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0
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1 args <- commandArgs(trailingOnly = TRUE)
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2
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3
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4 summaryfile = args[1]
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5 sequencesfile = args[2]
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6 mutationanalysisfile = args[3]
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7 mutationstatsfile = args[4]
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8 hotspotsfile = args[5]
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14
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9 aafile = args[6]
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10 gene_identification_file= args[7]
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11 output = args[8]
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12 before.unique.file = args[9]
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13 unmatchedfile = args[10]
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14 method=args[11]
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15 functionality=args[12]
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16 unique.type=args[13]
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17 filter.unique=args[14]
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18 class.filter=args[15]
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19 empty.region.filter=args[16]
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0
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20
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21 summ = read.table(summaryfile, header=T, sep="\t", fill=T, stringsAsFactors=F, quote="")
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22 sequences = read.table(sequencesfile, header=T, sep="\t", fill=T, stringsAsFactors=F, quote="")
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23 mutationanalysis = read.table(mutationanalysisfile, header=T, sep="\t", fill=T, stringsAsFactors=F, quote="")
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24 mutationstats = read.table(mutationstatsfile, header=T, sep="\t", fill=T, stringsAsFactors=F, quote="")
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25 hotspots = read.table(hotspotsfile, header=T, sep="\t", fill=T, stringsAsFactors=F, quote="")
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14
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26 AAs = read.table(aafile, header=T, sep="\t", fill=T, stringsAsFactors=F, quote="")
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0
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27 gene_identification = read.table(gene_identification_file, header=T, sep="\t", fill=T, stringsAsFactors=F, quote="")
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28
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29 if(method == "blastn"){
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10
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30 #"qseqid\tsseqid\tpident\tlength\tmismatch\tgapopen\tqstart\tqend\tsstart\tsend\tevalue\tbitscore"
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0
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31 gene_identification = gene_identification[!duplicated(gene_identification$qseqid),]
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32 ref_length = data.frame(sseqid=c("ca1", "ca2", "cg1", "cg2", "cg3", "cg4", "cm"), ref.length=c(81,81,141,141,141,141,52))
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33 gene_identification = merge(gene_identification, ref_length, by="sseqid", all.x=T)
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34 gene_identification$chunk_hit_percentage = (gene_identification$length / gene_identification$ref.length) * 100
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35 gene_identification = gene_identification[,c("qseqid", "chunk_hit_percentage", "pident", "qstart", "sseqid")]
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36 colnames(gene_identification) = c("Sequence.ID", "chunk_hit_percentage", "nt_hit_percentage", "start_locations", "best_match")
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37 }
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38
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39 input.sequence.count = nrow(summ)
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40 print(paste("Number of sequences in summary file:", input.sequence.count))
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41
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42 filtering.steps = data.frame(character(0), numeric(0))
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43
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44 filtering.steps = rbind(filtering.steps, c("Input", input.sequence.count))
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45
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46 filtering.steps[,1] = as.character(filtering.steps[,1])
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47 filtering.steps[,2] = as.character(filtering.steps[,2])
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48 #filtering.steps[,3] = as.numeric(filtering.steps[,3])
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49
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50 summ = merge(summ, gene_identification, by="Sequence.ID")
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51
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52 summ = summ[summ$Functionality != "No results",]
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53
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54 print(paste("Number of sequences after 'No results' filter:", nrow(summ)))
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55
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56 filtering.steps = rbind(filtering.steps, c("After 'No results' filter", nrow(summ)))
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57
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58 if(functionality == "productive"){
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59 summ = summ[summ$Functionality == "productive (see comment)" | summ$Functionality == "productive",]
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60 } else if (functionality == "unproductive"){
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61 summ = summ[summ$Functionality == "unproductive (see comment)" | summ$Functionality == "unproductive",]
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62 } else if (functionality == "remove_unknown"){
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63 summ = summ[summ$Functionality != "No results" & summ$Functionality != "unknown (see comment)" & summ$Functionality != "unknown",]
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64 }
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65
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1
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66 print(paste("Number of sequences after functionality filter:", nrow(summ)))
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0
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67
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1
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68 filtering.steps = rbind(filtering.steps, c("After functionality filter", nrow(summ)))
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0
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69
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70 result = merge(summ, mutationanalysis[,!(names(mutationanalysis) %in% names(summ)[-1])], by="Sequence.ID")
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71
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72 print(paste("Number of sequences after merging with mutation analysis file:", nrow(result)))
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73
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74 result = merge(result, mutationstats[,!(names(mutationstats) %in% names(result)[-1])], by="Sequence.ID")
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75
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76 print(paste("Number of sequences after merging with mutation stats file:", nrow(result)))
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77
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78 result = merge(result, hotspots[,!(names(hotspots) %in% names(result)[-1])], by="Sequence.ID")
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79
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80 print(paste("Number of sequences after merging with hotspots file:", nrow(result)))
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81
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82 sequences = sequences[,c("Sequence.ID", "FR1.IMGT", "CDR1.IMGT", "FR2.IMGT", "CDR2.IMGT", "FR3.IMGT", "CDR3.IMGT")]
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83 names(sequences) = c("Sequence.ID", "FR1.IMGT.seq", "CDR1.IMGT.seq", "FR2.IMGT.seq", "CDR2.IMGT.seq", "FR3.IMGT.seq", "CDR3.IMGT.seq")
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84 result = merge(result, sequences, by="Sequence.ID", all.x=T)
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85
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14
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86 AAs = AAs[,c("Sequence.ID", "CDR3.IMGT")]
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87 names(AAs) = c("Sequence.ID", "CDR3.IMGT.AA")
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88 result = merge(result, AAs, by="Sequence.ID", all.x=T)
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89
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0
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90 print(paste("Number of sequences in result after merging with sequences:", nrow(result)))
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91
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92 result$VGene = gsub("^Homsap ", "", result$V.GENE.and.allele)
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93 result$VGene = gsub("[*].*", "", result$VGene)
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94 result$DGene = gsub("^Homsap ", "", result$D.GENE.and.allele)
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95 result$DGene = gsub("[*].*", "", result$DGene)
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96 result$JGene = gsub("^Homsap ", "", result$J.GENE.and.allele)
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97 result$JGene = gsub("[*].*", "", result$JGene)
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98
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16
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99 print(result[result$Sequence.ID == "JY8QFUQ01C310D",c("Sequence.ID","best_match")])
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100
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12
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101 splt = strsplit(class.filter, "_")[[1]]
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102 chunk_hit_threshold = as.numeric(splt[1])
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103 nt_hit_threshold = as.numeric(splt[2])
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104
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105 higher_than=(result$chunk_hit_percentage >= chunk_hit_threshold & result$nt_hit_percentage >= nt_hit_threshold)
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106
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107 if(!all(higher_than, na.rm=T)){ #check for no unmatched
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108 result[!higher_than,"best_match"] = paste("unmatched,", result[!higher_than,"best_match"])
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109 }
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110
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111 if(class.filter == "101_101"){
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112 result$best_match = "all"
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113 }
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114
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16
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115 print(result[result$Sequence.ID == "JY8QFUQ01C310D",c("Sequence.ID","best_match")])
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116
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12
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117 write.table(x=result, file=gsub("merged.txt$", "before_filters.txt", output), sep="\t",quote=F,row.names=F,col.names=T)
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118
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0
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119 print(paste("Number of empty CDR1 sequences:", sum(result$CDR1.IMGT.seq == "")))
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120 print(paste("Number of empty FR2 sequences:", sum(result$FR2.IMGT.seq == "")))
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121 print(paste("Number of empty CDR2 sequences:", sum(result$CDR2.IMGT.seq == "")))
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122 print(paste("Number of empty FR3 sequences:", sum(result$FR3.IMGT.seq == "")))
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123
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1
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124 if(empty.region.filter == "leader"){
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125 result = result[result$FR1.IMGT.seq != "" & result$CDR1.IMGT.seq != "" & result$FR2.IMGT.seq != "" & result$CDR2.IMGT.seq != "" & result$FR3.IMGT.seq != "", ]
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126 print(paste("Number of sequences after empty FR1, CDR1, FR2, CDR2 and FR3 column filter:", nrow(result)))
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127 filtering.steps = rbind(filtering.steps, c("After empty FR1, CDR1, FR2, CDR2, FR3 filter", nrow(result)))
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128 } else if(empty.region.filter == "FR1"){
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0
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129 result = result[result$CDR1.IMGT.seq != "" & result$FR2.IMGT.seq != "" & result$CDR2.IMGT.seq != "" & result$FR3.IMGT.seq != "", ]
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130 print(paste("Number of sequences after empty CDR1, FR2, CDR2 and FR3 column filter:", nrow(result)))
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131 filtering.steps = rbind(filtering.steps, c("After empty CDR1, FR2, CDR2, FR3 filter", nrow(result)))
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132 } else if(empty.region.filter == "CDR1"){
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133 result = result[result$FR2.IMGT.seq != "" & result$CDR2.IMGT.seq != "" & result$FR3.IMGT.seq != "", ]
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134 print(paste("Number of sequences after empty FR2, CDR2 and FR3 column filter:", nrow(result)))
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135 filtering.steps = rbind(filtering.steps, c("After empty FR2, CDR2, FR3 filter", nrow(result)))
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136 } else if(empty.region.filter == "FR2"){
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137 result = result[result$CDR2.IMGT.seq != "" & result$FR3.IMGT.seq != "", ]
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138 print(paste("Number of sequences after empty CDR2 and FR3 column filter:", nrow(result)))
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139 filtering.steps = rbind(filtering.steps, c("After empty CDR2, FR3 filter", nrow(result)))
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140 }
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141
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16
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142 print(result[result$Sequence.ID == "JY8QFUQ01C310D",c("Sequence.ID","best_match")])
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143
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1
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144 if(empty.region.filter == "leader"){
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145 result = result[!(grepl("n|N", result$FR1.IMGT.seq) | grepl("n|N", result$FR2.IMGT.seq) | grepl("n|N", result$FR3.IMGT.seq) | grepl("n|N", result$CDR1.IMGT.seq) | grepl("n|N", result$CDR2.IMGT.seq) | grepl("n|N", result$CDR3.IMGT.seq)),]
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146 } else if(empty.region.filter == "FR1"){
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0
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147 result = result[!(grepl("n|N", result$FR2.IMGT.seq) | grepl("n|N", result$FR3.IMGT.seq) | grepl("n|N", result$CDR1.IMGT.seq) | grepl("n|N", result$CDR2.IMGT.seq) | grepl("n|N", result$CDR3.IMGT.seq)),]
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148 } else if(empty.region.filter == "CDR1"){
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149 result = result[!(grepl("n|N", result$FR2.IMGT.seq) | grepl("n|N", result$FR3.IMGT.seq) | grepl("n|N", result$CDR2.IMGT.seq) | grepl("n|N", result$CDR3.IMGT.seq)),]
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150 } else if(empty.region.filter == "FR2"){
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2
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151 result = result[!(grepl("n|N", result$FR3.IMGT.seq) | grepl("n|N", result$CDR2.IMGT.seq) | grepl("n|N", result$CDR3.IMGT.seq)),]
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0
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152 }
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153
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16
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154 print(result[result$Sequence.ID == "JY8QFUQ01C310D",c("Sequence.ID","best_match")])
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155 print(result[result$Sequence.ID == "JY8QFUQ01BNS72",c("Sequence.ID","best_match")])
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156
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0
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157 print(paste("Number of sequences in result after n filtering:", nrow(result)))
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158 filtering.steps = rbind(filtering.steps, c("After N filter", nrow(result)))
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159
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160 cleanup_columns = c("FR1.IMGT.Nb.of.mutations",
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161 "CDR1.IMGT.Nb.of.mutations",
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162 "FR2.IMGT.Nb.of.mutations",
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163 "CDR2.IMGT.Nb.of.mutations",
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164 "FR3.IMGT.Nb.of.mutations")
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165
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166 for(col in cleanup_columns){
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167 result[,col] = gsub("\\(.*\\)", "", result[,col])
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168 result[,col] = as.numeric(result[,col])
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169 result[is.na(result[,col]),] = 0
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170 }
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171
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5
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172 write.table(result, before.unique.file, sep="\t", quote=F,row.names=F,col.names=T)
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173
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174 if(filter.unique != "no"){
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175 clmns = names(result)
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176
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177 if(empty.region.filter == "leader"){
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178 result$unique.def = paste(result$FR1.IMGT.seq, result$CDR1.IMGT.seq, result$FR2.IMGT.seq, result$CDR2.IMGT.seq, result$FR3.IMGT.seq, result$CDR3.IMGT.seq)
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179 } else if(empty.region.filter == "FR1"){
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180 result$unique.def = paste(result$CDR1.IMGT.seq, result$FR2.IMGT.seq, result$CDR2.IMGT.seq, result$FR3.IMGT.seq, result$CDR3.IMGT.seq)
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181 } else if(empty.region.filter == "CDR1"){
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182 result$unique.def = paste(result$FR2.IMGT.seq, result$CDR2.IMGT.seq, result$FR3.IMGT.seq, result$CDR3.IMGT.seq)
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183 } else if(empty.region.filter == "FR2"){
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184 result$unique.def = paste(result$CDR2.IMGT.seq, result$FR3.IMGT.seq, result$CDR3.IMGT.seq)
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185 }
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12
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186
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15
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187 if(filter.unique == "remove"){
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5
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188 result = result[duplicated(result$unique.def) | duplicated(result$unique.def, fromLast=T),]
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189 }
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15
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190 result$unique.def = paste(result$unique.def, gsub(",.*", "", result$best_match)) #keep the unique sequences that are in multiple classes, gsub so the unmatched don't have a class after it
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191
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192 result = result[!duplicated(result$unique.def),]
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1
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193 }
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194
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16
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195 print(result[result$Sequence.ID == "JY8QFUQ01C310D",c("Sequence.ID","best_match")])
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196 print(result[result$Sequence.ID == "JY8QFUQ01BNS72",c("Sequence.ID","best_match")])
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197
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5
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198 write.table(result, gsub("before_unique_filter.txt", "after_unique_filter.txt", before.unique.file), sep="\t", quote=F,row.names=F,col.names=T)
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199
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200 filtering.steps = rbind(filtering.steps, c("After filter unique sequences", nrow(result)))
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201
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1
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202 if(nrow(summ) == 0){
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203 stop("No data remaining after filter")
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204 }
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205
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13
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206 result$best_match_class = gsub(",.*", "", result$best_match) #gsub so the unmatched don't have a class after it
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207
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1
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208 result$past = do.call(paste, c(result[unlist(strsplit(unique.type, ","))], sep = ":"))
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209
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15
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210
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211
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212
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213 result.matched = result[!grepl("unmatched", result$best_match),]
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214 result.unmatched = result[grepl("unmatched", result$best_match),]
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215
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216 result = rbind(result.matched, result.unmatched)
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217
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1
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218 result = result[!(duplicated(result$past)), ]
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219
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15
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220
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221
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16
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222 print(result[result$Sequence.ID == "JY8QFUQ01C310D",c("Sequence.ID","best_match")])
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15
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223
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224
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13
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225 result = result[,!(names(result) %in% c("past", "best_match_class"))]
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1
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226
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227 print(paste("Number of sequences in result after", unique.type, "filtering:", nrow(result)))
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228
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229 filtering.steps = rbind(filtering.steps, c("After remove duplicates based on filter", nrow(result)))
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0
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230
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10
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231 unmatched = result[grepl("^unmatched", result$best_match),c("Sequence.ID", "chunk_hit_percentage", "nt_hit_percentage", "start_locations", "best_match")]
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232
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0
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233 print(paste("Number of rows in result:", nrow(result)))
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234 print(paste("Number of rows in unmatched:", nrow(unmatched)))
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235
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236 matched.sequences = result[!grepl("^unmatched", result$best_match),]
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237
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238 write.table(x=matched.sequences, file=gsub("merged.txt$", "filtered.txt", output), sep="\t",quote=F,row.names=F,col.names=T)
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239
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240 matched.sequences.count = nrow(matched.sequences)
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241 unmatched.sequences.count = sum(grepl("^unmatched", result$best_match))
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242
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243 filtering.steps = rbind(filtering.steps, c("Number of matched sequences", matched.sequences.count))
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244 filtering.steps = rbind(filtering.steps, c("Number of unmatched sequences", unmatched.sequences.count))
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245 filtering.steps[,2] = as.numeric(filtering.steps[,2])
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246 filtering.steps$perc = round(filtering.steps[,2] / input.sequence.count * 100, 2)
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247
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248 write.table(x=filtering.steps, file=gsub("unmatched", "filtering_steps", unmatchedfile), sep="\t",quote=F,row.names=F,col.names=F)
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249
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250 write.table(x=result, file=output, sep="\t",quote=F,row.names=F,col.names=T)
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251 write.table(x=unmatched, file=unmatchedfile, sep="\t",quote=F,row.names=F,col.names=T)
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