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120 |
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121 #again for clonalityFrame but with sample+replicate |
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122 clonalityFrame$clonaltype = do.call(paste, c(clonalityFrame[unlist(strsplit(clonaltype, ","))], sep = ":")) |
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123 clonalityFrame$clonality_clonaltype = do.call(paste, c(clonalityFrame[unlist(strsplit(paste(clonaltype, ",Replicate", sep=""), ","))], sep = ":")) |
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124 clonalityFrame = clonalityFrame[!duplicated(clonalityFrame$clonality_clonaltype), ] |
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125 } |
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126 |
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127 if(nrow(PRODF) == 0){ |
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128 stop("No sequences left after filtering") |
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129 } |
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130 |
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131 prod.unique.sample.count = data.frame(data.table(PRODF)[, list(Productive_unique=.N), by=c("Sample")]) |
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132 prod.unique.rep.count = data.frame(data.table(PRODF)[, list(Productive_unique=.N), by=c("Sample", "Replicate")]) |
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133 |
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134 unprod.unique.sample.count = data.frame(data.table(UNPROD)[, list(Unproductive_unique=.N), by=c("Sample")]) |
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135 unprod.unique.rep.count = data.frame(data.table(UNPROD)[, list(Unproductive_unique=.N), by=c("Sample", "Replicate")]) |
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136 |
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137 |
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138 PRODF$freq = 1 |
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139 |
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140 if(any(grepl(pattern="_", x=PRODF$ID))){ #the frequency can be stored in the ID with the pattern ".*_freq_.*" |
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141 PRODF$freq = gsub("^[0-9]+_", "", PRODF$ID) |
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142 PRODF$freq = gsub("_.*", "", PRODF$freq) |
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143 PRODF$freq = as.numeric(PRODF$freq) |
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144 if(any(is.na(PRODF$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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145 PRODF$freq = 1 |
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146 } |
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147 } |
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148 |
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149 #make a names list with sample -> color |
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150 naive.colors = c('blue4', 'darkred', 'olivedrab3', 'red', 'gray74', 'darkviolet', 'lightblue1', 'gold', 'chartreuse2', 'pink', 'Paleturquoise3', 'Chocolate1', 'Yellow', 'Deeppink3', 'Mediumorchid1', 'Darkgreen', 'Blue', 'Gray36', 'Hotpink', 'Yellow4') |
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151 unique.samples = unique(PRODF$Sample) |
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152 |
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153 if(length(unique.samples) <= length(naive.colors)){ |
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154 sample.colors = naive.colors[1:length(unique.samples)] |
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155 } else { |
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156 sample.colors = rainbow(length(unique.samples)) |
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157 } |
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158 |
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159 names(sample.colors) = unique.samples |
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160 |
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161 print("Sample.colors") |
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162 print(sample.colors) |
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163 |
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164 |
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165 #write the complete dataset that is left over, will be the input if 'none' for clonaltype and 'no' for filterproductive |
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166 write.table(PRODF, "allUnique.txt", sep="\t",quote=F,row.names=F,col.names=T) |
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167 #write.table(PRODF, "allUnique.csv", sep=",",quote=F,row.names=F,col.names=T) |
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168 write.table(UNPROD, "allUnproductive.txt", sep="\t",quote=F,row.names=F,col.names=T) |
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169 |
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170 print("SAMPLE TABLE:") |
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171 print(table(PRODF$Sample)) |
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172 |
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173 #write the samples to a file |
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174 sampleFile <- file("samples.txt") |
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175 un = unique(inputdata$Sample) |
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176 un = paste(un, sep="\n") |
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177 writeLines(un, sampleFile) |
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178 close(sampleFile) |
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179 |
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180 # ---------------------- Counting the productive/unproductive and unique sequences ---------------------- |
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181 |
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182 print("Report Clonality - counting productive/unproductive/unique") |
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183 |
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184 #create the table on the overview page with the productive/unique counts per sample/replicate |
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185 #first for sample |
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186 |
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187 sample.count = merge(input.sample.count, prod.sample.count, by="Sample", all.x=T) |
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188 sample.count$perc_prod = round(sample.count$Productive / sample.count$All * 100) |
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189 sample.count = merge(sample.count, prod.unique.sample.count, by="Sample", all.x=T) |
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190 sample.count$perc_prod_un = round(sample.count$Productive_unique / sample.count$All * 100) |
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191 |
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192 sample.count = merge(sample.count , unprod.sample.count, by="Sample", all.x=T) |
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193 sample.count$perc_unprod = round(sample.count$Unproductive / sample.count$All * 100) |
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194 sample.count = merge(sample.count, unprod.unique.sample.count, by="Sample", all.x=T) |
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195 sample.count$perc_unprod_un = round(sample.count$Unproductive_unique / sample.count$All * 100) |
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196 |
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197 #then sample/replicate |
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198 rep.count = merge(input.rep.count, prod.rep.count, by=c("Sample", "Replicate"), all.x=T) |
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199 |
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200 print(rep.count) |
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201 |
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202 fltr = is.na(rep.count$Productive) |
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203 if(any(fltr)){ |
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204 rep.count[fltr,"Productive"] = 0 |
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205 } |
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206 |
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207 print(rep.count) |
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208 |
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209 rep.count$perc_prod = round(rep.count$Productive / rep.count$All * 100) |
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210 rep.count = merge(rep.count, prod.unique.rep.count, by=c("Sample", "Replicate"), all.x=T) |
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211 rep.count$perc_prod_un = round(rep.count$Productive_unique / rep.count$All * 100) |
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212 |
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213 rep.count = merge(rep.count, unprod.rep.count, by=c("Sample", "Replicate"), all.x=T) |
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214 rep.count$perc_unprod = round(rep.count$Unproductive / rep.count$All * 100) |
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215 rep.count = merge(rep.count, unprod.unique.rep.count, by=c("Sample", "Replicate"), all.x=T) |
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216 rep.count$perc_unprod_un = round(rep.count$Unproductive_unique / rep.count$All * 100) |
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217 |
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218 rep.count$Sample = paste(rep.count$Sample, rep.count$Replicate, sep="_") |
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219 rep.count = rep.count[,names(rep.count) != "Replicate"] |
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220 |
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221 count = rbind(sample.count, rep.count) |
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222 |
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223 |
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224 |
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225 write.table(x=count, file="productive_counting.txt", sep=",",quote=F,row.names=F,col.names=F) |
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226 |
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227 # ---------------------- V+J+CDR3 sequence count ---------------------- |
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228 |
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229 VJCDR3.count = data.frame(table(clonalityFrame$Top.V.Gene, clonalityFrame$Top.J.Gene, clonalityFrame$CDR3.Seq.DNA)) |
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230 names(VJCDR3.count) = c("Top.V.Gene", "Top.J.Gene", "CDR3.Seq.DNA", "Count") |
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231 |
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232 VJCDR3.count = VJCDR3.count[VJCDR3.count$Count > 0,] |
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233 VJCDR3.count = VJCDR3.count[order(-VJCDR3.count$Count),] |
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234 |
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235 write.table(x=VJCDR3.count, file="VJCDR3_count.txt", sep="\t",quote=F,row.names=F,col.names=T) |
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236 |
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237 # ---------------------- Frequency calculation for V, D and J ---------------------- |
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238 |
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239 print("Report Clonality - frequency calculation V, D and J") |
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240 |
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241 PRODFV = data.frame(data.table(PRODF)[, list(Length=sum(freq)), by=c("Sample", "Top.V.Gene")]) |
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242 Total = ddply(PRODFV, .(Sample), function(x) data.frame(Total = sum(x$Length))) |
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243 PRODFV = merge(PRODFV, Total, by.x='Sample', by.y='Sample', all.x=TRUE) |
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244 PRODFV = ddply(PRODFV, c("Sample", "Top.V.Gene"), summarise, relFreq= (Length*100 / Total)) |
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245 |
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246 PRODFD = data.frame(data.table(PRODF)[, list(Length=sum(freq)), by=c("Sample", "Top.D.Gene")]) |
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247 Total = ddply(PRODFD, .(Sample), function(x) data.frame(Total = sum(x$Length))) |
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248 PRODFD = merge(PRODFD, Total, by.x='Sample', by.y='Sample', all.x=TRUE) |
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249 PRODFD = ddply(PRODFD, c("Sample", "Top.D.Gene"), summarise, relFreq= (Length*100 / Total)) |
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250 |
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251 PRODFJ = data.frame(data.table(PRODF)[, list(Length=sum(freq)), by=c("Sample", "Top.J.Gene")]) |
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252 Total = ddply(PRODFJ, .(Sample), function(x) data.frame(Total = sum(x$Length))) |
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253 PRODFJ = merge(PRODFJ, Total, by.x='Sample', by.y='Sample', all.x=TRUE) |
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254 PRODFJ = ddply(PRODFJ, c("Sample", "Top.J.Gene"), summarise, relFreq= (Length*100 / Total)) |
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255 |
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256 # ---------------------- Setting up the gene names for the different species/loci ---------------------- |
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257 |
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258 print("Report Clonality - getting genes for species/loci") |
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259 |
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260 Vchain = "" |
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261 Dchain = "" |
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262 Jchain = "" |
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263 |
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264 if(species == "custom"){ |
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265 print("Custom genes: ") |
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266 splt = unlist(strsplit(locus, ";")) |
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267 print(paste("V:", splt[1])) |
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268 print(paste("D:", splt[2])) |
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269 print(paste("J:", splt[3])) |
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270 |
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271 Vchain = unlist(strsplit(splt[1], ",")) |
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272 Vchain = data.frame(v.name = Vchain, chr.orderV = 1:length(Vchain)) |
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273 |
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274 Dchain = unlist(strsplit(splt[2], ",")) |
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275 if(length(Dchain) > 0){ |
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276 Dchain = data.frame(v.name = Dchain, chr.orderD = 1:length(Dchain)) |
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277 } else { |
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278 Dchain = data.frame(v.name = character(0), chr.orderD = numeric(0)) |
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279 } |
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280 |
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281 Jchain = unlist(strsplit(splt[3], ",")) |
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282 Jchain = data.frame(v.name = Jchain, chr.orderJ = 1:length(Jchain)) |
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283 |
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284 } else { |
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285 genes = read.table("genes.txt", sep="\t", header=TRUE, fill=T, comment.char="") |
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286 |
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287 Vchain = genes[grepl(species, genes$Species) & genes$locus == locus & genes$region == "V",c("IMGT.GENE.DB", "chr.order")] |
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288 colnames(Vchain) = c("v.name", "chr.orderV") |
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289 Dchain = genes[grepl(species, genes$Species) & genes$locus == locus & genes$region == "D",c("IMGT.GENE.DB", "chr.order")] |
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290 colnames(Dchain) = c("v.name", "chr.orderD") |
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291 Jchain = genes[grepl(species, genes$Species) & genes$locus == locus & genes$region == "J",c("IMGT.GENE.DB", "chr.order")] |
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292 colnames(Jchain) = c("v.name", "chr.orderJ") |
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293 } |
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294 useD = TRUE |
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295 if(nrow(Dchain) == 0){ |
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296 useD = FALSE |
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297 cat("No D Genes in this species/locus") |
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298 } |
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299 print(paste(nrow(Vchain), "genes in V")) |
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300 print(paste(nrow(Dchain), "genes in D")) |
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301 print(paste(nrow(Jchain), "genes in J")) |
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302 |
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303 # ---------------------- merge with the frequency count ---------------------- |
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304 |
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305 PRODFV = merge(PRODFV, Vchain, by.x='Top.V.Gene', by.y='v.name', all.x=TRUE) |
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306 |
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307 PRODFD = merge(PRODFD, Dchain, by.x='Top.D.Gene', by.y='v.name', all.x=TRUE) |
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308 |
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309 PRODFJ = merge(PRODFJ, Jchain, by.x='Top.J.Gene', by.y='v.name', all.x=TRUE) |
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310 |
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311 # ---------------------- Create the V, D and J frequency plots and write the data.frame for every plot to a file ---------------------- |
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312 |
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313 print("Report Clonality - V, D and J frequency plots") |
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314 |
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315 pV = ggplot(PRODFV) |
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316 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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317 pV = pV + xlab("Summary of V gene") + ylab("Frequency") + ggtitle("Relative frequency of V gene usage") + scale_fill_manual(values=sample.colors) |
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318 pV = pV + theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), axis.text.x = element_text(angle = 45, hjust = 1), panel.grid.major.y = element_line(colour = "black"), panel.grid.major.x = element_blank()) |
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319 write.table(x=PRODFV, file="VFrequency.txt", sep="\t",quote=F,row.names=F,col.names=T) |
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320 |
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321 png("VPlot.png",width = 1280, height = 720) |
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322 pV |
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323 dev.off() |
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324 |
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325 ggsave("VPlot.pdf", pV, width=13, height=7) |
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326 |
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327 if(useD){ |
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328 pD = ggplot(PRODFD) |
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329 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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330 pD = pD + xlab("Summary of D gene") + ylab("Frequency") + ggtitle("Relative frequency of D gene usage") + scale_fill_manual(values=sample.colors) |
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331 pD = pD + theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), axis.text.x = element_text(angle = 45, hjust = 1), panel.grid.major.y = element_line(colour = "black"), panel.grid.major.x = element_blank()) |
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332 write.table(x=PRODFD, file="DFrequency.txt", sep="\t",quote=F,row.names=F,col.names=T) |
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333 |
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334 png("DPlot.png",width = 800, height = 600) |
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335 print(pD) |
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336 dev.off() |
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337 |
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338 ggsave("DPlot.pdf", pD, width=10, height=7) |
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339 } |
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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") + scale_fill_manual(values=sample.colors) |
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344 pJ = pJ + theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), axis.text.x = element_text(angle = 45, hjust = 1), panel.grid.major.y = element_line(colour = "black"), panel.grid.major.x = element_blank()) |
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345 write.table(x=PRODFJ, file="JFrequency.txt", sep="\t",quote=F,row.names=F,col.names=T) |
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346 |
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347 png("JPlot.png",width = 800, height = 600) |
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348 pJ |
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349 dev.off() |
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350 |
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351 ggsave("JPlot.pdf", pJ) |
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352 |
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353 # ---------------------- Now the frequency plots of the V, D and J families ---------------------- |
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354 |
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355 print("Report Clonality - V, D and J family plots") |
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356 |
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357 VGenes = PRODF[,c("Sample", "Top.V.Gene")] |
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358 VGenes$Top.V.Gene = gsub("-.*", "", VGenes$Top.V.Gene) |
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359 VGenes = data.frame(data.table(VGenes)[, list(Count=.N), by=c("Sample", "Top.V.Gene")]) |
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360 TotalPerSample = data.frame(data.table(VGenes)[, list(total=sum(.SD$Count)), by=Sample]) |
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361 VGenes = merge(VGenes, TotalPerSample, by="Sample") |
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362 VGenes$Frequency = VGenes$Count * 100 / VGenes$total |
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363 VPlot = ggplot(VGenes) |
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364 VPlot = VPlot + geom_bar(aes( x = Top.V.Gene, y = Frequency, fill = Sample), stat='identity', position='dodge' ) + theme(axis.text.x = element_text(angle = 90, hjust = 1)) + |
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365 ggtitle("Distribution of V gene families") + |
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366 ylab("Percentage of sequences") + |
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367 scale_fill_manual(values=sample.colors) + |
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368 theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), axis.text.x = element_text(angle = 45, hjust = 1), panel.grid.major.y = element_line(colour = "black"), panel.grid.major.x = element_blank()) |
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369 png("VFPlot.png") |
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370 VPlot |
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371 dev.off() |
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372 ggsave("VFPlot.pdf", VPlot) |
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373 |
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374 write.table(x=VGenes, file="VFFrequency.txt", sep="\t",quote=F,row.names=F,col.names=T) |
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375 |
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376 if(useD){ |
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377 DGenes = PRODF[,c("Sample", "Top.D.Gene")] |
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378 DGenes$Top.D.Gene = gsub("-.*", "", DGenes$Top.D.Gene) |
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379 DGenes = data.frame(data.table(DGenes)[, list(Count=.N), by=c("Sample", "Top.D.Gene")]) |
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380 TotalPerSample = data.frame(data.table(DGenes)[, list(total=sum(.SD$Count)), by=Sample]) |
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381 DGenes = merge(DGenes, TotalPerSample, by="Sample") |
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382 DGenes$Frequency = DGenes$Count * 100 / DGenes$total |
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383 DPlot = ggplot(DGenes) |
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384 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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385 ggtitle("Distribution of D gene families") + |
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386 ylab("Percentage of sequences") + |
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387 scale_fill_manual(values=sample.colors) + |
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388 theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), axis.text.x = element_text(angle = 45, hjust = 1), panel.grid.major.y = element_line(colour = "black"), panel.grid.major.x = element_blank()) |
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389 png("DFPlot.png") |
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390 print(DPlot) |
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391 dev.off() |
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392 |
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393 ggsave("DFPlot.pdf", DPlot) |
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394 write.table(x=DGenes, file="DFFrequency.txt", sep="\t",quote=F,row.names=F,col.names=T) |
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395 } |
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396 |
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397 # ---------------------- Plotting the cdr3 length ---------------------- |
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398 |
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399 print("Report Clonality - CDR3 length plot") |
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400 |
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401 CDR3Length = data.frame(data.table(PRODF)[, list(Count=.N), by=c("Sample", "CDR3.Length")]) |
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402 TotalPerSample = data.frame(data.table(CDR3Length)[, list(total=sum(.SD$Count)), by=Sample]) |
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403 CDR3Length = merge(CDR3Length, TotalPerSample, by="Sample") |
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404 CDR3Length$Frequency = CDR3Length$Count * 100 / CDR3Length$total |
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405 CDR3LengthPlot = ggplot(CDR3Length) |
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406 CDR3LengthPlot = CDR3LengthPlot + geom_bar(aes( x = factor(reorder(CDR3.Length, as.numeric(CDR3.Length))), y = Frequency, fill = Sample), stat='identity', position='dodge' ) + theme(axis.text.x = element_text(angle = 90, hjust = 1)) + |
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407 ggtitle("Length distribution of CDR3") + |
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408 xlab("CDR3 Length") + |
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409 ylab("Percentage of sequences") + |
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410 scale_fill_manual(values=sample.colors) + |
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411 theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), axis.text.x = element_text(angle = 45, hjust = 1), panel.grid.major.y = element_line(colour = "black"), panel.grid.major.x = element_blank()) |
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412 png("CDR3LengthPlot.png",width = 1280, height = 720) |
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413 CDR3LengthPlot |
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414 dev.off() |
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415 |
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416 ggsave("CDR3LengthPlot.pdf", CDR3LengthPlot, width=12, height=7) |
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417 |
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418 write.table(x=CDR3Length, file="CDR3LengthPlot.txt", sep="\t",quote=F,row.names=F,col.names=T) |
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419 |
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420 # ---------------------- Plot the heatmaps ---------------------- |
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421 |
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422 #get the reverse order for the V and D genes |
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423 revVchain = Vchain |
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424 revDchain = Dchain |
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425 revVchain$chr.orderV = rev(revVchain$chr.orderV) |
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426 revDchain$chr.orderD = rev(revDchain$chr.orderD) |
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427 |
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428 if(useD){ |
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429 print("Report Clonality - Heatmaps VD") |
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430 plotVD <- function(dat){ |
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431 if(length(dat[,1]) == 0){ |
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432 return() |
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433 } |
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434 |
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435 img = ggplot() + |
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436 geom_tile(data=dat, aes(x=factor(reorder(Top.D.Gene, chr.orderD)), y=factor(reorder(Top.V.Gene, chr.orderV)), fill=relLength)) + |
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437 theme(axis.text.x = element_text(angle = 90, hjust = 1)) + |
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438 scale_fill_gradient(low="gold", high="blue", na.value="white") + |
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439 ggtitle(paste(unique(dat$Sample), " (N=" , sum(dat$Length, na.rm=T) ,")", sep="")) + |
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440 xlab("D genes") + |
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441 ylab("V Genes") + |
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442 theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), panel.grid.major = element_line(colour = "gainsboro")) |
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443 |
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444 png(paste("HeatmapVD_", unique(dat[3])[1,1] , ".png", sep=""), width=200+(15*length(Dchain$v.name)), height=100+(15*length(Vchain$v.name))) |
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445 print(img) |
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446 dev.off() |
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447 |
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448 ggsave(paste("HeatmapVD_", unique(dat[3])[1,1] , ".pdf", sep=""), img, height=13, width=8) |
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449 |
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450 write.table(x=acast(dat, Top.V.Gene~Top.D.Gene, value.var="Length"), file=paste("HeatmapVD_", unique(dat[3])[1,1], ".txt", sep=""), sep="\t",quote=F,row.names=T,col.names=NA) |
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451 } |
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452 |
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453 VandDCount = data.frame(data.table(PRODF)[, list(Length=.N), by=c("Top.V.Gene", "Top.D.Gene", "Sample")]) |
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454 |
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455 VandDCount$l = log(VandDCount$Length) |
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456 maxVD = data.frame(data.table(VandDCount)[, list(max=max(l)), by=c("Sample")]) |
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457 VandDCount = merge(VandDCount, maxVD, by.x="Sample", by.y="Sample", all.x=T) |
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458 VandDCount$relLength = VandDCount$l / VandDCount$max |
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459 check = is.nan(VandDCount$relLength) |
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460 if(any(check)){ |
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461 VandDCount[check,"relLength"] = 0 |
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462 } |
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463 |
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464 completeVD = merge(VandDCount, revVchain, by.x="Top.V.Gene", by.y="v.name", all=TRUE) |
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465 completeVD = merge(completeVD, Dchain, by.x="Top.D.Gene", by.y="v.name", all=TRUE) |
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466 |
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467 fltr = is.nan(completeVD$relLength) |
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468 if(all(fltr)){ |
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469 completeVD[fltr,"relLength"] = 0 |
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470 } |
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471 |
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472 VDList = split(completeVD, f=completeVD[,"Sample"]) |
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473 lapply(VDList, FUN=plotVD) |
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474 } |
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475 |
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476 print("Report Clonality - Heatmaps VJ") |
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477 |
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478 plotVJ <- function(dat){ |
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479 if(length(dat[,1]) == 0){ |
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480 return() |
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481 } |
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482 cat(paste(unique(dat[3])[1,1])) |
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483 img = ggplot() + |
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484 geom_tile(data=dat, aes(x=factor(reorder(Top.J.Gene, chr.orderJ)), y=factor(reorder(Top.V.Gene, chr.orderV)), fill=relLength)) + |
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485 theme(axis.text.x = element_text(angle = 90, hjust = 1)) + |
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486 scale_fill_gradient(low="gold", high="blue", na.value="white") + |
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487 ggtitle(paste(unique(dat$Sample), " (N=" , sum(dat$Length, na.rm=T) ,")", sep="")) + |
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488 xlab("J genes") + |
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489 ylab("V Genes") + |
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490 theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), panel.grid.major = element_line(colour = "gainsboro")) |
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491 |
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492 png(paste("HeatmapVJ_", unique(dat[3])[1,1] , ".png", sep=""), width=200+(15*length(Jchain$v.name)), height=100+(15*length(Vchain$v.name))) |
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493 print(img) |
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494 dev.off() |
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495 |
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496 ggsave(paste("HeatmapVJ_", unique(dat[3])[1,1] , ".pdf", sep=""), img, height=11, width=4) |
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497 |
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498 write.table(x=acast(dat, Top.V.Gene~Top.J.Gene, value.var="Length"), file=paste("HeatmapVJ_", unique(dat[3])[1,1], ".txt", sep=""), sep="\t",quote=F,row.names=T,col.names=NA) |
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499 } |
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500 |
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501 |
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502 |
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503 VandJCount = data.frame(data.table(PRODF)[, list(Length=.N), by=c("Top.V.Gene", "Top.J.Gene", "Sample")]) |
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504 |
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505 VandJCount$l = log(VandJCount$Length) |
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506 maxVJ = data.frame(data.table(VandJCount)[, list(max=max(l)), by=c("Sample")]) |
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507 VandJCount = merge(VandJCount, maxVJ, by.x="Sample", by.y="Sample", all.x=T) |
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508 VandJCount$relLength = VandJCount$l / VandJCount$max |
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509 |
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510 check = is.nan(VandJCount$relLength) |
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511 if(any(check)){ |
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512 VandJCount[check,"relLength"] = 0 |
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513 } |
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514 |
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515 completeVJ = merge(VandJCount, revVchain, by.x="Top.V.Gene", by.y="v.name", all=TRUE) |
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516 completeVJ = merge(completeVJ, Jchain, by.x="Top.J.Gene", by.y="v.name", all=TRUE) |
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517 |
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518 fltr = is.nan(completeVJ$relLength) |
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519 if(any(fltr)){ |
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520 completeVJ[fltr,"relLength"] = 1 |
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521 } |
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522 |
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523 VJList = split(completeVJ, f=completeVJ[,"Sample"]) |
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524 lapply(VJList, FUN=plotVJ) |
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525 |
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526 |
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527 |
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528 if(useD){ |
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529 print("Report Clonality - Heatmaps DJ") |
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530 plotDJ <- function(dat){ |
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531 if(length(dat[,1]) == 0){ |
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532 return() |
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533 } |
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534 img = ggplot() + |
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535 geom_tile(data=dat, aes(x=factor(reorder(Top.J.Gene, chr.orderJ)), y=factor(reorder(Top.D.Gene, chr.orderD)), fill=relLength)) + |
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536 theme(axis.text.x = element_text(angle = 90, hjust = 1)) + |
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537 scale_fill_gradient(low="gold", high="blue", na.value="white") + |
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538 ggtitle(paste(unique(dat$Sample), " (N=" , sum(dat$Length, na.rm=T) ,")", sep="")) + |
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539 xlab("J genes") + |
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540 ylab("D Genes") + |
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541 theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), panel.grid.major = element_line(colour = "gainsboro")) |
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542 |
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543 png(paste("HeatmapDJ_", unique(dat[3])[1,1] , ".png", sep=""), width=200+(15*length(Jchain$v.name)), height=100+(15*length(Dchain$v.name))) |
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544 print(img) |
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545 dev.off() |
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546 |
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547 ggsave(paste("HeatmapDJ_", unique(dat[3])[1,1] , ".pdf", sep=""), img, width=4, height=7) |
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548 |
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549 write.table(x=acast(dat, Top.D.Gene~Top.J.Gene, value.var="Length"), file=paste("HeatmapDJ_", unique(dat[3])[1,1], ".txt", sep=""), sep="\t",quote=F,row.names=T,col.names=NA) |
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550 } |
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551 |
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552 |
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553 DandJCount = data.frame(data.table(PRODF)[, list(Length=.N), by=c("Top.D.Gene", "Top.J.Gene", "Sample")]) |
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554 |
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555 DandJCount$l = log(DandJCount$Length) |
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556 maxDJ = data.frame(data.table(DandJCount)[, list(max=max(l)), by=c("Sample")]) |
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557 DandJCount = merge(DandJCount, maxDJ, by.x="Sample", by.y="Sample", all.x=T) |
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558 DandJCount$relLength = DandJCount$l / DandJCount$max |
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559 |
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560 check = is.nan(DandJCount$relLength) |
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561 if(any(check)){ |
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562 DandJCount[check,"relLength"] = 0 |
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563 } |
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564 |
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565 cartegianProductDJ = expand.grid(Top.D.Gene = Dchain$v.name, Top.J.Gene = Jchain$v.name) |
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566 |
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567 completeDJ = merge(DandJCount, revDchain, by.x="Top.D.Gene", by.y="v.name", all=TRUE) |
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568 completeDJ = merge(completeDJ, Jchain, by.x="Top.J.Gene", by.y="v.name", all=TRUE) |
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569 |
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570 fltr = is.nan(completeDJ$relLength) |
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571 if(any(fltr)){ |
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572 completeDJ[fltr, "relLength"] = 1 |
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573 } |
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574 |
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575 DJList = split(completeDJ, f=completeDJ[,"Sample"]) |
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576 lapply(DJList, FUN=plotDJ) |
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577 } |
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578 |
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579 |
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580 # ---------------------- output tables for the circos plots ---------------------- |
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581 |
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582 print("Report Clonality - Circos data") |
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583 |
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584 for(smpl in unique(PRODF$Sample)){ |
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585 PRODF.sample = PRODF[PRODF$Sample == smpl,] |
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586 |
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587 fltr = PRODF.sample$Top.V.Gene == "" |
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588 if(any(fltr, na.rm=T)){ |
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589 PRODF.sample[fltr, "Top.V.Gene"] = "NA" |
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590 } |
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591 |
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592 fltr = PRODF.sample$Top.D.Gene == "" |
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593 if(any(fltr, na.rm=T)){ |
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594 PRODF.sample[fltr, "Top.D.Gene"] = "NA" |
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595 } |
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596 |
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597 fltr = PRODF.sample$Top.J.Gene == "" |
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598 if(any(fltr, na.rm=T)){ |
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599 PRODF.sample[fltr, "Top.J.Gene"] = "NA" |
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600 } |
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601 |
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602 v.d = table(PRODF.sample$Top.V.Gene, PRODF.sample$Top.D.Gene) |
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603 v.j = table(PRODF.sample$Top.V.Gene, PRODF.sample$Top.J.Gene) |
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604 d.j = table(PRODF.sample$Top.D.Gene, PRODF.sample$Top.J.Gene) |
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605 |
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606 write.table(v.d, file=paste(smpl, "_VD_circos.txt", sep=""), sep="\t", quote=F, row.names=T, col.names=NA) |
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607 write.table(v.j, file=paste(smpl, "_VJ_circos.txt", sep=""), sep="\t", quote=F, row.names=T, col.names=NA) |
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608 write.table(d.j, file=paste(smpl, "_DJ_circos.txt", sep=""), sep="\t", quote=F, row.names=T, col.names=NA) |
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609 } |
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610 |
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611 # ---------------------- calculating the clonality score ---------------------- |
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612 |
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613 if("Replicate" %in% colnames(inputdata)) #can only calculate clonality score when replicate information is available |
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614 { |
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615 print("Report Clonality - Clonality") |
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616 write.table(clonalityFrame, "clonalityComplete.txt", sep="\t",quote=F,row.names=F,col.names=T) |
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617 if(clonality_method == "boyd"){ |
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618 samples = split(clonalityFrame, clonalityFrame$Sample, drop=T) |
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619 |
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620 for (sample in samples){ |
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621 res = data.frame(paste=character(0)) |
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622 sample_id = unique(sample$Sample)[[1]] |
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623 for(replicate in unique(sample$Replicate)){ |
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624 tmp = sample[sample$Replicate == replicate,] |
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625 clone_table = data.frame(table(tmp$clonaltype)) |
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626 clone_col_name = paste("V", replicate, sep="") |
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627 colnames(clone_table) = c("paste", clone_col_name) |
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628 res = merge(res, clone_table, by="paste", all=T) |
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629 } |
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630 |
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631 res[is.na(res)] = 0 |
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632 |
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633 write.table(res, file=paste("raw_clonality_", sample_id, ".txt", sep=""), sep="\t",quote=F,row.names=F,col.names=F) |
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634 write.table(as.matrix(res[,2:ncol(res)]), file=paste("raw_clonality2_", sample_id, ".txt", sep=""), sep="\t",quote=F,row.names=F,col.names=F) |
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635 |
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636 res = read.table(paste("raw_clonality_", sample_id, ".txt", sep=""), header=F, sep="\t", quote="", stringsAsFactors=F, fill=T, comment.char="") |
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637 |
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638 infer.result = infer.clonality(as.matrix(res[,2:ncol(res)])) |
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639 |
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640 #print(infer.result) |
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641 |
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642 write.table(data.table(infer.result[[12]]), file=paste("lymphclon_clonality_", sample_id, ".txt", sep=""), sep="\t",quote=F,row.names=F,col.names=F) |
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643 |
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644 res$type = rowSums(res[,2:ncol(res)]) |
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645 |
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646 coincidence.table = data.frame(table(res$type)) |
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647 colnames(coincidence.table) = c("Coincidence Type", "Raw Coincidence Freq") |
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648 write.table(coincidence.table, file=paste("lymphclon_coincidences_", sample_id, ".txt", sep=""), sep="\t",quote=F,row.names=F,col.names=T) |
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649 } |
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650 } |
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651 clonalFreq = data.frame(data.table(clonalityFrame)[, list(Type=.N), by=c("Sample", "clonaltype")]) |
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652 |
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653 #write files for every coincidence group of >1 |
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654 samples = unique(clonalFreq$Sample) |
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655 for(sample in samples){ |
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656 clonalFreqSample = clonalFreq[clonalFreq$Sample == sample,] |
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657 if(max(clonalFreqSample$Type) > 1){ |
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658 for(i in 2:max(clonalFreqSample$Type)){ |
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659 clonalFreqSampleType = clonalFreqSample[clonalFreqSample$Type == i,] |
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660 clonalityFrame.sub = clonalityFrame[clonalityFrame$clonaltype %in% clonalFreqSampleType$clonaltype,] |
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661 clonalityFrame.sub = clonalityFrame.sub[order(clonalityFrame.sub$clonaltype),] |
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662 write.table(clonalityFrame.sub, file=paste("coincidences_", sample, "_", i, ".txt", sep=""), sep="\t",quote=F,row.names=F,col.names=T) |
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663 } |
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664 } |
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665 } |
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666 |
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667 clonalFreqCount = data.frame(data.table(clonalFreq)[, list(Count=.N), by=c("Sample", "Type")]) |
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668 clonalFreqCount$realCount = clonalFreqCount$Type * clonalFreqCount$Count |
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669 clonalSum = data.frame(data.table(clonalFreqCount)[, list(Reads=sum(realCount)), by=c("Sample")]) |
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670 clonalFreqCount = merge(clonalFreqCount, clonalSum, by.x="Sample", by.y="Sample") |
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671 |
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672 ct = c('Type\tWeight\n2\t1\n3\t3\n4\t6\n5\t10\n6\t15') |
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673 tcct = textConnection(ct) |
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674 CT = read.table(tcct, sep="\t", header=TRUE) |
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675 close(tcct) |
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676 clonalFreqCount = merge(clonalFreqCount, CT, by.x="Type", by.y="Type", all.x=T) |
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677 clonalFreqCount$WeightedCount = clonalFreqCount$Count * clonalFreqCount$Weight |
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678 |
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679 ReplicateReads = data.frame(data.table(clonalityFrame)[, list(Type=.N), by=c("Sample", "Replicate", "clonaltype")]) |
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680 ReplicateReads = data.frame(data.table(ReplicateReads)[, list(Reads=.N), by=c("Sample", "Replicate")]) |
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681 clonalFreqCount$Reads = as.numeric(clonalFreqCount$Reads) |
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682 ReplicateReads$Reads = as.numeric(ReplicateReads$Reads) |
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683 ReplicateReads$squared = as.numeric(ReplicateReads$Reads * ReplicateReads$Reads) |
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684 |
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685 ReplicatePrint <- function(dat){ |
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686 write.table(dat[-1], paste("ReplicateReads_", unique(dat[1])[1,1] , ".txt", sep=""), sep="\t",quote=F,na="-",row.names=F,col.names=F) |
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687 } |
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688 |
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689 ReplicateSplit = split(ReplicateReads, f=ReplicateReads[,"Sample"]) |
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690 lapply(ReplicateSplit, FUN=ReplicatePrint) |
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691 |
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692 ReplicateReads = data.frame(data.table(ReplicateReads)[, list(ReadsSum=sum(as.numeric(Reads)), ReadsSquaredSum=sum(as.numeric(squared))), by=c("Sample")]) |
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693 clonalFreqCount = merge(clonalFreqCount, ReplicateReads, by.x="Sample", by.y="Sample", all.x=T) |
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694 |
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695 ReplicateSumPrint <- function(dat){ |
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696 write.table(dat[-1], paste("ReplicateSumReads_", unique(dat[1])[1,1] , ".txt", sep=""), sep="\t",quote=F,na="-",row.names=F,col.names=F) |
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697 } |
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698 |
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699 ReplicateSumSplit = split(ReplicateReads, f=ReplicateReads[,"Sample"]) |
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700 lapply(ReplicateSumSplit, FUN=ReplicateSumPrint) |
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701 |
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702 clonalFreqCountSum = data.frame(data.table(clonalFreqCount)[, list(Numerator=sum(WeightedCount, na.rm=T)), by=c("Sample")]) |
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703 clonalFreqCount = merge(clonalFreqCount, clonalFreqCountSum, by.x="Sample", by.y="Sample", all.x=T) |
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704 clonalFreqCount$ReadsSum = as.numeric(clonalFreqCount$ReadsSum) #prevent integer overflow |
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705 clonalFreqCount$Denominator = (((clonalFreqCount$ReadsSum * clonalFreqCount$ReadsSum) - clonalFreqCount$ReadsSquaredSum) / 2) |
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706 clonalFreqCount$Result = (clonalFreqCount$Numerator + 1) / (clonalFreqCount$Denominator + 1) |
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707 |
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708 ClonalityScorePrint <- function(dat){ |
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709 write.table(dat$Result, paste("ClonalityScore_", unique(dat[1])[1,1] , ".txt", sep=""), sep="\t",quote=F,na="-",row.names=F,col.names=F) |
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710 } |
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711 |
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712 clonalityScore = clonalFreqCount[c("Sample", "Result")] |
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713 clonalityScore = unique(clonalityScore) |
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714 |
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715 clonalityScoreSplit = split(clonalityScore, f=clonalityScore[,"Sample"]) |
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716 lapply(clonalityScoreSplit, FUN=ClonalityScorePrint) |
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717 |
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718 clonalityOverview = clonalFreqCount[c("Sample", "Type", "Count", "Weight", "WeightedCount")] |
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719 |
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720 |
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721 |
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722 ClonalityOverviewPrint <- function(dat){ |
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723 dat = dat[order(dat[,2]),] |
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724 write.table(dat[-1], paste("ClonalityOverView_", unique(dat[1])[1,1] , ".txt", sep=""), sep="\t",quote=F,na="-",row.names=F,col.names=F) |
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725 } |
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726 |
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727 clonalityOverviewSplit = split(clonalityOverview, f=clonalityOverview$Sample) |
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728 lapply(clonalityOverviewSplit, FUN=ClonalityOverviewPrint) |
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729 |
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730 } |
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731 |
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732 bak = PRODF |
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733 bakun = UNPROD |
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734 |
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735 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") |
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736 if(all(imgtcolumns %in% colnames(inputdata))) |
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737 { |
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738 print("found IMGT columns, running junction analysis") |
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739 |
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740 #ensure certain columns are in the data (files generated with older versions of IMGT Loader) |
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741 col.checks = c("N.REGION.nt.nb", "N1.REGION.nt.nb", "N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb") |
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742 for(col.check in col.checks){ |
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743 if(!(col.check %in% names(PRODF))){ |
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744 print(paste(col.check, "not found adding new column")) |
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745 if(nrow(PRODF) > 0){ #because R is anoying... |
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746 PRODF[,col.check] = 0 |
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747 } else { |
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748 PRODF = cbind(PRODF, data.frame(N3.REGION.nt.nb=numeric(0), N4.REGION.nt.nb=numeric(0))) |
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749 } |
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750 if(nrow(UNPROD) > 0){ |
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751 UNPROD[,col.check] = 0 |
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752 } else { |
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753 UNPROD = cbind(UNPROD, data.frame(N3.REGION.nt.nb=numeric(0), N4.REGION.nt.nb=numeric(0))) |
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754 } |
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755 } |
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756 } |
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757 |
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758 PRODF.with.D = PRODF[nchar(PRODF$Top.D.Gene, keepNA=F) > 2,] |
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759 PRODF.no.D = PRODF[nchar(PRODF$Top.D.Gene, keepNA=F) < 4,] |
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760 write.table(PRODF.no.D, "productive_no_D.txt" , sep="\t",quote=F,na="-",row.names=F,col.names=T) |
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761 |
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762 UNPROD.with.D = UNPROD[nchar(UNPROD$Top.D.Gene, keepNA=F) > 2,] |
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763 UNPROD.no.D = UNPROD[nchar(UNPROD$Top.D.Gene, keepNA=F) < 4,] |
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764 write.table(UNPROD.no.D, "unproductive_no_D.txt" , sep="\t",quote=F,na="-",row.names=F,col.names=T) |
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765 |
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766 num_median = function(x, na.rm=T) { as.numeric(median(x, na.rm=na.rm)) } |
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767 |
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768 newData = data.frame(data.table(PRODF.with.D)[,list(unique=.N, |
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769 VH.DEL=mean(.SD$X3V.REGION.trimmed.nt.nb, na.rm=T), |
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770 P1=mean(.SD$P3V.nt.nb, na.rm=T), |
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771 N1=mean(rowSums(.SD[,c("N.REGION.nt.nb", "N1.REGION.nt.nb"), with=F], na.rm=T)), |
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772 P2=mean(.SD$P5D.nt.nb, na.rm=T), |
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773 DEL.DH=mean(.SD$X5D.REGION.trimmed.nt.nb, na.rm=T), |
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774 DH.DEL=mean(.SD$X3D.REGION.trimmed.nt.nb, na.rm=T), |
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775 P3=mean(.SD$P3D.nt.nb, na.rm=T), |
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776 N2=mean(rowSums(.SD[,c("N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb"), with=F], na.rm=T)), |
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777 P4=mean(.SD$P5J.nt.nb, na.rm=T), |
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778 DEL.JH=mean(.SD$X5J.REGION.trimmed.nt.nb, na.rm=T), |
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779 Total.Del=mean(rowSums(.SD[,c("X3V.REGION.trimmed.nt.nb", "X5D.REGION.trimmed.nt.nb", "X3D.REGION.trimmed.nt.nb", "X5J.REGION.trimmed.nt.nb"), with=F], na.rm=T)), |
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780 Total.N=mean(rowSums(.SD[,c("N.REGION.nt.nb", "N1.REGION.nt.nb", "N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb"), with=F], na.rm=T)), |
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781 Total.P=mean(rowSums(.SD[,c("P3V.nt.nb", "P5D.nt.nb", "P3D.nt.nb", "P5J.nt.nb"), with=F], na.rm=T)), |
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782 Median.CDR3.l=as.double(median(as.numeric(.SD$CDR3.Length), na.rm=T))), |
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783 by=c("Sample")]) |
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784 newData[,sapply(newData, is.numeric)] = round(newData[,sapply(newData, is.numeric)],1) |
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785 write.table(newData, "junctionAnalysisProd_mean_wD.txt" , sep="\t",quote=F,na="-",row.names=F,col.names=F) |
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786 |
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787 newData = data.frame(data.table(PRODF.with.D)[,list(unique=.N, |
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788 VH.DEL=num_median(.SD$X3V.REGION.trimmed.nt.nb, na.rm=T), |
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789 P1=num_median(.SD$P3V.nt.nb, na.rm=T), |
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790 N1=num_median(rowSums(.SD[,c("N.REGION.nt.nb", "N1.REGION.nt.nb"), with=F], na.rm=T)), |
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791 P2=num_median(.SD$P5D.nt.nb, na.rm=T), |
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792 DEL.DH=num_median(.SD$X5D.REGION.trimmed.nt.nb, na.rm=T), |
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793 DH.DEL=num_median(.SD$X3D.REGION.trimmed.nt.nb, na.rm=T), |
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794 P3=num_median(.SD$P3D.nt.nb, na.rm=T), |
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795 N2=num_median(rowSums(.SD[,c("N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb"), with=F], na.rm=T)), |
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796 P4=num_median(.SD$P5J.nt.nb, na.rm=T), |
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797 DEL.JH=num_median(.SD$X5J.REGION.trimmed.nt.nb, na.rm=T), |
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798 Total.Del=num_median(rowSums(.SD[,c("X3V.REGION.trimmed.nt.nb", "X5D.REGION.trimmed.nt.nb", "X3D.REGION.trimmed.nt.nb", "X5J.REGION.trimmed.nt.nb"), with=F], na.rm=T)), |
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799 Total.N=num_median(rowSums(.SD[,c("N.REGION.nt.nb", "N1.REGION.nt.nb", "N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb"), with=F], na.rm=T)), |
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800 Total.P=num_median(rowSums(.SD[,c("P3V.nt.nb", "P5D.nt.nb", "P3D.nt.nb", "P5J.nt.nb"), with=F], na.rm=T)), |
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801 Median.CDR3.l=as.double(median(as.numeric(.SD$CDR3.Length), na.rm=T))), |
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802 by=c("Sample")]) |
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803 newData[,sapply(newData, is.numeric)] = round(newData[,sapply(newData, is.numeric)],1) |
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804 write.table(newData, "junctionAnalysisProd_median_wD.txt" , sep="\t",quote=F,na="-",row.names=F,col.names=F) |
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805 |
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806 newData = data.frame(data.table(UNPROD.with.D)[,list(unique=.N, |
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807 VH.DEL=mean(.SD$X3V.REGION.trimmed.nt.nb, na.rm=T), |
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808 P1=mean(.SD$P3V.nt.nb, na.rm=T), |
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809 N1=mean(rowSums(.SD[,c("N.REGION.nt.nb", "N1.REGION.nt.nb"), with=F], na.rm=T)), |
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810 P2=mean(.SD$P5D.nt.nb, na.rm=T), |
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811 DEL.DH=mean(.SD$X5D.REGION.trimmed.nt.nb, na.rm=T), |
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812 DH.DEL=mean(.SD$X3D.REGION.trimmed.nt.nb, na.rm=T), |
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813 P3=mean(.SD$P3D.nt.nb, na.rm=T), |
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814 N2=mean(rowSums(.SD[,c("N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb"), with=F], na.rm=T)), |
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815 P4=mean(.SD$P5J.nt.nb, na.rm=T), |
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816 DEL.JH=mean(.SD$X5J.REGION.trimmed.nt.nb, na.rm=T), |
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817 Total.Del=mean(rowSums(.SD[,c("X3V.REGION.trimmed.nt.nb", "X5D.REGION.trimmed.nt.nb", "X3D.REGION.trimmed.nt.nb", "X5J.REGION.trimmed.nt.nb"), with=F], na.rm=T)), |
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818 Total.N=mean(rowSums(.SD[,c("N.REGION.nt.nb", "N1.REGION.nt.nb", "N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb"), with=F], na.rm=T)), |
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819 Total.P=mean(rowSums(.SD[,c("P3V.nt.nb", "P5D.nt.nb", "P3D.nt.nb", "P5J.nt.nb"), with=F], na.rm=T)), |
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820 Median.CDR3.l=as.double(median(as.numeric(.SD$CDR3.Length), na.rm=T))), |
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821 by=c("Sample")]) |
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822 newData[,sapply(newData, is.numeric)] = round(newData[,sapply(newData, is.numeric)],1) |
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823 write.table(newData, "junctionAnalysisUnProd_mean_wD.txt" , sep="\t",quote=F,na="-",row.names=F,col.names=F) |
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824 |
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825 newData = data.frame(data.table(UNPROD.with.D)[,list(unique=.N, |
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826 VH.DEL=num_median(.SD$X3V.REGION.trimmed.nt.nb, na.rm=T), |
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827 P1=num_median(.SD$P3V.nt.nb, na.rm=T), |
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828 N1=num_median(rowSums(.SD[,c("N.REGION.nt.nb", "N1.REGION.nt.nb"), with=F], na.rm=T)), |
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829 P2=num_median(.SD$P5D.nt.nb, na.rm=T), |
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830 DEL.DH=num_median(.SD$X5D.REGION.trimmed.nt.nb, na.rm=T), |
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831 DH.DEL=num_median(.SD$X3D.REGION.trimmed.nt.nb, na.rm=T), |
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832 P3=num_median(.SD$P3D.nt.nb, na.rm=T), |
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833 N2=num_median(rowSums(.SD[,c("N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb"), with=F], na.rm=T)), |
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834 P4=num_median(.SD$P5J.nt.nb, na.rm=T), |
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835 DEL.JH=num_median(.SD$X5J.REGION.trimmed.nt.nb, na.rm=T), |
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836 Total.Del=num_median(rowSums(.SD[,c("X3V.REGION.trimmed.nt.nb", "X5D.REGION.trimmed.nt.nb", "X3D.REGION.trimmed.nt.nb", "X5J.REGION.trimmed.nt.nb"), with=F], na.rm=T)), |
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837 Total.N=num_median(rowSums(.SD[,c("N.REGION.nt.nb", "N1.REGION.nt.nb", "N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb"), with=F], na.rm=T)), |
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838 Total.P=num_median(rowSums(.SD[,c("P3V.nt.nb", "P5D.nt.nb", "P3D.nt.nb", "P5J.nt.nb"), with=F], na.rm=T)), |
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839 Median.CDR3.l=as.double(median(as.numeric(.SD$CDR3.Length), na.rm=T))), |
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840 by=c("Sample")]) |
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841 newData[,sapply(newData, is.numeric)] = round(newData[,sapply(newData, is.numeric)],1) |
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842 write.table(newData, "junctionAnalysisUnProd_median_wD.txt" , sep="\t",quote=F,na="-",row.names=F,col.names=F) |
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843 |
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844 #---------------- again for no-D |
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845 |
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846 newData = data.frame(data.table(PRODF.no.D)[,list(unique=.N, |
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847 VH.DEL=mean(.SD$X3V.REGION.trimmed.nt.nb, na.rm=T), |
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848 P1=mean(.SD$P3V.nt.nb, na.rm=T), |
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849 N1=mean(.SD$N.REGION.nt.nb, na.rm=T), |
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850 P2=mean(.SD$P5J.nt.nb, na.rm=T), |
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851 DEL.JH=mean(.SD$X5J.REGION.trimmed.nt.nb, na.rm=T), |
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852 Total.Del=mean(rowSums(.SD[,c("X3V.REGION.trimmed.nt.nb", "X5J.REGION.trimmed.nt.nb"), with=F], na.rm=T)), |
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853 Total.N=mean(.SD$N.REGION.nt.nb, na.rm=T), |
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854 Total.P=mean(rowSums(.SD[,c("P3V.nt.nb", "P5J.nt.nb"), with=F], na.rm=T)), |
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855 Median.CDR3.l=as.double(median(as.numeric(.SD$CDR3.Length), na.rm=T))), |
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856 by=c("Sample")]) |
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857 newData[,sapply(newData, is.numeric)] = round(newData[,sapply(newData, is.numeric)],1) |
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858 write.table(newData, "junctionAnalysisProd_mean_nD.txt" , sep="\t",quote=F,na="-",row.names=F,col.names=F) |
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859 |
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860 newData = data.frame(data.table(PRODF.no.D)[,list(unique=.N, |
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861 VH.DEL=num_median(.SD$X3V.REGION.trimmed.nt.nb, na.rm=T), |
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862 P1=num_median(.SD$P3V.nt.nb, na.rm=T), |
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863 N1=num_median(.SD$N.REGION.nt.nb, na.rm=T), |
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864 P2=num_median(.SD$P5J.nt.nb, na.rm=T), |
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865 DEL.JH=num_median(.SD$X5J.REGION.trimmed.nt.nb, na.rm=T), |
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866 Total.Del=num_median(rowSums(.SD[,c("X3V.REGION.trimmed.nt.nb", "X5J.REGION.trimmed.nt.nb"), with=F], na.rm=T)), |
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867 Total.N=num_median(.SD$N.REGION.nt.nb, na.rm=T), |
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868 Total.P=num_median(rowSums(.SD[,c("P3V.nt.nb", "P5J.nt.nb"), with=F], na.rm=T)), |
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869 Median.CDR3.l=as.double(median(as.numeric(.SD$CDR3.Length), na.rm=T))), |
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870 by=c("Sample")]) |
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871 newData[,sapply(newData, is.numeric)] = round(newData[,sapply(newData, is.numeric)],1) |
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872 write.table(newData, "junctionAnalysisProd_median_nD.txt" , sep="\t",quote=F,na="-",row.names=F,col.names=F) |
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873 |
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874 newData = data.frame(data.table(UNPROD.no.D)[,list(unique=.N, |
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875 VH.DEL=mean(.SD$X3V.REGION.trimmed.nt.nb, na.rm=T), |
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876 P1=mean(.SD$P3V.nt.nb, na.rm=T), |
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877 N1=mean(.SD$N.REGION.nt.nb, na.rm=T), |
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878 P2=mean(.SD$P5J.nt.nb, na.rm=T), |
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879 DEL.JH=mean(.SD$X5J.REGION.trimmed.nt.nb, na.rm=T), |
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880 Total.Del=mean(rowSums(.SD[,c("X3V.REGION.trimmed.nt.nb", "X5J.REGION.trimmed.nt.nb"), with=F], na.rm=T)), |
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881 Total.N=mean(.SD$N.REGION.nt.nb, na.rm=T), |
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882 Total.P=mean(rowSums(.SD[,c("P3V.nt.nb", "P5J.nt.nb"), with=F], na.rm=T)), |
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883 Median.CDR3.l=as.double(median(as.numeric(.SD$CDR3.Length), na.rm=T))), |
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884 by=c("Sample")]) |
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885 newData[,sapply(newData, is.numeric)] = round(newData[,sapply(newData, is.numeric)],1) |
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886 write.table(newData, "junctionAnalysisUnProd_mean_nD.txt" , sep="\t",quote=F,na="-",row.names=F,col.names=F) |
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887 |
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888 |
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889 newData = data.frame(data.table(UNPROD.no.D)[,list(unique=.N, |
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890 VH.DEL=num_median(.SD$X3V.REGION.trimmed.nt.nb, na.rm=T), |
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891 P1=num_median(.SD$P3V.nt.nb, na.rm=T), |
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892 N1=num_median(.SD$N.REGION.nt.nb, na.rm=T), |
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893 P2=num_median(.SD$P5J.nt.nb, na.rm=T), |
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894 DEL.JH=num_median(.SD$X5J.REGION.trimmed.nt.nb, na.rm=T), |
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895 Total.Del=num_median(rowSums(.SD[,c("X3V.REGION.trimmed.nt.nb", "X5J.REGION.trimmed.nt.nb"), with=F], na.rm=T)), |
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896 Total.N=num_median(.SD$N.REGION.nt.nb, na.rm=T), |
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897 Total.P=num_median(rowSums(.SD[,c("P3V.nt.nb", "P5J.nt.nb"), with=F], na.rm=T)), |
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898 Median.CDR3.l=as.double(median(as.numeric(.SD$CDR3.Length), na.rm=T))), |
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899 by=c("Sample")]) |
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900 newData[,sapply(newData, is.numeric)] = round(newData[,sapply(newData, is.numeric)],1) |
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901 write.table(newData, "junctionAnalysisUnProd_median_nD.txt" , sep="\t",quote=F,na="-",row.names=F,col.names=F) |
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902 } |
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903 |
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904 PRODF = bak |
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905 UNPROD = bakun |
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906 |
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907 |
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908 # ---------------------- D reading frame ---------------------- |
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909 |
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910 D.REGION.reading.frame = PRODF[,c("Sample", "D.REGION.reading.frame")] |
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911 |
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912 chck = is.na(D.REGION.reading.frame$D.REGION.reading.frame) |
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913 if(any(chck)){ |
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914 D.REGION.reading.frame[chck,"D.REGION.reading.frame"] = "No D" |
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915 } |
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916 |
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917 D.REGION.reading.frame.1 = data.frame(data.table(D.REGION.reading.frame)[, list(Freq=.N), by=c("Sample", "D.REGION.reading.frame")]) |
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918 |
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919 D.REGION.reading.frame.2 = data.frame(data.table(D.REGION.reading.frame)[, list(sample.sum=sum(as.numeric(.SD$D.REGION.reading.frame), na.rm=T)), by=c("Sample")]) |
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920 |
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921 D.REGION.reading.frame = merge(D.REGION.reading.frame.1, D.REGION.reading.frame.2, by="Sample") |
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922 |
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923 D.REGION.reading.frame$percentage = round(D.REGION.reading.frame$Freq / D.REGION.reading.frame$sample.sum * 100, 1) |
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924 |
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925 write.table(D.REGION.reading.frame, "DReadingFrame.txt" , sep="\t",quote=F,row.names=F,col.names=T) |
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926 |
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927 D.REGION.reading.frame = ggplot(D.REGION.reading.frame) |
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928 D.REGION.reading.frame = D.REGION.reading.frame + geom_bar(aes( x = D.REGION.reading.frame, y = percentage, fill=Sample), stat='identity', position='dodge' ) + ggtitle("D reading frame") + xlab("Frame") + ylab("Frequency") |
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929 D.REGION.reading.frame = D.REGION.reading.frame + scale_fill_manual(values=sample.colors) |
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930 D.REGION.reading.frame = D.REGION.reading.frame + theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), axis.text.x = element_text(angle = 45, hjust = 1), panel.grid.major.y = element_line(colour = "black"), panel.grid.major.x = element_blank()) |
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931 |
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932 png("DReadingFrame.png") |
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933 D.REGION.reading.frame |
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934 dev.off() |
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935 |
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936 ggsave("DReadingFrame.pdf", D.REGION.reading.frame) |
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937 |
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938 # ---------------------- AA composition in CDR3 ---------------------- |
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939 |
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940 AACDR3 = PRODF[,c("Sample", "CDR3.Seq")] |
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941 |
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942 TotalPerSample = data.frame(data.table(AACDR3)[, list(total=sum(nchar(as.character(.SD$CDR3.Seq)))), by=Sample]) |
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943 |
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944 AAfreq = list() |
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945 |
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946 for(i in 1:nrow(TotalPerSample)){ |
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947 sample = TotalPerSample$Sample[i] |
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948 AAfreq[[i]] = data.frame(table(unlist(strsplit(as.character(AACDR3[AACDR3$Sample == sample,c("CDR3.Seq")]), "")))) |
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949 AAfreq[[i]]$Sample = sample |
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950 } |
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951 |
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952 AAfreq = ldply(AAfreq, data.frame) |
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953 AAfreq = merge(AAfreq, TotalPerSample, by="Sample", all.x = T) |
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954 AAfreq$freq_perc = as.numeric(AAfreq$Freq / AAfreq$total * 100) |
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955 |
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956 |
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957 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") |
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958 AAfreq = merge(AAfreq, AAorder, by.x='Var1', by.y='AA', all.x=TRUE) |
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959 |
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960 AAfreq = AAfreq[!is.na(AAfreq$order.aa),] |
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961 |
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962 AAfreqplot = ggplot(AAfreq) |
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963 AAfreqplot = AAfreqplot + geom_bar(aes( x=factor(reorder(Var1, order.aa)), y = freq_perc, fill = Sample), stat='identity', position='dodge' ) |
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964 AAfreqplot = AAfreqplot + annotate("rect", xmin = 0.5, xmax = 2.5, ymin = 0, ymax = Inf, fill = "red", alpha = 0.2) |
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965 AAfreqplot = AAfreqplot + annotate("rect", xmin = 3.5, xmax = 4.5, ymin = 0, ymax = Inf, fill = "blue", alpha = 0.2) |
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966 AAfreqplot = AAfreqplot + annotate("rect", xmin = 5.5, xmax = 6.5, ymin = 0, ymax = Inf, fill = "blue", alpha = 0.2) |
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967 AAfreqplot = AAfreqplot + annotate("rect", xmin = 6.5, xmax = 7.5, ymin = 0, ymax = Inf, fill = "red", alpha = 0.2) |
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968 AAfreqplot = AAfreqplot + ggtitle("Amino Acid Composition in the CDR3") + xlab("Amino Acid, from Hydrophilic (left) to Hydrophobic (right)") + ylab("Percentage") + scale_fill_manual(values=sample.colors) |
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969 AAfreqplot = AAfreqplot + theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), panel.grid.major.y = element_line(colour = "black"), panel.grid.major.x = element_blank()) |
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970 |
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971 png("AAComposition.png",width = 1280, height = 720) |
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972 AAfreqplot |
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973 dev.off() |
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974 |
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975 ggsave("AAComposition.pdf", AAfreqplot, width=12, height=7) |
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976 |
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977 write.table(AAfreq, "AAComposition.txt" , sep="\t",quote=F,na="-",row.names=F,col.names=T) |
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978 |
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979 # ---------------------- AA median CDR3 length ---------------------- |
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980 |
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981 median.aa.l = data.frame(data.table(PRODF)[, list(median=as.double(median(as.numeric(.SD$CDR3.Length, na.rm=T), na.rm=T))), by=c("Sample")]) |
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982 write.table(median.aa.l, "AAMedianBySample.txt" , sep="\t",quote=F,na="-",row.names=F,col.names=F) |
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983 |
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984 if(clonaltype != "none"){ |
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985 #generate the "Sequences that are present in more than one replicate" dataset |
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986 clonaltype.in.replicates = inputdata |
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987 clonaltype.in.replicates = clonaltype.in.replicates[clonaltype.in.replicates$Functionality %in% c("productive (see comment)","productive"),] |
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988 clonaltype.in.replicates = clonaltype.in.replicates[!(is.na(clonaltype.in.replicates$ID) | is.na(clonaltype.in.replicates$Top.V.Gene) | is.na(clonaltype.in.replicates$Top.J.Gene)),] |
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989 clonaltype = unlist(strsplit(clonaltype, ",")) |
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990 |
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991 clonaltype.in.replicates$clonaltype = do.call(paste, c(clonaltype.in.replicates[clonaltype], sep = ":")) |
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992 |
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993 clonaltype.in.replicates = clonaltype.in.replicates[!duplicated(clonaltype.in.replicates$clonaltype),] |
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994 |
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995 clonaltype = clonaltype[-which(clonaltype == "Sample")] |
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996 |
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997 clonaltype.in.replicates$clonaltype = do.call(paste, c(clonaltype.in.replicates[clonaltype], sep = ":")) |
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998 clonaltype.in.replicates = clonaltype.in.replicates[,c("clonaltype","Replicate", "ID", "Sequence", "Sample")] |
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999 |
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1000 |
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1001 write.table(clonaltype.in.replicates, "clonaltypes_replicates_before_table.txt" , sep="\t",quote=F,na="-",row.names=F,col.names=T) |
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1002 |
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1003 clonaltype.counts = data.frame(table(clonaltype.in.replicates$clonaltype)) |
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1004 |
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1005 write.table(clonaltype.counts, "clonaltypes_counts.txt" , sep="\t",quote=F,na="-",row.names=F,col.names=T) |
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1006 |
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1007 names(clonaltype.counts) = c("clonaltype", "coincidence") |
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1008 |
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1009 clonaltype.counts = clonaltype.counts[clonaltype.counts$coincidence > 1,] |
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1010 |
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1011 clonaltype.in.replicates = clonaltype.in.replicates[clonaltype.in.replicates$clonaltype %in% clonaltype.counts$clonaltype,] |
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1012 clonaltype.in.replicates = merge(clonaltype.in.replicates, clonaltype.counts, by="clonaltype") |
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1013 clonaltype.in.replicates = clonaltype.in.replicates[order(-clonaltype.in.replicates$coincidence, clonaltype.in.replicates$clonaltype, clonaltype.in.replicates$Replicate),c("coincidence","clonaltype", "Sample", "Replicate", "ID", "Sequence")] |
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1014 |
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1015 |
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1016 write.table(clonaltype.in.replicates, "clonaltypes_replicates.txt" , sep="\t",quote=F,na="-",row.names=F,col.names=T) |
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1017 } else { |
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1018 cat("No clonaltype", file="clonaltypes_replicates_before_table.txt") |
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1019 cat("No clonaltype", file="clonaltypes_counts.txt") |
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1020 cat("No clonaltype", file="clonaltypes_replicates.txt") |
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1021 } |
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1022 |
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1023 |
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1024 |
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1025 |
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1026 |
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1027 |
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1028 |
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1029 |
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1030 |
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1031 |
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1032 |
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1033 |
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1034 |
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1035 |
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1036 |
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1037 |
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1038 |
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1039 |
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1040 |
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1041 |
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1042 |
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1043 |
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1044 |