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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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49
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18 filter.unique.count=as.numeric(args[15])
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19 class.filter=args[16]
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20 empty.region.filter=args[17]
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21
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22 print(paste("filter.unique.count:", filter.unique.count))
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0
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23
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24 summ = read.table(summaryfile, header=T, sep="\t", fill=T, stringsAsFactors=F, quote="")
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25 sequences = read.table(sequencesfile, header=T, sep="\t", fill=T, stringsAsFactors=F, quote="")
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26 mutationanalysis = read.table(mutationanalysisfile, header=T, sep="\t", fill=T, stringsAsFactors=F, quote="")
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27 mutationstats = read.table(mutationstatsfile, header=T, sep="\t", fill=T, stringsAsFactors=F, quote="")
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28 hotspots = read.table(hotspotsfile, header=T, sep="\t", fill=T, stringsAsFactors=F, quote="")
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14
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29 AAs = read.table(aafile, header=T, sep="\t", fill=T, stringsAsFactors=F, quote="")
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0
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30 gene_identification = read.table(gene_identification_file, header=T, sep="\t", fill=T, stringsAsFactors=F, quote="")
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31
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47
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32 fix_column_names = function(df){
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33 if("V.DOMAIN.Functionality" %in% names(df)){
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34 names(df)[names(df) == "V.DOMAIN.Functionality"] = "Functionality"
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35 print("found V.DOMAIN.Functionality, changed")
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36 }
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37 if("V.DOMAIN.Functionality.comment" %in% names(df)){
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38 names(df)[names(df) == "V.DOMAIN.Functionality.comment"] = "Functionality.comment"
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39 print("found V.DOMAIN.Functionality.comment, changed")
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40 }
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41 return(df)
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42 }
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43
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44 summ = fix_column_names(summ)
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45 sequences = fix_column_names(sequences)
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46 mutationanalysis = fix_column_names(mutationanalysis)
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47 mutationstats = fix_column_names(mutationstats)
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48 hotspots = fix_column_names(hotspots)
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49 AAs = fix_column_names(AAs)
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50
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0
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51 if(method == "blastn"){
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10
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52 #"qseqid\tsseqid\tpident\tlength\tmismatch\tgapopen\tqstart\tqend\tsstart\tsend\tevalue\tbitscore"
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0
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53 gene_identification = gene_identification[!duplicated(gene_identification$qseqid),]
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54 ref_length = data.frame(sseqid=c("ca1", "ca2", "cg1", "cg2", "cg3", "cg4", "cm"), ref.length=c(81,81,141,141,141,141,52))
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55 gene_identification = merge(gene_identification, ref_length, by="sseqid", all.x=T)
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56 gene_identification$chunk_hit_percentage = (gene_identification$length / gene_identification$ref.length) * 100
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57 gene_identification = gene_identification[,c("qseqid", "chunk_hit_percentage", "pident", "qstart", "sseqid")]
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58 colnames(gene_identification) = c("Sequence.ID", "chunk_hit_percentage", "nt_hit_percentage", "start_locations", "best_match")
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59 }
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60
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47
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61 #print("Summary analysis files columns")
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62 #print(names(summ))
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41
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63
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64
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65
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0
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66 input.sequence.count = nrow(summ)
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67 print(paste("Number of sequences in summary file:", input.sequence.count))
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68
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69 filtering.steps = data.frame(character(0), numeric(0))
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70
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71 filtering.steps = rbind(filtering.steps, c("Input", input.sequence.count))
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72
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73 filtering.steps[,1] = as.character(filtering.steps[,1])
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74 filtering.steps[,2] = as.character(filtering.steps[,2])
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75 #filtering.steps[,3] = as.numeric(filtering.steps[,3])
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76
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40
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77 #print("summary files columns")
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78 #print(names(summ))
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38
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79
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0
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80 summ = merge(summ, gene_identification, by="Sequence.ID")
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81
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82 summ = summ[summ$Functionality != "No results",]
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83
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84 print(paste("Number of sequences after 'No results' filter:", nrow(summ)))
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85
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86 filtering.steps = rbind(filtering.steps, c("After 'No results' filter", nrow(summ)))
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87
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88 if(functionality == "productive"){
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89 summ = summ[summ$Functionality == "productive (see comment)" | summ$Functionality == "productive",]
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90 } else if (functionality == "unproductive"){
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91 summ = summ[summ$Functionality == "unproductive (see comment)" | summ$Functionality == "unproductive",]
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92 } else if (functionality == "remove_unknown"){
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93 summ = summ[summ$Functionality != "No results" & summ$Functionality != "unknown (see comment)" & summ$Functionality != "unknown",]
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94 }
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95
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1
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96 print(paste("Number of sequences after functionality filter:", nrow(summ)))
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0
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97
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1
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98 filtering.steps = rbind(filtering.steps, c("After functionality filter", nrow(summ)))
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0
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99
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47
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100 if(FALSE){ #to speed up debugging
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101 set.seed(1)
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49
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102 summ = summ[sample(nrow(summ), floor(nrow(summ) * 0.1)),]
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47
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103 print(paste("Number of sequences after sampling 5%:", nrow(summ)))
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104
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105 filtering.steps = rbind(filtering.steps, c("Number of sequences after sampling 5%", nrow(summ)))
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106 }
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107
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41
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108 print("mutation analysis files columns")
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109 print(names(mutationanalysis[,!(names(mutationanalysis) %in% names(summ)[-1])]))
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38
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110
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0
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111 result = merge(summ, mutationanalysis[,!(names(mutationanalysis) %in% names(summ)[-1])], by="Sequence.ID")
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112
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113 print(paste("Number of sequences after merging with mutation analysis file:", nrow(result)))
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114
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47
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115 #print("mutation stats files columns")
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116 #print(names(mutationstats[,!(names(mutationstats) %in% names(result)[-1])]))
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38
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117
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0
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118 result = merge(result, mutationstats[,!(names(mutationstats) %in% names(result)[-1])], by="Sequence.ID")
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119
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120 print(paste("Number of sequences after merging with mutation stats file:", nrow(result)))
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121
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41
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122 print("hotspots files columns")
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123 print(names(hotspots[,!(names(hotspots) %in% names(result)[-1])]))
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38
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124
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0
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125 result = merge(result, hotspots[,!(names(hotspots) %in% names(result)[-1])], by="Sequence.ID")
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126
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127 print(paste("Number of sequences after merging with hotspots file:", nrow(result)))
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128
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41
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129 print("sequences files columns")
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130 print(c("FR1.IMGT", "CDR1.IMGT", "FR2.IMGT", "CDR2.IMGT", "FR3.IMGT", "CDR3.IMGT"))
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131
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0
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132 sequences = sequences[,c("Sequence.ID", "FR1.IMGT", "CDR1.IMGT", "FR2.IMGT", "CDR2.IMGT", "FR3.IMGT", "CDR3.IMGT")]
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133 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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134 result = merge(result, sequences, by="Sequence.ID", all.x=T)
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135
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41
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136 print("sequences files columns")
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137 print("CDR3.IMGT")
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138
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14
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139 AAs = AAs[,c("Sequence.ID", "CDR3.IMGT")]
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140 names(AAs) = c("Sequence.ID", "CDR3.IMGT.AA")
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141 result = merge(result, AAs, by="Sequence.ID", all.x=T)
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142
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0
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143 print(paste("Number of sequences in result after merging with sequences:", nrow(result)))
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144
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145 result$VGene = gsub("^Homsap ", "", result$V.GENE.and.allele)
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146 result$VGene = gsub("[*].*", "", result$VGene)
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147 result$DGene = gsub("^Homsap ", "", result$D.GENE.and.allele)
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148 result$DGene = gsub("[*].*", "", result$DGene)
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149 result$JGene = gsub("^Homsap ", "", result$J.GENE.and.allele)
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150 result$JGene = gsub("[*].*", "", result$JGene)
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151
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12
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152 splt = strsplit(class.filter, "_")[[1]]
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153 chunk_hit_threshold = as.numeric(splt[1])
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154 nt_hit_threshold = as.numeric(splt[2])
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155
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156 higher_than=(result$chunk_hit_percentage >= chunk_hit_threshold & result$nt_hit_percentage >= nt_hit_threshold)
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157
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158 if(!all(higher_than, na.rm=T)){ #check for no unmatched
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159 result[!higher_than,"best_match"] = paste("unmatched,", result[!higher_than,"best_match"])
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160 }
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161
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162 if(class.filter == "101_101"){
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163 result$best_match = "all"
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164 }
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165
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166 write.table(x=result, file=gsub("merged.txt$", "before_filters.txt", output), sep="\t",quote=F,row.names=F,col.names=T)
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167
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47
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168 print(paste("Number of empty CDR1 sequences:", sum(result$CDR1.IMGT.seq == "", na.rm=T)))
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169 print(paste("Number of empty FR2 sequences:", sum(result$FR2.IMGT.seq == "", na.rm=T)))
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170 print(paste("Number of empty CDR2 sequences:", sum(result$CDR2.IMGT.seq == "", na.rm=T)))
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171 print(paste("Number of empty FR3 sequences:", sum(result$FR3.IMGT.seq == "", na.rm=T)))
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0
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172
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1
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173 if(empty.region.filter == "leader"){
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174 result = result[result$FR1.IMGT.seq != "" & result$CDR1.IMGT.seq != "" & result$FR2.IMGT.seq != "" & result$CDR2.IMGT.seq != "" & result$FR3.IMGT.seq != "", ]
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175 } else if(empty.region.filter == "FR1"){
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0
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176 result = result[result$CDR1.IMGT.seq != "" & result$FR2.IMGT.seq != "" & result$CDR2.IMGT.seq != "" & result$FR3.IMGT.seq != "", ]
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177 } else if(empty.region.filter == "CDR1"){
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178 result = result[result$FR2.IMGT.seq != "" & result$CDR2.IMGT.seq != "" & result$FR3.IMGT.seq != "", ]
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179 } else if(empty.region.filter == "FR2"){
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180 result = result[result$CDR2.IMGT.seq != "" & result$FR3.IMGT.seq != "", ]
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181 }
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182
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30
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183 print(paste("After removal sequences that are missing a gene region:", nrow(result)))
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184 filtering.steps = rbind(filtering.steps, c("After removal sequences that are missing a gene region", nrow(result)))
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185
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1
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186 if(empty.region.filter == "leader"){
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187 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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188 } else if(empty.region.filter == "FR1"){
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0
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189 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$CDR3.IMGT.seq)),]
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190 } else if(empty.region.filter == "CDR1"){
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191 result = result[!(grepl("n|N", result$FR2.IMGT.seq) | grepl("n|N", result$FR3.IMGT.seq) | grepl("n|N", result$CDR2.IMGT.seq) | grepl("n|N", result$CDR3.IMGT.seq)),]
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192 } else if(empty.region.filter == "FR2"){
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2
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193 result = result[!(grepl("n|N", result$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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194 }
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195
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196 print(paste("Number of sequences in result after n filtering:", nrow(result)))
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197 filtering.steps = rbind(filtering.steps, c("After N filter", nrow(result)))
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198
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199 cleanup_columns = c("FR1.IMGT.Nb.of.mutations",
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200 "CDR1.IMGT.Nb.of.mutations",
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201 "FR2.IMGT.Nb.of.mutations",
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202 "CDR2.IMGT.Nb.of.mutations",
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203 "FR3.IMGT.Nb.of.mutations")
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204
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205 for(col in cleanup_columns){
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206 result[,col] = gsub("\\(.*\\)", "", result[,col])
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207 result[,col] = as.numeric(result[,col])
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208 result[is.na(result[,col]),] = 0
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209 }
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210
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5
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211 write.table(result, before.unique.file, sep="\t", quote=F,row.names=F,col.names=T)
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212
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213 if(filter.unique != "no"){
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214 clmns = names(result)
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215 if(empty.region.filter == "leader"){
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216 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$CDR3.IMGT.seq)
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217 } else if(empty.region.filter == "FR1"){
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218 result$unique.def = paste(result$CDR1.IMGT.seq, result$FR2.IMGT.seq, result$CDR2.IMGT.seq, result$FR3.IMGT.seq, result$CDR3.IMGT.seq)
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219 } else if(empty.region.filter == "CDR1"){
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220 result$unique.def = paste(result$FR2.IMGT.seq, result$CDR2.IMGT.seq, result$FR3.IMGT.seq, result$CDR3.IMGT.seq)
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221 } else if(empty.region.filter == "FR2"){
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222 result$unique.def = paste(result$CDR2.IMGT.seq, result$FR3.IMGT.seq, result$CDR3.IMGT.seq)
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223 }
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12
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224
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15
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225 if(filter.unique == "remove"){
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5
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226 result = result[duplicated(result$unique.def) | duplicated(result$unique.def, fromLast=T),]
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227 }
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40
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228
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15
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229 result$unique.def = paste(result$unique.def, gsub(",.*", "", result$best_match)) #keep the unique sequences that are in multiple classes, gsub so the unmatched don't have a class after it
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230
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49
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231 if(filter.unique == "remove"){
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232 unique.defs = data.frame(table(result$unique.def))
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233 unique.defs = unique.defs[unique.defs$Freq >= filter.unique.count,]
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234 result = result[result$unique.def %in% unique.defs$Var1,]
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235 }
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236
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15
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237 result = result[!duplicated(result$unique.def),]
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1
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238 }
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239
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5
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240 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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241
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242 filtering.steps = rbind(filtering.steps, c("After filter unique sequences", nrow(result)))
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243
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40
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244 print(paste("Number of sequences in result after unique filtering:", nrow(result)))
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245
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1
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246 if(nrow(summ) == 0){
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247 stop("No data remaining after filter")
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248 }
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249
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13
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250 result$best_match_class = gsub(",.*", "", result$best_match) #gsub so the unmatched don't have a class after it
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251
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40
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252 #result$past = ""
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253 #cls = unlist(strsplit(unique.type, ","))
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254 #for (i in 1:nrow(result)){
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255 # result[i,"past"] = paste(result[i,cls], collapse=":")
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256 #}
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1
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257
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47
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258
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259
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40
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260 result$past = do.call(paste, c(result[unlist(strsplit(unique.type, ","))], sep = ":"))
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15
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261
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262 result.matched = result[!grepl("unmatched", result$best_match),]
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263 result.unmatched = result[grepl("unmatched", result$best_match),]
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264
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265 result = rbind(result.matched, result.unmatched)
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266
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1
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267 result = result[!(duplicated(result$past)), ]
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268
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13
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269 result = result[,!(names(result) %in% c("past", "best_match_class"))]
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1
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270
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271 print(paste("Number of sequences in result after", unique.type, "filtering:", nrow(result)))
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272
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273 filtering.steps = rbind(filtering.steps, c("After remove duplicates based on filter", nrow(result)))
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0
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274
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10
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275 unmatched = result[grepl("^unmatched", result$best_match),c("Sequence.ID", "chunk_hit_percentage", "nt_hit_percentage", "start_locations", "best_match")]
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276
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0
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277 print(paste("Number of rows in result:", nrow(result)))
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278 print(paste("Number of rows in unmatched:", nrow(unmatched)))
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279
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280 matched.sequences = result[!grepl("^unmatched", result$best_match),]
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281
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282 write.table(x=matched.sequences, file=gsub("merged.txt$", "filtered.txt", output), sep="\t",quote=F,row.names=F,col.names=T)
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283
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284 matched.sequences.count = nrow(matched.sequences)
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285 unmatched.sequences.count = sum(grepl("^unmatched", result$best_match))
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286
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287 filtering.steps = rbind(filtering.steps, c("Number of matched sequences", matched.sequences.count))
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288 filtering.steps = rbind(filtering.steps, c("Number of unmatched sequences", unmatched.sequences.count))
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289 filtering.steps[,2] = as.numeric(filtering.steps[,2])
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290 filtering.steps$perc = round(filtering.steps[,2] / input.sequence.count * 100, 2)
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291
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292 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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293
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294 write.table(x=result, file=output, sep="\t",quote=F,row.names=F,col.names=T)
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295 write.table(x=unmatched, file=unmatchedfile, sep="\t",quote=F,row.names=F,col.names=T)
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