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1 #!/usr/bin/perl
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2
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3 ## needs twoBitToFa v285, or other versions supporting the -bed option.
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4
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5 # load modules
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6 use Getopt::Std;
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7
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8 ##########
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9 ## opts ##
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10 ##########
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11 ## input files
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12 # i : input fastq 1
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13 # I : input fastq 2 (if paired)
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14 # b : bed file with amplicon positions
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15 # r : read length (default 250)
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16 # o : output fastq1
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17 # O : output fastq2
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18 # F : failed readpairs
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19 # R : short report
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20 # t : 2bit file location
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21 # w : working directory (defaults to tmp)
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22
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23 getopts('i:I:b:r:o:O:F:R:t:', \%opts) ;
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24
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25 # check input values
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26 if (!exists($opts{'i'}) || !-e $opts{'i'}) {
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27 die('Fastq File not found');
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28 }
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29 if (!exists($opts{'I'}) || !-e $opts{'I'}) {
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30 die('FastQ for paired end reads not found');
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31 }
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32 if (!exists($opts{'o'})) {
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33 die('No output file specified for forward reads');
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34 }
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35 if (!exists($opts{'O'})) {
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36 die('No output file specified for reverse reads');
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37 }
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38 if (!exists($opts{'F'})) {
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39 die('No output file specified for failed pairs');
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40 }
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41
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42 if (!exists($opts{'b'}) || !-e $opts{'b'}) {
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43 die('BED-File not found');
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44 }
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45
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46 #if (exists($opts{'m'})) {
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47 # $minmap = $opts{'m'};
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48 #}
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49 #else {
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50 # $minmap = 3;
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51 #}
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52 if (exists($opts{'r'})) {
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53 $readl = $opts{'r'};
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54 }
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55 else {
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56 print "Assuming default read length of 2x250bp\n";
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57 $readl = 250;
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58 }
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59 if (exists($opts{'t'}) && -e $opts{'t'}) {
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60 $tobit = $opts{'t'};
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61 }
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62 else {
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63 die("2BIT reference not found");
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64 }
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65
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66 my $tbtf = `which twoBitToFa`;
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67 chomp($tbtf) ;
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68 if ($tbtf eq '') {
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69 if (-e "/opt/software/bin/twoBitToFa") {
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70 $tbtf = "/opt/software/bin/twoBitToFa";
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71 }
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72 else {
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73 die("Could not find a twoBitToFa executable.\n");
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74 }
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75 }
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76
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77
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78 # make output directory in (tmp) working dir
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79 if (exists($opts{'w'}) && -d $opts{'w'}) {
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80 our $wd = $opts{'w'};
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81 }
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82 else {
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83 our $wd = "/tmp/Trim.".int(rand(1000));
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84 while (-d $wd) {
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85 $wd = "/tmp/Trim.".int(rand(1000));
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86 }
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87 system("mkdir $wd");
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88 }
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89 #print "Using wd : $wd\n";
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90
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91
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92 ## build sequence hash.
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93 my %alen = %flen = %rlen = ();
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94 open BED, $opts{'b'};
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95 open OUT, ">$wd/bedfile.zero.bed";
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96 my $minf = 999;
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97 my $minr = 999;
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98 while (<BED>) {
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99 if ($_ !~ m/^(chr.{1,2}\t)(\d+)(\t.*)/) {
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100 next;
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101 }
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102 chomp($_);
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103 my @p = split(/\t/,$_);
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104 my $fl = $p[6] - $p[1]; # p6 holds first non-primer position
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105 my $rl = $p[2] - $p[7]; # p7 hold last non-primer position
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106 ## lengths
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107 $alen{"$p[0]:$p[1]-$p[2]"} = $p[7] - $p[6] + 1;
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108 $flen{"F:$p[0]:$p[1]-$p[2]"} = $fl;
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109 if ($fl < $minf) {
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110 $minf = $fl;
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111 }
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112 if ($rl < $minr) {
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113 $minr = $rl;
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114 }
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115 $rlen{"R:$p[0]:$p[1]-$p[2]"} = $rl;
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116 print OUT "$p[0]\t".($p[1]-1)."\t".($p[6]-1)."\tF:$p[0]:$p[1]-$p[2]\n";
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117 print OUT "$p[0]\t".$p[7]."\t".$p[2]."\tR:$p[0]:$p[1]-$p[2]\n";
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118
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119 }
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120 close BED;
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121 close OUT;
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122
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123 system("cd $wd && $tbtf -noMask -bed=bedfile.zero.bed $tobit amplicons.zero.fa");
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124
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125 open IN, "$wd/amplicons.zero.fa";
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126 my %fseq = %rseq = ();
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127 my %rmm = %fmm = ();
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128 my @nts = ('A','C','T','G');
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129
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130 while(<IN>) {
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131 my $pr = $_;
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132 my $seq = <IN>;
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133 chomp($pr);
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134 chomp($seq);
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135 $pr = substr($pr,1);
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136 if (substr($pr,0,1) eq 'F') {
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137 $fseq{$pr} = $seq;
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138 for ($i = 0; $i < 10; $i++) {
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139 foreach(@nts) {
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140 my $mut = substr($fseq{$pr},0,$i).$_.substr($rseq{$pr},$i+1,9-$i);
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141 $fmm{$pr}{$mut} = $fseq{$pr};
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142 }
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143 }
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144 }
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145 else {
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146 $rseq{$pr} = rc($seq);
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147 for ($i = 0; $i< 10;$i++){
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148 foreach(@nts) {
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149 my $mut = substr($rseq{$pr},0,$i).$_.substr($rseq{$pr},$i+1,9-$i);
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150 $rmm{$pr}{$mut} = $rseq{$pr};
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151 }
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152 }
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153
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154 }
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155 }
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156 close IN;
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157
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158 ###############################
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159 ## generate smallest overlap F##
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160 ###############################
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161 $ntf = $minf;
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162 BUILDMIN:
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163 my %fmin = ();
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164 my %fpairs = ();
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165 foreach( keys(%fseq)) {
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166 my $sub = substr($fseq{$_},0,$ntf);
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167 ## clash => increase nt.
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168 if (exists($fmin{$sub})) {
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169 ## check if not identical (yes, this is possible...) (same start + same length.)
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170 $_ =~ m/F:chr(.{1,2}):(\d+)-(\d+)/;
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171 my $cchr = $1;
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172 my $cstart = $2;
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173 my $cl = $flen{$_};
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174 my @prev = split(/\|/,$fmin{$sub});
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175 my $pprim = $prev[0];
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176 $pprim =~ m/F:chr(.{1,2}):(\d+)-(\d+)/;
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177 my $pchr = $1;
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178 my $pstart = $2;
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179 my $pl = $flen{$pprim};
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180 if ("$cchr" ne "$pchr" || "$cstart" ne "$pstart" || "$cl" ne "$pl") {
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181 $ntf++;
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182 goto BUILDMIN;
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183 }
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184 else {
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185 $fmin{$sub} .= "|$_";
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186 my $rn = $_;
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187 $rn =~ s/F:/R:/;
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188 $fpairs{$_} .= "|$rn";
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189 }
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190 }
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191 else {
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192 $fmin{$sub} = $_;
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193 my $rn = $_;
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194 $rn =~ s/F:/R:/;
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195 $fpairs{$_} = "$rn";
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196
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197 }
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198 }
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199
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200 print "Minimal number of nucleotides needed for forward: $ntf\n";
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201
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202 ## allow one mismatch.
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203 my %mmhash =();
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204 foreach (keys(%fmin)) {
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205 my $orig = $_;
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206 for ($i = 0; $i< length($orig);$i++){
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207 foreach(@nts) {
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208 if ($_ eq substr($orig,$i,1)) {
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209 $mmhash{$orig} = $orig;
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210 next;
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211 }
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212 my $mut = substr($orig,0,$i).$_.substr($orig,$i+1);
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213 ## if in mmhash && not in $fmin => clash after mutation => delete from mmhash.
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214 if (exists($mmhash{$mut}) ) {
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215 if (!exists($fmin{$mut})) {
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216 delete($mmhash{$mut});
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217 next;
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218 }
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219 else {
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220 ## mut == original from oth primer => do not add reference.
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221 next;
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222 }
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223 }
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224 else {
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225 $mmhash{$mut} = $orig;
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226 }
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227 }
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228 }
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229 }
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230
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231 ###############################
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232 ## generate smallest overlap R##
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233 ###############################
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234 $ntr = $minr;
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235 BUILDMINR:
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236 my %rmin = ();
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237 my %frairs = ();
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238 foreach( keys(%rseq)) {
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239 my $sub = substr($rseq{$_},0,$ntr);
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240 ## clash => increase nt.
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241 if (exists($rmin{$sub})) {
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242 ## check if not identical (yes, this is possible...) (same start + same length.)
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243 $_ =~ m/R:chr(.{1,2}):(\d+)-(\d+)/;
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244 my $cchr = $1;
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245 my $cstart = $2;
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246 my $cl = $rlen{$_};
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247 my @prev = split(/\|/,$rmin{$sub});
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248 my $pprim = $prev[0];
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249 $pprim =~ m/R:chr(.{1,2}):(\d+)-(\d+)/;
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250 my $pchr = $1;
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251 my $pstart = $2;
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252 my $pl = $Rlen{$pprim};
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253 if ("$cchr" ne "$pchr" || "$cstart" ne "$pstart" || "$cl" ne "$pl") {
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254 $ntr++;
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255 goto BUILDMINR;
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256 }
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257 else {
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258 $Rmin{$sub} .= "|$_";
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259 my $fn = $_;
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260 $fn =~ s/R:/F:/;
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261 $rpairs{$_} .= "|$fn";
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262 }
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263 }
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264 else {
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265 $rmin{$sub} = $_;
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266 my $fn = $_;
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267 $fn =~ s/R:/F:/;
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268 $rpairs{$_} = "$fn";
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269
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270 }
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271 }
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272
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273 print "Minimal number of nucleotides needed for reverse: $ntr\n";
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274
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275 ## allow one mismatch.
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276 my %rmmhash =();
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277 foreach (keys(%rmin)) {
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278 my $orig = $_;
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279 for ($i = 0; $i< length($orig);$i++){
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280 foreach(@nts) {
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281 if ($_ eq substr($orig,$i,1)) {
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282 $rmmhash{$orig} = $orig;
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283 next;
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284 }
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285 my $mut = substr($orig,0,$i).$_.substr($orig,$i+1);
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286 ## if in mmhash && not in $fmin => clash after mutation => delete from mmhash.
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287 if (exists($rmmhash{$mut}) ) {
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288 if (!exists($rmin{$mut})) {
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289 delete($rmmhash{$mut});
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290 next;
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291 }
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292 else {
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293 ## mut == original from oth primer => do not add reference.
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294 next;
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295 }
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296 }
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297 else {
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298 $rmmhash{$mut} = $orig;
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299 }
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300 }
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301 }
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302 }
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303
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304 ##########################
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305 ## process fastq files. ##
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306 ##########################
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307 open IN, $opts{'i'}; # open forward reads
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308 open INR, $opts{'I'}; # open reverse reads
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309 open OUTF, ">".$opts{'o'}; # where to put trimmed forward reads
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310 open OUTR, ">".$opts{'O'}; ## where to put trimmed Reverse reads
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311 open FAIL, ">$opts{'F'}"; #$wd/failed.interlaced.fq"; # keep non-matches to seperate file => for debug?
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312 my $outf = $outr = $failout =''; # buffer output to speedup I/O
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313 my $count = $failcount = 0 ; # track output buffer
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314 my $nrmissed = $nrfound = 0; # statistics
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315 my $foundboth = $byf = $byr = $toolongf = $toolongr = 0; # statistics
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316 my $bptrimmed = $totalbp = 0; # statistics
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317 while (my $r1 = <IN>) {
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318 # read in forward
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319 my $s1 = <IN>;
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320 chomp($s1);
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321 $totalbp += length($s1);
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322 my $d = <IN>;
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323 my $q1 = <IN>;
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324 chomp($q1);
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325 # read in reverse
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326 my $r2 = <INR>;
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327 my $s2 = <INR>;
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328 chomp($s2);
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329 $totalbp += length($s2);
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330 my $d = <INR>;
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331 my $q2 = <INR>;
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332 chomp($q2);
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333 ## the seeds : first positions of reads, lenght is determined above.
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334 my $fseed = substr($s1,0,$ntf);
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335 my $rseed = substr($s2,0,$ntr);
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336 ## check if the seed exists in the "mutated" forward primer list
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337 if (!exists($mmhash{$fseed})) {
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338 ## not found : try the reverse primers.
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339 if (!exists($rmmhash{$rseed})) {
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340 # not found either : print out to failed reads in interlaced format
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341 $nrmissed++;
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342 $failout .= "$r1$s1\n+\n$q1\n$r2$s2\n+\n$q2\n";
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343 $failcount++;
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344 if ($failcount > 50000) {
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345 chomp($failout);
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346 print FAIL $failout;
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347 $failout = "\n";
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348 $failcount = 0;
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349 }
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350 next;
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351 }
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352 ## trim reverse 5'
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353 $s2 = substr($s2,$rlen{$rmin{$rmmhash{$rseed}}}); # from position *length of reverse primer* : rseed points to the original primer in the rmm hash
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354 $q2 = substr($q2,$rlen{$rmin{$rmmhash{$rseed}}});
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355 ## statistics
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356 $bptrimmed += $rlen{$rmin{$rmmhash{$rseed}}};
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357 ## trim if readlength > amplicon size (without primers)
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358 if ($readl > $rlen{$rmin{$rmmhash{$rseed}}} + $alen{substr($rmin{$rmmhash{$rseed}},2)} ) {
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359 # trim to alength (incorrect if indels !)
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360 # statistics
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361 $toolongr++;
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362 $bptrimmed += length($s2) - $alen{substr($rmin{$rmmhash{$rseed}},2)};
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363 ## 5' primer is already trimmed. This removes fragments of 3' primer.
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364 $s2 = substr($s2,0,$alen{substr($rmin{$rmmhash{$rseed}},2)});
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365 $q2 = substr($q2,0,$alen{substr($rmin{$rmmhash{$rseed}},2)});
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366 }
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367 $byr++;
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368 ## trim forward 5'
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369 my @fps = split(/\|/,$rpairs{$rmin{$rmmhash{$rseed}}});
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370 my $forok = 0;
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371 my $forl = 0;
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372 foreach(@fps) {
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373 if (exists($fmm{$_}{substr($s1,0,10)})) {
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374 $s1 = substr($s1,$flen{$_});
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375 $q1 = substr($q1,$flen{$_});
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376 #statistics
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377 $bptrimmed += $flen{$_};
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378 if ($readl > $flen{$_} + $alen{substr($rmin{$rmmhash{$rseed}},2)} ) {
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379 # trim to alength (incorrect if indels !)
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380 # statistics
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381 $toolongf++;
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382 $bptrimmed += length($s1) - $alen{substr($rmin{$rmmhash{$rseed}},2)};
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383
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384 $s1 = substr($s1,0,$alen{substr($rmin{$rmmhash{$rseed}},2)});
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385 $q1 = substr($q1,0,$alen{substr($rmin{$rmmhash{$rseed}},2)});
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386 }
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387
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388 $forok++;
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389 $foundboth++;
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390 last;
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391 }
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392 else {
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393 if ($forl < $flen{$_}) {
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394 $forl = $flen{$_};
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395 }
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396 }
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397 }
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398 if ($forok == 0) {
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399 ## trim by max length of should be forwards.
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400 $s1 = substr($s1,$forl);
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401 $q1 = substr($q1,$forl);
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402 # statistics
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403 $bptrimmed += $forl;
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404 if ($readl > $forl + $alen{substr($rmin{$rmmhash{$rseed}},2)} ) {
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405 # trim to alength (incorrect if indels !)
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406 # statistics
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407 $toolongf++;
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408 $bptrimmed += length($s1) - $alen{substr($rmin{$rmmhash{$rseed}},2)};
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409 $s1 = substr($s1,0,$alen{substr($rmin{$rmmhash{$rseed}},2)});
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410 $q1 = substr($q1,0,$alen{substr($rmin{$rmmhash{$rseed}},2)});
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411 }
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412
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413 }
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414 $nrfound++;
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415 }
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416 else {
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417 ## trim forward 5'
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418 $s1 = substr($s1,$flen{$fmin{$mmhash{$fseed}}});
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419 $q1 = substr($q1,$flen{$fmin{$mmhash{$fseed}}});
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420 # statistics
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421 $bptrimmed += $flen{$fmin{$mmhash{$fseed}}};
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422
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423 if ($readl > $flen{$fmin{$mmhash{$fseed}}} + $alen{substr($fmin{$mmhash{$fseed}},2)}) {
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424 # trim to alength (incorrect if indels !)
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425 # statistics
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426 $toolongf++;
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427 $bptrimmed += length($s1) - $alen{substr($fmin{$mmhash{$fseed}},2)};
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428
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429 $s1 = substr($s1,0,$alen{substr($fmin{$mmhash{$fseed}},2)});
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430 $q1 = substr($q1,0,$alen{substr($fmin{$mmhash{$fseed}},2)});
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431 }
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|
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432 $byf++;
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|
|
433 ## trim reverse 5'
|
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434 my @rps = split(/\|/,$fpairs{$fmin{$mmhash{$fseed}}});
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435 $revok = 0;
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436 my $revl = 0;
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437 foreach(@rps) {
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438 if (exists($rmm{$_}{substr($s2,0,10)})) {
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439 $s2 = substr($s2,$rlen{$_});
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|
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440 $q2 = substr($q2,$rlen{$_});
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|
|
441 # statistics
|
|
|
442 $bptrimmed += $rlen{$_};
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|
|
443 if ($readl > $rlen{$_} + $alen{substr($rmin{$rmmhash{$rseed}},2)} ) {
|
|
|
444 # trim to alength (incorrect if indels !)
|
|
|
445 # statistics
|
|
|
446 $toolongr++;
|
|
|
447 $bptrimmed += length($s2) - $alen{substr($fmin{$mmhash{$fseed}},2)};
|
|
|
448
|
|
|
449 $s2 = substr($s2,0,$alen{substr($fmin{$mmhash{$fseed}},2)});
|
|
|
450 $q2 = substr($q2,0,$alen{substr($fmin{$mmhash{$fseed}},2)});
|
|
|
451 }
|
|
|
452
|
|
|
453 $revok++;
|
|
|
454 $foundboth++;
|
|
|
455 last;
|
|
|
456 }
|
|
|
457 else {
|
|
|
458 if ($revl < $rlen{$_}) {
|
|
|
459 $revl = $rlen{$_};
|
|
|
460 }
|
|
|
461 }
|
|
|
462 }
|
|
|
463 if ($revok == 0) {
|
|
|
464 # trim by max length of should be reverses.
|
|
|
465 $s2 = substr($s2,$revl);
|
|
|
466 $q2 = substr($q2,$revl);
|
|
|
467 # statistics
|
|
|
468 $bptrimmed += $revl;
|
|
|
469 if ($readl > $revl + $alen{substr($rmin{$rmmhash{$rseed}},2)} ) {
|
|
|
470 # trim to alength (incorrect if indels !)
|
|
|
471 # statistics
|
|
|
472 $toolongr++;
|
|
|
473 $bptrimmed += length($s2) - $alen{substr($fmin{$mmhash{$fseed}},2)};
|
|
|
474 $s2 = substr($s2,0,$alen{substr($fmin{$mmhash{$fseed}},2)});
|
|
|
475 $q2 = substr($q2,0,$alen{substr($fmin{$mmhash{$fseed}},2)});
|
|
|
476 }
|
|
|
477
|
|
|
478 }
|
|
|
479 $nrfound++;
|
|
|
480 }
|
|
|
481 $outf .= "$r1$s1\n+\n$q1\n";
|
|
|
482 $outr .= "$r2$s2\n+\n$q2\n";
|
|
|
483 $count++;
|
|
|
484 if ($count > 100000) {
|
|
|
485 print OUTF $outf;
|
|
|
486 print OUTR $outr;
|
|
|
487
|
|
|
488 $outf = "\n";
|
|
|
489 $outr = "\n";
|
|
|
490 $count = 0;
|
|
|
491 }
|
|
|
492
|
|
|
493
|
|
|
494 }
|
|
|
495 chomp($outf);
|
|
|
496 chomp($outr);
|
|
|
497 chomp($failout);
|
|
|
498 print OUTF $outf;
|
|
|
499 print OUTR $outr;
|
|
|
500 print FAIL $failout;
|
|
|
501 close IN;
|
|
|
502 close INR;
|
|
|
503 close OUTF;
|
|
|
504 close OUTR;
|
|
|
505 close FAIL;
|
|
|
506 open REPORT, ">$opts{'R'}" or die ("Could not open report file");
|
|
|
507 print REPORT "Results: \n";
|
|
|
508 print REPORT "########\n";
|
|
|
509 print REPORT " Read pairs without match: $nrmissed\n";
|
|
|
510 print REPORT " Read pairs with a valid match: $nrfound\n";
|
|
|
511 print REPORT " Initial match on Forward: $byf\n";
|
|
|
512 print REPORT " Initial match on Reverse: $byr\n";
|
|
|
513 print REPORT " Both F and R Matched: $foundboth\n";
|
|
|
514 print REPORT " Forward reads trimmed to amplicon length: $toolongf\n";
|
|
|
515 print REPORT " Reverse reads trimmed to amplicon length: $toolongr\n";
|
|
|
516 print REPORT " Total basepairs in fastq files: $totalbp\n";
|
|
|
517 print REPORT " Total basepairs trimmed: $bptrimmed (".sprintf("%.2f",($bptrimmed/$totalbp)*100)."%)\n";
|
|
|
518 close REPORT;
|
|
|
519
|
|
|
520
|
|
|
521
|
|
|
522
|
|
|
523
|
|
|
524
|
|
|
525 if (!exists($opts{'w'})) {
|
|
|
526 ## clean up
|
|
|
527 system("rm -Rf $wd");
|
|
|
528 }
|
|
|
529
|
|
|
530
|
|
|
531 sub rc {
|
|
|
532 my $seq = shift;
|
|
|
533 $seq =~ tr/ACGT/TGCA/;
|
|
|
534 $seq = reverse($seq);
|
|
|
535 return $seq;
|
|
|
536
|
|
|
537 }
|
