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0
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1 args <- commandArgs(trailingOnly = TRUE)
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2
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3 inFile = args[1]
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4 outDir = args[2]
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3
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5 logfile = args[3]
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6 min_freq = as.numeric(args[4])
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7 min_cells = as.numeric(args[5])
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8
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9 cat("<html><table><tr><td>Starting analysis</td></tr>", file=logfile, append=F)
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0
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10
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4
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11 library(ggplot2)
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12 library(reshape2)
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13 library(data.table)
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14 library(grid)
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15 library(parallel)
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0
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16 #require(xtable)
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3
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17 cat("<tr><td>Reading input</td></tr>", file=logfile, append=T)
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13
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18 dat = read.table(inFile, header=T, sep="\t", dec=".", fill=T, stringsAsFactors=F)
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9
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19 dat = dat[!is.na(dat$Patient),]
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13
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20 dat$Related_to_leukemia_clone = F
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9
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21
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0
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22 setwd(outDir)
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3
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23 cat("<tr><td>Selecting first V/J Genes</td></tr>", file=logfile, append=T)
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2
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24 dat$V_Segment_Major_Gene = as.factor(as.character(lapply(strsplit(as.character(dat$V_Segment_Major_Gene), "; "), "[[", 1)))
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25 dat$J_Segment_Major_Gene = as.factor(as.character(lapply(strsplit(as.character(dat$J_Segment_Major_Gene), "; "), "[[", 1)))
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26
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3
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27 cat("<tr><td>Calculating Frequency</td></tr>", file=logfile, append=T)
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12
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28
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13
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29 dat$Frequency = ((10^dat$Log10_Frequency)*100)
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2
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30
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3
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31 dat = dat[dat$Frequency >= min_freq,]
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32
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13
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33 triplets = dat[grepl("VanDongen_cALL_14696", dat$Patient) | grepl("(16278)|(26402)|(26759)", dat$Sample),]
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34
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35 cat("<tr><td>Normalizing to lowest cell count within locus</td></tr>", file=logfile, append=T)
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36
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37 dat$locus_V = substring(dat$V_Segment_Major_Gene, 0, 4)
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38 dat$locus_J = substring(dat$J_Segment_Major_Gene, 0, 4)
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39 min_cell_count = data.frame(data.table(dat)[, list(min_cell_count=min(.SD$Cell_Count)), by=c("Patient", "locus_V", "locus_J")])
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40
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41 dat$min_cell_paste = paste(dat$Patient, dat$locus_V, dat$locus_J)
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42 min_cell_count$min_cell_paste = paste(min_cell_count$Patient, min_cell_count$locus_V, min_cell_count$locus_J)
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43
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44 min_cell_count = min_cell_count[,c("min_cell_paste", "min_cell_count")]
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45
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46 dat = merge(dat, min_cell_count, by="min_cell_paste")
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47
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48 dat$normalized_read_count = round(dat$Clone_Molecule_Count_From_Spikes / dat$Cell_Count * dat$min_cell_count / 2, digits=2) #??????????????????????????????????? wel of geen / 2
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49
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3
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50 dat = dat[dat$normalized_read_count >= min_cells,]
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13
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51
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52 dat$paste = paste(dat$Sample, dat$Clone_Sequence)
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53
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22
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54 patients = split(dat, dat$Patient, drop=T)
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9
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55 intervalReads = rev(c(0,10,25,50,100,250,500,750,1000,10000))
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6
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56 intervalFreq = rev(c(0,0.01,0.05,0.1,0.5,1,5))
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0
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57 V_Segments = c(".*", "IGHV", "IGHD", "IGKV", "IGKV", "IgKINTR", "TRGV", "TRDV", "TRDD" , "TRBV")
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58 J_Segments = c(".*", ".*", ".*", "IGKJ", "KDE", ".*", ".*", ".*", ".*", ".*")
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59 Titles = c("Total", "IGH-Vh-Jh", "IGH-Dh-Jh", "Vk-Jk", "Vk-Kde" , "Intron-Kde", "TCRG", "TCRD-Vd-Dd", "TCRD-Dd-Dd", "TCRB-Vb-Jb")
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60 Titles = factor(Titles, levels=Titles)
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61 TitlesOrder = data.frame("Title"=Titles, "TitlesOrder"=1:length(Titles))
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62
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63 patientCountOnColumn <- function(x, product, interval, on, appendtxt=F){
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64 x$Sample = factor(x$Sample, levels=unique(x$Sample))
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65 onShort = "reads"
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66 if(on == "Frequency"){
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67 onShort = "freq"
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68 }
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18
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69 onx = paste(on, ".x", sep="")
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70 ony = paste(on, ".y", sep="")
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0
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71 splt = split(x, x$Sample, drop=T)
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4
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72 type="pair"
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2
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73 if(length(splt) == 1){
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3
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74 print(paste(paste(x[1,which(colnames(x) == "Patient")]), "has one sample"))
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4
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75 splt[[2]] = data.frame("Patient" = character(0), "Receptor" = character(0), "Sample" = character(0), "Cell_Count" = numeric(0), "Clone_Molecule_Count_From_Spikes" = numeric(0), "Log10_Frequency" = numeric(0), "Total_Read_Count" = numeric(0), "dsMol_per_1e6_cells" = numeric(0), "J_Segment_Major_Gene" = character(0), "V_Segment_Major_Gene" = character(0), "Clone_Sequence" = character(0), "CDR3_Sense_Sequence" = character(0), "Related_to_leukemia_clone" = logical(0), "Frequency"= numeric(0), "normalized_read_count" = numeric(0), "paste" = character(0))
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76 type="single"
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2
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77 }
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0
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78 patient1 = splt[[1]]
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79 patient2 = splt[[2]]
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80
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81 threshholdIndex = which(colnames(product) == "interval")
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82 V_SegmentIndex = which(colnames(product) == "V_Segments")
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83 J_SegmentIndex = which(colnames(product) == "J_Segments")
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84 titleIndex = which(colnames(product) == "Titles")
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85 sampleIndex = which(colnames(x) == "Sample")
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86 patientIndex = which(colnames(x) == "Patient")
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87 oneSample = paste(patient1[1,sampleIndex], sep="")
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88 twoSample = paste(patient2[1,sampleIndex], sep="")
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89 patient = paste(x[1,patientIndex])
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3
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90
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0
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91 switched = F
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92 if(length(grep(".*_Right$", twoSample)) == 1 || length(grep(".*_Dx_BM$", twoSample)) == 1 || length(grep(".*_Dx$", twoSample)) == 1 ){
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93 tmp = twoSample
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94 twoSample = oneSample
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95 oneSample = tmp
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96 tmp = patient1
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97 patient1 = patient2
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98 patient2 = tmp
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99 switched = T
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100 }
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101 if(appendtxt){
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4
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102 cat(paste(patient, oneSample, twoSample, type, sep="\t"), file="patients.txt", append=T, sep="", fill=3)
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0
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103 }
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3
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104 cat(paste("<tr><td>", patient, "</td></tr>", sep=""), file=logfile, append=T)
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105
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12
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106 #patient1$merge = paste(patient1$V_Segment_Major_Gene, patient1$J_Segment_Major_Gene, patient1$CDR3_Sense_Sequence)
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107 #patient2$merge = paste(patient2$V_Segment_Major_Gene, patient2$J_Segment_Major_Gene, patient2$CDR3_Sense_Sequence)
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13
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108 patient1$merge = paste(patient1$Clone_Sequence)
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109 patient2$merge = paste(patient2$Clone_Sequence)
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9
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110
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12
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111 #patientMerge = merge(patient1, patient2, by.x="merge", by.y="merge")
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112 patientMerge = merge(patient1, patient2, by.x="merge", by.y="merge")
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18
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113 patientMerge$thresholdValue = pmax(patientMerge[,onx], patientMerge[,ony])
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0
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114 res1 = vector()
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115 res2 = vector()
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116 resBoth = vector()
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117 read1Count = vector()
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118 read2Count = vector()
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2
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119 locussum1 = vector()
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120 locussum2 = vector()
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9
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121
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122 print(patient)
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0
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123 #for(iter in 1){
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124 for(iter in 1:length(product[,1])){
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125 threshhold = product[iter,threshholdIndex]
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126 V_Segment = paste(".*", as.character(product[iter,V_SegmentIndex]), ".*", sep="")
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127 J_Segment = paste(".*", as.character(product[iter,J_SegmentIndex]), ".*", sep="")
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18
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128 #both = (grepl(V_Segment, patientMerge$V_Segment_Major_Gene.x) & grepl(J_Segment, patientMerge$J_Segment_Major_Gene.x) & patientMerge[,onx] > threshhold & patientMerge[,ony] > threshhold) #both higher than threshold
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129 both = (grepl(V_Segment, patientMerge$V_Segment_Major_Gene.x) & grepl(J_Segment, patientMerge$J_Segment_Major_Gene.x) & patientMerge$thresholdValue > threshhold) #highest of both higher than threshold
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19
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130 one = (grepl(V_Segment, patient1$V_Segment_Major_Gene) & grepl(J_Segment, patient1$J_Segment_Major_Gene) & patient1[,on] > threshhold & !(patient1$Clone_Sequence %in% patientMerge[both,]$merge))
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131 two = (grepl(V_Segment, patient2$V_Segment_Major_Gene) & grepl(J_Segment, patient2$J_Segment_Major_Gene) & patient2[,on] > threshhold & !(patient2$Clone_Sequence %in% patientMerge[both,]$merge))
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14
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132 read1Count = append(read1Count, sum(patient1[one,]$normalized_read_count))
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133 read2Count = append(read2Count, sum(patient2[two,]$normalized_read_count))
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0
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134 res1 = append(res1, sum(one))
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2
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135 res2 = append(res2, sum(two))
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0
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136 resBoth = append(resBoth, sum(both))
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2
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137 locussum1 = append(locussum1, sum(patient1[(grepl(V_Segment, patient1$V_Segment_Major_Gene) & grepl(J_Segment, patient1$J_Segment_Major_Gene)),]$normalized_read_count))
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138 locussum2 = append(locussum2, sum(patient2[(grepl(V_Segment, patient2$V_Segment_Major_Gene) & grepl(J_Segment, patient2$J_Segment_Major_Gene)),]$normalized_read_count))
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0
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139 #threshhold = 0
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140 if(threshhold != 0){
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141 if(sum(one) > 0){
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15
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142 dfOne = patient1[one,c("V_Segment_Major_Gene", "J_Segment_Major_Gene", "normalized_read_count", "Frequency", "Clone_Sequence", "Related_to_leukemia_clone")]
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143 colnames(dfOne) = c("Proximal segment", "Distal segment", "normalized_read_count", "Frequency", "Clone Sequence", "Related_to_leukemia_clone")
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0
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144 filenameOne = paste(oneSample, "_", product[iter, titleIndex], "_", threshhold, sep="")
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145 write.table(dfOne, file=paste(filenameOne, ".txt", sep=""), quote=F, sep="\t", dec=",", row.names=F, col.names=T)
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146 }
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147 if(sum(two) > 0){
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15
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148 dfTwo = patient2[two,c("V_Segment_Major_Gene", "J_Segment_Major_Gene", "normalized_read_count", "Frequency", "Clone_Sequence", "Related_to_leukemia_clone")]
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149 colnames(dfTwo) = c("Proximal segment", "Distal segment", "normalized_read_count", "Frequency", "Clone Sequence", "Related_to_leukemia_clone")
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0
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150 filenameTwo = paste(twoSample, "_", product[iter, titleIndex], "_", threshhold, sep="")
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151 write.table(dfTwo, file=paste(filenameTwo, ".txt", sep=""), quote=F, sep="\t", dec=",", row.names=F, col.names=T)
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152 }
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153 }
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154 if(sum(both) > 0){
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15
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155 dfBoth = patientMerge[both,c("V_Segment_Major_Gene.x", "J_Segment_Major_Gene.x", "normalized_read_count.x", "Frequency.x", "Related_to_leukemia_clone.x", "Clone_Sequence.x", "V_Segment_Major_Gene.y", "J_Segment_Major_Gene.y", "normalized_read_count.y", "Frequency.y", "Related_to_leukemia_clone.y")]
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156 colnames(dfBoth) = c(paste("Proximal segment", oneSample), paste("Distal segment", oneSample), paste("Normalized_Read_Count", oneSample), paste("Frequency", oneSample), paste("Related_to_leukemia_clone", oneSample),"Clone Sequence", paste("Proximal segment", twoSample), paste("Distal segment", twoSample), paste("Normalized_Read_Count", twoSample), paste("Frequency", twoSample), paste("Related_to_leukemia_clone", twoSample))
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0
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157 filenameBoth = paste(oneSample, "_", twoSample, "_", product[iter, titleIndex], "_", threshhold, sep="")
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158 write.table(dfBoth, file=paste(filenameBoth, ".txt", sep=""), quote=F, sep="\t", dec=",", row.names=F, col.names=T)
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159 }
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160 }
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2
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161 patientResult = data.frame("Locus"=product$Titles, "J_Segment"=product$J_Segments, "V_Segment"=product$V_Segments, "cut_off_value"=paste(">", product$interval, sep=""), "Both"=resBoth, "tmp1"=res1, "read_count1" = round(read1Count), "tmp2"=res2, "read_count2"= round(read2Count), "Sum"=res1 + res2 + resBoth, "percentage" = round((resBoth/(res1 + res2 + resBoth)) * 100, digits=2), "Locus_sum1"=locussum1, "Locus_sum2"=locussum2)
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0
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162 if(sum(is.na(patientResult$percentage)) > 0){
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163 patientResult[is.na(patientResult$percentage),]$percentage = 0
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164 }
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165 colnames(patientResult)[6] = oneSample
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166 colnames(patientResult)[8] = twoSample
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167 colnamesBak = colnames(patientResult)
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2
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168 colnames(patientResult) = c("Ig/TCR gene rearrangement type", "Distal Gene segment", "Proximal gene segment", "cut_off_value", paste("Number of sequences ", patient, "_Both", sep=""), paste("Number of sequences", oneSample, sep=""), paste("Normalized Read Count", oneSample), paste("Number of sequences", twoSample, sep=""), paste("Normalized Read Count", twoSample), paste("Sum number of sequences", patient), paste("Percentage of sequences ", patient, "_Both", sep=""), paste("Locus Sum", oneSample), paste("Locus Sum", twoSample))
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0
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169 write.table(patientResult, file=paste(patient, "_", onShort, ".txt", sep=""), quote=F, sep="\t", dec=",", row.names=F, col.names=T)
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170 colnames(patientResult) = colnamesBak
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171
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172 patientResult$Locus = factor(patientResult$Locus, Titles)
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173 patientResult$cut_off_value = factor(patientResult$cut_off_value, paste(">", interval, sep=""))
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174
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175 plt = ggplot(patientResult[,c("Locus", "cut_off_value", "Both")])
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176 plt = plt + geom_bar( aes( x=factor(cut_off_value), y=Both), stat='identity', position="dodge", fill="#79c36a")
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177 plt = plt + facet_grid(.~Locus) + theme(axis.text.x = element_text(angle = 45, hjust = 1))
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178 plt = plt + geom_text(aes(ymax=max(Both), x=cut_off_value,y=Both,label=Both), angle=90, hjust=0)
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179 plt = plt + xlab("Reads per locus") + ylab("Count") + ggtitle("Number of clones in both")
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180 plt = plt + theme(plot.margin = unit(c(1,8.8,0.5,1.5), "lines"))
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181 png(paste(patient, "_", onShort, ".png", sep=""), width=1920, height=1080)
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182 print(plt)
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183 dev.off()
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184 #(t,r,b,l)
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185 plt = ggplot(patientResult[,c("Locus", "cut_off_value", "percentage")])
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186 plt = plt + geom_bar( aes( x=factor(cut_off_value), y=percentage), stat='identity', position="dodge", fill="#79c36a")
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187 plt = plt + facet_grid(.~Locus) + theme(axis.text.x = element_text(angle = 45, hjust = 1))
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188 plt = plt + geom_text(aes(ymax=max(percentage), x=cut_off_value,y=percentage,label=percentage), angle=90, hjust=0)
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189 plt = plt + xlab("Reads per locus") + ylab("Count") + ggtitle("% clones in both left and right")
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190 plt = plt + theme(plot.margin = unit(c(1,8.8,0.5,1.5), "lines"))
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191 png(paste(patient, "_percent_", onShort, ".png", sep=""), width=1920, height=1080)
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192 print(plt)
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193 dev.off()
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194
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195 patientResult = melt(patientResult[,c('Locus','cut_off_value', oneSample, twoSample)] ,id.vars=1:2)
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196 patientResult$relativeValue = patientResult$value * 10
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197 patientResult[patientResult$relativeValue == 0,]$relativeValue = 1
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198 plt = ggplot(patientResult)
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199 plt = plt + geom_bar( aes( x=factor(cut_off_value), y=relativeValue, fill=variable), stat='identity', position="dodge")
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200 plt = plt + facet_grid(.~Locus) + theme(axis.text.x = element_text(angle = 45, hjust = 1))
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201 plt = plt + scale_y_continuous(trans="log", breaks=10^c(0:10), labels=c(0, 10^c(0:9)))
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202 plt = plt + geom_text(data=patientResult[patientResult$variable == oneSample,], aes(ymax=max(value), x=cut_off_value,y=relativeValue,label=value), angle=90, position=position_dodge(width=0.9), hjust=0, vjust=-0.2)
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203 plt = plt + geom_text(data=patientResult[patientResult$variable == twoSample,], aes(ymax=max(value), x=cut_off_value,y=relativeValue,label=value), angle=90, position=position_dodge(width=0.9), hjust=0, vjust=0.8)
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204 plt = plt + xlab("Reads per locus") + ylab("Count") + ggtitle(paste("Number of clones in only ", oneSample, " and only ", twoSample, sep=""))
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205 png(paste(patient, "_", onShort, "_both.png", sep=""), width=1920, height=1080)
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206 print(plt)
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207 dev.off()
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208 }
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209
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3
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210 cat("<tr><td>Starting Frequency analysis</td></tr>", file=logfile, append=T)
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211
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0
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212 interval = intervalFreq
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213 intervalOrder = data.frame("interval"=paste(">", interval, sep=""), "intervalOrder"=1:length(interval))
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4
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214 product = data.frame("Titles"=rep(Titles, each=length(interval)), "interval"=rep(interval, times=10), "V_Segments"=rep(V_Segments, each=length(interval)), "J_Segments"=rep(J_Segments, each=length(interval)))
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215 mclapply(patients, FUN=patientCountOnColumn, product = product, interval=interval, on="Frequency", appendtxt=T)
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0
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216
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3
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217 cat("<tr><td>Starting Cell Count analysis</td></tr>", file=logfile, append=T)
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218
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0
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219 interval = intervalReads
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220 intervalOrder = data.frame("interval"=paste(">", interval, sep=""), "intervalOrder"=1:length(interval))
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4
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221 product = data.frame("Titles"=rep(Titles, each=length(interval)), "interval"=rep(interval, times=10), "V_Segments"=rep(V_Segments, each=length(interval)), "J_Segments"=rep(J_Segments, each=length(interval)))
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9
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222 mclapply(patients, FUN=patientCountOnColumn, product = product, interval=interval, on="normalized_read_count")
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0
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223
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3
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224 cat("</table></html>", file=logfile, append=T)
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225
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7
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226
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13
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227
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7
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228 tripletAnalysis <- function(patient1, label1, patient2, label2, patient3, label3, product, interval, on, appendTriplets= FALSE){
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229 onShort = "reads"
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230 if(on == "Frequency"){
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231 onShort = "freq"
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232 }
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18
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233 onx = paste(on, ".x", sep="")
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234 ony = paste(on, ".y", sep="")
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235 onz = paste(on, ".z", sep="")
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7
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236 type="triplet"
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237
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238 threshholdIndex = which(colnames(product) == "interval")
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239 V_SegmentIndex = which(colnames(product) == "V_Segments")
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240 J_SegmentIndex = which(colnames(product) == "J_Segments")
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241 titleIndex = which(colnames(product) == "Titles")
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242 sampleIndex = which(colnames(patient1) == "Sample")
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243 patientIndex = which(colnames(patient1) == "Patient")
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244 oneSample = paste(patient1[1,sampleIndex], sep="")
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245 twoSample = paste(patient2[1,sampleIndex], sep="")
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246 threeSample = paste(patient3[1,sampleIndex], sep="")
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247
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12
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248 #patient1$merge = paste(patient1$V_Segment_Major_Gene, patient1$J_Segment_Major_Gene, patient1$CDR3_Sense_Sequence)
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249 #patient2$merge = paste(patient2$V_Segment_Major_Gene, patient2$J_Segment_Major_Gene, patient2$CDR3_Sense_Sequence)
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250 #patient3$merge = paste(patient3$V_Segment_Major_Gene, patient3$J_Segment_Major_Gene, patient3$CDR3_Sense_Sequence)
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251
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|
15
|
252 patient1$merge = paste(patient1$Clone_Sequence)
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253 patient2$merge = paste(patient2$Clone_Sequence)
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|
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254 patient3$merge = paste(patient3$Clone_Sequence)
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9
|
255
|
|
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256 patientMerge = merge(patient1, patient2, by="merge")
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257 patientMerge = merge(patientMerge, patient3, by="merge")
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13
|
258 colnames(patientMerge)[30:length(colnames(patientMerge))] = paste(colnames(patientMerge)[30:length(colnames(patientMerge))], ".z", sep="")
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18
|
259 patientMerge$thresholdValue = pmax(patientMerge[,onx], patientMerge[,ony], patientMerge[,onz])
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20
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260
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|
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261 patientMerge12 = merge(patient1, patient2, by="merge")
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262 patientMerge12$thresholdValue = pmax(patientMerge12[,onx], patientMerge12[,ony])
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263 patientMerge13 = merge(patient1, patient3, by="merge")
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|
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264 patientMerge13$thresholdValue = pmax(patientMerge13[,onx], patientMerge13[,ony])
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265 patientMerge23 = merge(patient2, patient3, by="merge")
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266 patientMerge23$thresholdValue = pmax(patientMerge23[,onx], patientMerge23[,ony])
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267
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24
|
268 scatterplot_data_columns = c("Clone_Sequence", "Frequency", "normalized_read_count", "V_Segment_Major_Gene", "J_Segment_Major_Gene")
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20
|
269 scatterplot_data = rbind(patient1[,scatterplot_data_columns], patient2[,scatterplot_data_columns], patient3[,scatterplot_data_columns])
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270 scatterplot_data = scatterplot_data[!duplicated(scatterplot_data$Clone_Sequence),]
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|
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271 scatterplot_data$type = factor(x="single", levels=c("In one", "In two", "In three"))
|
|
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272
|
|
7
|
273 res1 = vector()
|
|
|
274 res2 = vector()
|
|
|
275 res3 = vector()
|
|
20
|
276 res12 = vector()
|
|
|
277 res13 = vector()
|
|
|
278 res23 = vector()
|
|
7
|
279 resAll = vector()
|
|
|
280 read1Count = vector()
|
|
|
281 read2Count = vector()
|
|
|
282 read3Count = vector()
|
|
|
283
|
|
|
284 if(appendTriplets){
|
|
9
|
285 cat(paste(label1, label2, label3, sep="\t"), file="triplets.txt", append=T, sep="", fill=3)
|
|
7
|
286 }
|
|
|
287 for(iter in 1:length(product[,1])){
|
|
|
288 threshhold = product[iter,threshholdIndex]
|
|
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289 V_Segment = paste(".*", as.character(product[iter,V_SegmentIndex]), ".*", sep="")
|
|
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290 J_Segment = paste(".*", as.character(product[iter,J_SegmentIndex]), ".*", sep="")
|
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18
|
291 #all = (grepl(V_Segment, patientMerge$V_Segment_Major_Gene.x) & grepl(J_Segment, patientMerge$J_Segment_Major_Gene.x) & patientMerge[,onx] > threshhold & patientMerge[,ony] > threshhold & patientMerge[,onz] > threshhold)
|
|
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292 all = (grepl(V_Segment, patientMerge$V_Segment_Major_Gene.x) & grepl(J_Segment, patientMerge$J_Segment_Major_Gene.x) & patientMerge$thresholdValue > threshhold)
|
|
7
|
293
|
|
25
|
294 one_two = (grepl(V_Segment, patientMerge12$V_Segment_Major_Gene.x) & grepl(J_Segment, patientMerge12$J_Segment_Major_Gene.x) & patientMerge12$thresholdValue > threshhold & !(patientMerge12$Clone_Sequence.x %in% patientMerge[all,]$merge))
|
|
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295 one_three = (grepl(V_Segment, patientMerge13$V_Segment_Major_Gene.x) & grepl(J_Segment, patientMerge13$J_Segment_Major_Gene.x) & patientMerge13$thresholdValue > threshhold & !(patientMerge13$Clone_Sequence.x %in% patientMerge[all,]$merge))
|
|
|
296 two_three = (grepl(V_Segment, patientMerge23$V_Segment_Major_Gene.x) & grepl(J_Segment, patientMerge23$J_Segment_Major_Gene.x) & patientMerge23$thresholdValue > threshhold & !(patientMerge23$Clone_Sequence.x %in% patientMerge[all,]$merge))
|
|
24
|
297
|
|
25
|
298 one = (grepl(V_Segment, patient1$V_Segment_Major_Gene) & grepl(J_Segment, patient1$J_Segment_Major_Gene) & patient1[,on] > threshhold & !(patient1$Clone_Sequence %in% patientMerge[all,]$merge) & !(patient1$Clone_Sequence %in% patientMerge12[one_two,]$merge) & !(patient1$Clone_Sequence %in% patientMerge13[one_three,]$merge))
|
|
|
299 two = (grepl(V_Segment, patient2$V_Segment_Major_Gene) & grepl(J_Segment, patient2$J_Segment_Major_Gene) & patient2[,on] > threshhold & !(patient2$Clone_Sequence %in% patientMerge[all,]$merge) & !(patient2$Clone_Sequence %in% patientMerge12[one_two,]$merge) & !(patient2$Clone_Sequence %in% patientMerge23[two_three,]$merge))
|
|
|
300 three = (grepl(V_Segment, patient3$V_Segment_Major_Gene) & grepl(J_Segment, patient3$J_Segment_Major_Gene) & patient3[,on] > threshhold & !(patient3$Clone_Sequence %in% patientMerge[all,]$merge) & !(patient3$Clone_Sequence %in% patientMerge13[one_three,]$merge) & !(patient3$Clone_Sequence %in% patientMerge23[two_three,]$merge))
|
|
20
|
301
|
|
18
|
302 read1Count = append(read1Count, sum(patient1[one,]$normalized_read_count) + sum(patientMerge[all,]$normalized_read_count.x))
|
|
|
303 read2Count = append(read2Count, sum(patient2[two,]$normalized_read_count) + sum(patientMerge[all,]$normalized_read_count.y))
|
|
|
304 read3Count = append(read3Count, sum(patient3[three,]$normalized_read_count) + sum(patientMerge[all,]$normalized_read_count.z))
|
|
7
|
305 res1 = append(res1, sum(one))
|
|
|
306 res2 = append(res2, sum(two))
|
|
|
307 res3 = append(res3, sum(three))
|
|
|
308 resAll = append(resAll, sum(all))
|
|
20
|
309 res12 = append(res12, sum(one_two))
|
|
|
310 res13 = append(res13, sum(one_three))
|
|
|
311 res23 = append(res23, sum(two_three))
|
|
7
|
312 #threshhold = 0
|
|
|
313 if(threshhold != 0){
|
|
|
314 if(sum(one) > 0){
|
|
20
|
315 dfOne = patient1[one,c("V_Segment_Major_Gene", "J_Segment_Major_Gene", "normalized_read_count", "Frequency", "Clone_Sequence", "Related_to_leukemia_clone")]
|
|
|
316 colnames(dfOne) = c("Proximal segment", "Distal segment", "normalized_read_count", "Frequency", "Clone_Sequence", "Related_to_leukemia_clone")
|
|
7
|
317 filenameOne = paste(label1, "_", product[iter, titleIndex], "_", threshhold, sep="")
|
|
|
318 write.table(dfOne, file=paste(filenameOne, ".txt", sep=""), quote=F, sep="\t", dec=",", row.names=F, col.names=T)
|
|
|
319 }
|
|
|
320 if(sum(two) > 0){
|
|
20
|
321 dfTwo = patient2[two,c("V_Segment_Major_Gene", "J_Segment_Major_Gene", "normalized_read_count", "Frequency", "Clone_Sequence", "Related_to_leukemia_clone")]
|
|
|
322 colnames(dfTwo) = c("Proximal segment", "Distal segment", "normalized_read_count", "Frequency", "Clone_Sequence", "Related_to_leukemia_clone")
|
|
7
|
323 filenameTwo = paste(label2, "_", product[iter, titleIndex], "_", threshhold, sep="")
|
|
|
324 write.table(dfTwo, file=paste(filenameTwo, ".txt", sep=""), quote=F, sep="\t", dec=",", row.names=F, col.names=T)
|
|
|
325 }
|
|
|
326 if(sum(three) > 0){
|
|
20
|
327 dfThree = patient3[three,c("V_Segment_Major_Gene", "J_Segment_Major_Gene", "normalized_read_count", "Frequency", "Clone_Sequence", "Related_to_leukemia_clone")]
|
|
|
328 colnames(dfThree) = c("Proximal segment", "Distal segment", "normalized_read_count", "Frequency", "Clone_Sequence", "Related_to_leukemia_clone")
|
|
7
|
329 filenameThree = paste(label3, "_", product[iter, titleIndex], "_", threshhold, sep="")
|
|
|
330 write.table(dfThree, file=paste(filenameThree, ".txt", sep=""), quote=F, sep="\t", dec=",", row.names=F, col.names=T)
|
|
|
331 }
|
|
20
|
332 if(sum(one_two) > 0){
|
|
|
333 dfOne_two = patientMerge12[one_two,c("V_Segment_Major_Gene.x", "J_Segment_Major_Gene.x", "normalized_read_count.x", "Frequency.x", "Related_to_leukemia_clone.x", "Clone_Sequence.x", "V_Segment_Major_Gene.y", "J_Segment_Major_Gene.y", "normalized_read_count.y", "Frequency.y", "Related_to_leukemia_clone.y")]
|
|
|
334 colnames(dfOne_two) = c(paste("Proximal segment", oneSample), paste("Distal segment", oneSample), paste("Normalized_Read_Count", oneSample), paste("Frequency", oneSample), paste("Related_to_leukemia_clone", oneSample),"Clone_Sequence", paste("Proximal segment", twoSample), paste("Distal segment", twoSample), paste("Normalized_Read_Count", twoSample), paste("Frequency", twoSample), paste("Related_to_leukemia_clone", twoSample))
|
|
|
335 filenameOne_two = paste(label1, "_", label2, "_", product[iter, titleIndex], "_", threshhold, onShort, sep="")
|
|
|
336 write.table(dfOne_two, file=paste(filenameOne_two, ".txt", sep=""), quote=F, sep="\t", dec=",", row.names=F, col.names=T)
|
|
|
337 }
|
|
|
338 if(sum(one_three) > 0){
|
|
|
339 dfOne_three = patientMerge13[one_three,c("V_Segment_Major_Gene.x", "J_Segment_Major_Gene.x", "normalized_read_count.x", "Frequency.x", "Related_to_leukemia_clone.x", "Clone_Sequence.x", "V_Segment_Major_Gene.y", "J_Segment_Major_Gene.y", "normalized_read_count.y", "Frequency.y", "Related_to_leukemia_clone.y")]
|
|
|
340 colnames(dfOne_three) = c(paste("Proximal segment", oneSample), paste("Distal segment", oneSample), paste("Normalized_Read_Count", oneSample), paste("Frequency", oneSample), paste("Related_to_leukemia_clone", oneSample),"Clone_Sequence", paste("Proximal segment", threeSample), paste("Distal segment", threeSample), paste("Normalized_Read_Count", threeSample), paste("Frequency", threeSample), paste("Related_to_leukemia_clone", threeSample))
|
|
|
341 filenameOne_three = paste(label1, "_", label3, "_", product[iter, titleIndex], "_", threshhold, onShort, sep="")
|
|
|
342 write.table(dfOne_three, file=paste(filenameOne_three, ".txt", sep=""), quote=F, sep="\t", dec=",", row.names=F, col.names=T)
|
|
|
343 }
|
|
|
344 if(sum(two_three) > 0){
|
|
|
345 dfTwo_three = patientMerge23[two_three,c("V_Segment_Major_Gene.x", "J_Segment_Major_Gene.x", "normalized_read_count.x", "Frequency.x", "Related_to_leukemia_clone.x", "Clone_Sequence.x", "V_Segment_Major_Gene.y", "J_Segment_Major_Gene.y", "normalized_read_count.y", "Frequency.y", "Related_to_leukemia_clone.y")]
|
|
|
346 colnames(dfTwo_three) = c(paste("Proximal segment", twoSample), paste("Distal segment", twoSample), paste("Normalized_Read_Count", twoSample), paste("Frequency", twoSample), paste("Related_to_leukemia_clone", twoSample),"Clone_Sequence", paste("Proximal segment", threeSample), paste("Distal segment", threeSample), paste("Normalized_Read_Count", threeSample), paste("Frequency", threeSample), paste("Related_to_leukemia_clone", threeSample))
|
|
|
347 filenameTwo_three = paste(label2, "_", label3, "_", product[iter, titleIndex], "_", threshhold, onShort, sep="")
|
|
|
348 write.table(dfTwo_three, file=paste(filenameTwo_three, ".txt", sep=""), quote=F, sep="\t", dec=",", row.names=F, col.names=T)
|
|
|
349 }
|
|
|
350 } else { #scatterplot data
|
|
24
|
351 scatterplot_locus_data = scatterplot_data[grepl(V_Segment, scatterplot_data$V_Segment_Major_Gene) & grepl(J_Segment, scatterplot_data$J_Segment_Major_Gene),]
|
|
20
|
352 scatterplot_locus_data$type = ifelse(scatterplot_locus_data$Clone_Sequence %in% patientMerge12[one_two,]$Clone_Sequence.x, "In two", "In one")
|
|
|
353 scatterplot_locus_data$type = ifelse(scatterplot_locus_data$Clone_Sequence %in% patientMerge13[one_three,]$Clone_Sequence.x, "In two", "In one")
|
|
|
354 scatterplot_locus_data$type = ifelse(scatterplot_locus_data$Clone_Sequence %in% patientMerge23[two_three,]$Clone_Sequence.x, "In two", "In one")
|
|
|
355 scatterplot_locus_data$type = ifelse(scatterplot_locus_data$type == "In two", ifelse(scatterplot_locus_data$Clone_Sequence %in% patientMerge[all,]$Clone_Sequence.x, "In three", "In two"), "In one")
|
|
|
356 scatterplot_locus_data$type = ifelse(scatterplot_locus_data$type == "In one", "In one", "In multiple")
|
|
|
357
|
|
|
358 p = NULL
|
|
|
359 if(nrow(scatterplot_locus_data) != 0){
|
|
|
360 if(on == "normalized_read_count"){
|
|
|
361 scales = 10^(0:ceiling(log10(max(scatterplot_locus_data$normalized_read_count))))
|
|
|
362 p = ggplot(scatterplot_locus_data, aes(type, normalized_read_count)) + scale_y_log10(breaks=scales,labels=scales)
|
|
|
363 } else {
|
|
|
364 p = ggplot(scatterplot_locus_data, aes(type, Frequency))
|
|
|
365 }
|
|
|
366 p = p + geom_point(aes(colour=type), position="jitter")
|
|
25
|
367 p = p + xlab("In one or in multiple samples") + ylab(onShort) + ggtitle(paste(label1, label2, label3, onShort, product[iter, titleIndex]))
|
|
20
|
368 } else {
|
|
|
369 p = ggplot(NULL, aes(x=c("In one", "In multiple"),y=0)) + geom_blank(NULL) + xlab("In two or in three of the samples") + ylab(onShort) + ggtitle(paste(label1, label2, label3, onShort, product[iter, titleIndex]))
|
|
|
370 }
|
|
|
371 png(paste(label1, "_", label2, "_", label3, "_", onShort, "_", product[iter, titleIndex],"_scatter.png", sep=""))
|
|
|
372 print(p)
|
|
|
373 dev.off()
|
|
|
374 }
|
|
7
|
375 if(sum(all) > 0){
|
|
20
|
376 dfAll = patientMerge[all,c("V_Segment_Major_Gene.x", "J_Segment_Major_Gene.x", "normalized_read_count.x", "Frequency.x", "Related_to_leukemia_clone.x", "Clone_Sequence.x", "V_Segment_Major_Gene.y", "J_Segment_Major_Gene.y", "normalized_read_count.y", "Frequency.y", "Related_to_leukemia_clone.y", "V_Segment_Major_Gene.z", "J_Segment_Major_Gene.z", "normalized_read_count.z", "Frequency.z", "Related_to_leukemia_clone.z")]
|
|
|
377 colnames(dfAll) = c(paste("Proximal segment", oneSample), paste("Distal segment", oneSample), paste("Normalized_Read_Count", oneSample), paste("Frequency", oneSample), paste("Related_to_leukemia_clone", oneSample),"Clone_Sequence", paste("Proximal segment", twoSample), paste("Distal segment", twoSample), paste("Normalized_Read_Count", twoSample), paste("Frequency", twoSample), paste("Related_to_leukemia_clone", twoSample), paste("Proximal segment", threeSample), paste("Distal segment", threeSample), paste("Normalized_Read_Count", threeSample), paste("Frequency", threeSample), paste("Related_to_leukemia_clone", threeSample))
|
|
7
|
378 filenameAll = paste(label1, "_", label2, "_", label3, "_", product[iter, titleIndex], "_", threshhold, sep="")
|
|
|
379 write.table(dfAll, file=paste(filenameAll, ".txt", sep=""), quote=F, sep="\t", dec=",", row.names=F, col.names=T)
|
|
|
380 }
|
|
|
381 }
|
|
20
|
382 #patientResult = data.frame("Locus"=product$Titles, "J_Segment"=product$J_Segments, "V_Segment"=product$V_Segments, "cut_off_value"=paste(">", product$interval, sep=""), "All"=resAll, "tmp1"=res1, "read_count1" = round(read1Count), "tmp2"=res2, "read_count2"= round(read2Count), "tmp3"=res3, "read_count3"=round(read3Count))
|
|
|
383 patientResult = data.frame("Locus"=product$Titles, "J_Segment"=product$J_Segments, "V_Segment"=product$V_Segments, "cut_off_value"=paste(">", product$interval, sep=""), "All"=resAll, "tmp1"=res1, "tmp2"=res2, "tmp3"=res3, "tmp12"=res12, "tmp13"=res13, "tmp23"=res23)
|
|
7
|
384 colnames(patientResult)[6] = oneSample
|
|
20
|
385 colnames(patientResult)[7] = twoSample
|
|
|
386 colnames(patientResult)[8] = threeSample
|
|
|
387 colnames(patientResult)[9] = paste(oneSample, twoSample, sep="_")
|
|
|
388 colnames(patientResult)[10] = paste(oneSample, twoSample, sep="_")
|
|
|
389 colnames(patientResult)[11] = paste(oneSample, twoSample, sep="_")
|
|
7
|
390
|
|
|
391 colnamesBak = colnames(patientResult)
|
|
20
|
392 colnames(patientResult) = c("Ig/TCR gene rearrangement type", "Distal Gene segment", "Proximal gene segment", "cut_off_value", "Number of sequences All", paste("Number of sequences", oneSample), paste("Number of sequences", twoSample), paste("Number of sequences", threeSample), paste("Number of sequences", oneSample, twoSample), paste("Number of sequences", oneSample, threeSample), paste("Number of sequences", twoSample, threeSample))
|
|
7
|
393 write.table(patientResult, file=paste(label1, "_", label2, "_", label3, "_", onShort, ".txt", sep=""), quote=F, sep="\t", dec=",", row.names=F, col.names=T)
|
|
|
394 colnames(patientResult) = colnamesBak
|
|
|
395
|
|
|
396 patientResult$Locus = factor(patientResult$Locus, Titles)
|
|
|
397 patientResult$cut_off_value = factor(patientResult$cut_off_value, paste(">", interval, sep=""))
|
|
|
398
|
|
|
399 plt = ggplot(patientResult[,c("Locus", "cut_off_value", "All")])
|
|
|
400 plt = plt + geom_bar( aes( x=factor(cut_off_value), y=All), stat='identity', position="dodge", fill="#79c36a")
|
|
|
401 plt = plt + facet_grid(.~Locus) + theme(axis.text.x = element_text(angle = 45, hjust = 1))
|
|
|
402 plt = plt + geom_text(aes(ymax=max(All), x=cut_off_value,y=All,label=All), angle=90, hjust=0)
|
|
|
403 plt = plt + xlab("Reads per locus") + ylab("Count") + ggtitle("Number of clones in All")
|
|
|
404 plt = plt + theme(plot.margin = unit(c(1,8.8,0.5,1.5), "lines"))
|
|
|
405 png(paste(label1, "_", label2, "_", label3, "_", onShort, "_total_all.png", sep=""), width=1920, height=1080)
|
|
|
406 print(plt)
|
|
|
407 dev.off()
|
|
|
408
|
|
|
409 fontSize = 4
|
|
|
410
|
|
|
411 bak = patientResult
|
|
|
412 patientResult = melt(patientResult[,c('Locus','cut_off_value', oneSample, twoSample, threeSample)] ,id.vars=1:2)
|
|
|
413 patientResult$relativeValue = patientResult$value * 10
|
|
|
414 patientResult[patientResult$relativeValue == 0,]$relativeValue = 1
|
|
|
415 plt = ggplot(patientResult)
|
|
|
416 plt = plt + geom_bar( aes( x=factor(cut_off_value), y=relativeValue, fill=variable), stat='identity', position="dodge")
|
|
|
417 plt = plt + facet_grid(.~Locus) + theme(axis.text.x = element_text(angle = 45, hjust = 1))
|
|
|
418 plt = plt + scale_y_continuous(trans="log", breaks=10^c(0:10), labels=c(0, 10^c(0:9)))
|
|
|
419 plt = plt + geom_text(data=patientResult[patientResult$variable == oneSample,], aes(ymax=max(value), x=cut_off_value,y=relativeValue,label=value), angle=90, position=position_dodge(width=0.9), hjust=0, vjust=-0.7, size=fontSize)
|
|
|
420 plt = plt + geom_text(data=patientResult[patientResult$variable == twoSample,], aes(ymax=max(value), x=cut_off_value,y=relativeValue,label=value), angle=90, position=position_dodge(width=0.9), hjust=0, vjust=0.4, size=fontSize)
|
|
|
421 plt = plt + geom_text(data=patientResult[patientResult$variable == threeSample,], aes(ymax=max(value), x=cut_off_value,y=relativeValue,label=value), angle=90, position=position_dodge(width=0.9), hjust=0, vjust=1.5, size=fontSize)
|
|
|
422 plt = plt + xlab("Reads per locus") + ylab("Count") + ggtitle("Number of clones in only one sample")
|
|
|
423 png(paste(label1, "_", label2, "_", label3, "_", onShort, "_indiv_all.png", sep=""), width=1920, height=1080)
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424 print(plt)
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425 dev.off()
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426 }
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427
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9
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428 triplets$uniqueID = "ID"
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429
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430 triplets[grepl("16278_Left", triplets$Sample),]$uniqueID = "16278_26402_26759_Left"
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431 triplets[grepl("26402_Left", triplets$Sample),]$uniqueID = "16278_26402_26759_Left"
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432 triplets[grepl("26759_Left", triplets$Sample),]$uniqueID = "16278_26402_26759_Left"
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433
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434 triplets[grepl("16278_Right", triplets$Sample),]$uniqueID = "16278_26402_26759_Right"
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435 triplets[grepl("26402_Right", triplets$Sample),]$uniqueID = "16278_26402_26759_Right"
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436 triplets[grepl("26759_Right", triplets$Sample),]$uniqueID = "16278_26402_26759_Right"
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437
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438 triplets[grepl("14696", triplets$Patient),]$uniqueID = "14696"
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439
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13
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440 triplets$locus_V = substring(triplets$V_Segment_Major_Gene, 0, 4)
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441 triplets$locus_J = substring(triplets$J_Segment_Major_Gene, 0, 4)
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442 min_cell_count = data.frame(data.table(triplets)[, list(min_cell_count=min(.SD$Cell_Count)), by=c("uniqueID", "locus_V", "locus_J")])
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443
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444 triplets$min_cell_paste = paste(triplets$uniqueID, triplets$locus_V, triplets$locus_J)
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445 min_cell_count$min_cell_paste = paste(min_cell_count$uniqueID, min_cell_count$locus_V, min_cell_count$locus_J)
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446
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447 min_cell_count = min_cell_count[,c("min_cell_paste", "min_cell_count")]
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9
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448
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13
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449 triplets = merge(triplets, min_cell_count, by="min_cell_paste")
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450
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451 triplets$normalized_read_count = round(triplets$Clone_Molecule_Count_From_Spikes / triplets$Cell_Count * triplets$min_cell_count / 2, digits=2) #??????????????????????????????????? wel of geen / 2
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452
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453 triplets = triplets[triplets$normalized_read_count >= min_cells,]
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454
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455 column_drops = c("locus_V", "locus_J", "min_cell_count", "min_cell_paste")
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456
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457 triplets = triplets[,!(colnames(triplets) %in% column_drops)]
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9
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458
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7
|
459 interval = intervalReads
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460 intervalOrder = data.frame("interval"=paste(">", interval, sep=""), "intervalOrder"=1:length(interval))
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461 product = data.frame("Titles"=rep(Titles, each=length(interval)), "interval"=rep(interval, times=10), "V_Segments"=rep(V_Segments, each=length(interval)), "J_Segments"=rep(J_Segments, each=length(interval)))
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462
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9
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463 one = triplets[triplets$Sample == "14696_reg_BM",]
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464 two = triplets[triplets$Sample == "24536_reg_BM",]
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465 three = triplets[triplets$Sample == "24062_reg_BM",]
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8
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466 tripletAnalysis(one, "14696_1", two, "14696_2", three, "14696_3", product=product, interval=interval, on="normalized_read_count", T)
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7
|
467
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9
|
468 one = triplets[triplets$Sample == "16278_Left",]
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469 two = triplets[triplets$Sample == "26402_Left",]
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470 three = triplets[triplets$Sample == "26759_Left",]
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8
|
471 tripletAnalysis(one, "16278_Left", two, "26402_Left", three, "26759_Left", product=product, interval=interval, on="normalized_read_count", T)
|
|
7
|
472
|
|
9
|
473 one = triplets[triplets$Sample == "16278_Right",]
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474 two = triplets[triplets$Sample == "26402_Right",]
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475 three = triplets[triplets$Sample == "26759_Right",]
|
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8
|
476 tripletAnalysis(one, "16278_Right", two, "26402_Right", three, "26759_Right", product=product, interval=interval, on="normalized_read_count", T)
|
|
7
|
477
|
|
|
478
|
|
|
479 interval = intervalFreq
|
|
|
480 intervalOrder = data.frame("interval"=paste(">", interval, sep=""), "intervalOrder"=1:length(interval))
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481 product = data.frame("Titles"=rep(Titles, each=length(interval)), "interval"=rep(interval, times=10), "V_Segments"=rep(V_Segments, each=length(interval)), "J_Segments"=rep(J_Segments, each=length(interval)))
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|
|
482
|
|
9
|
483 one = triplets[triplets$Sample == "14696_reg_BM",]
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|
|
484 two = triplets[triplets$Sample == "24536_reg_BM",]
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|
|
485 three = triplets[triplets$Sample == "24062_reg_BM",]
|
|
8
|
486 tripletAnalysis(one, "14696_1", two, "14696_2", three, "14696_3", product=product, interval=interval, on="Frequency", F)
|
|
7
|
487
|
|
9
|
488 one = triplets[triplets$Sample == "16278_Left",]
|
|
|
489 two = triplets[triplets$Sample == "26402_Left",]
|
|
|
490 three = triplets[triplets$Sample == "26759_Left",]
|
|
8
|
491 tripletAnalysis(one, "16278_Left", two, "26402_Left", three, "26759_Left", product=product, interval=interval, on="Frequency", F)
|
|
7
|
492
|
|
9
|
493 one = triplets[triplets$Sample == "16278_Right",]
|
|
|
494 two = triplets[triplets$Sample == "26402_Right",]
|
|
|
495 three = triplets[triplets$Sample == "26759_Right",]
|
|
8
|
496 tripletAnalysis(one, "16278_Right", two, "26402_Right", three, "26759_Right", product=product, interval=interval, on="Frequency", F)
|
