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7
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1 #!/usr/bin/perl
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2 #V1.0.1 added log, option parameters
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3 use strict;
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4 use warnings;
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5 use Getopt::Long;
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6
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7 my $read1_file;
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8 my $read2_file;
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9 my $log_file;
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10 my $output1_file;
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11 my $output2_file;
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12
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13 my $TYPE="sanger";
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14 my $MIN_LENGTH=30;
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15 my $MIN_QUALITY=30;
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16
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17 my $VERBOSE = "OFF";
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18
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19 GetOptions (
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20 "read1_file=s" => \$read1_file,
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21 "read2_file=s" => \$read2_file,
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22 "log_file=s" => \$log_file,
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23 "output1_file=s" => \$output1_file,
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24 "output2_file=s" => \$output2_file,
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25 "type=s" => \$TYPE,
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26 "min_length=i" => \$MIN_LENGTH,
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27 "min_quality=i" => \$MIN_QUALITY,
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28 "verbose=s" => \$VERBOSE
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29 ) or die("Error in command line arguments\n");
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30
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31
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32 my $nb_read1=0;
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33 my $nb_base_read1=0;
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34 my $nb_read2=0;
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35 my $nb_base_read2=0;
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36
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37 my $nb_read1_t=0;
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38 my $nb_base_read1_t=0;
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39 my $nb_read2_t=0;
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40 my $nb_base_read2_t=0;
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41
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42 my $nb_base_current_t=0;
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43
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44
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45 open(READ1, $read1_file) or die ("Can't open $read1_file\n");
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46 open(READ2, $read2_file) or die ("Can't open $read2_file\n");
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47 open(OUT1, ">$output1_file") or die ("Can't open $output1_file\n");
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48 open(OUT2, ">$output2_file") or die ("Can't open $output2_file\n");
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49
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50 my $error1=0;
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51 my $error2=0;
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52 my $error3=0;
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53 my $error4=0;
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54 my $error5=0;
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55 my $error6=0;
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56 my $error7=0;
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57 my $error8=0;
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58 my $error9=0;
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59 my $error10=0;
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60
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61 while (my $ligne1_r1 =<READ1>){
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62 my $ligne2_r1 =<READ1>;
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63 my $ligne3_r1 =<READ1>;
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64 my $ligne4_r1 =<READ1>;
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65 my $ligne1_r2 =<READ2>;
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66 my $ligne2_r2 =<READ2>;
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67 my $ligne3_r2 =<READ2>;
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68 my $ligne4_r2 =<READ2>;
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69
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70 $nb_read1++;
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71 $nb_read2++;
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72
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73 #@ 1 sec
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74 if ((!$ligne1_r1)||(!$ligne2_r1)||(!$ligne3_r1)||(!$ligne4_r1)||(!$ligne1_r2)||(!$ligne2_r2)||(!$ligne3_r2)||(!$ligne4_r2)){
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75 if ($VERBOSE eq "ON"){
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76 print "Error in file format";
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77 if ($ligne1_r1){print $ligne1_r1;}
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78 if ($ligne2_r1){print $ligne2_r1;}
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79 if ($ligne3_r1){print $ligne3_r1;}
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80 if ($ligne4_r1){print $ligne4_r1;}
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81 if ($ligne1_r2){print $ligne1_r2;}
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82 if ($ligne2_r2){print $ligne2_r2;}
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83 if ($ligne3_r2){print $ligne3_r2;}
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84 if ($ligne4_r2){print $ligne4_r2;}
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85 print "\n";
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86 }
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87 $error1++;
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88 }
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89 elsif(($ligne1_r1 !~/^\@/)||($ligne1_r2 !~/^\@/)||($ligne3_r1 !~/^\+/)||($ligne3_r2 !~/^\+/)){
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90 if ($VERBOSE eq "ON"){
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91 print "Error in header : format\n";
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92 print $ligne1_r1;
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93 print $ligne2_r1;
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94 print $ligne3_r1;
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95 print $ligne4_r1;
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96 print $ligne1_r2;
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97 print $ligne2_r2;
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98 print $ligne3_r2;
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99 print $ligne4_r2;
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100 print "\n";
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101 }
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102 $error2++;
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103 }
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104 #@ 1 - 2 sec
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105 else {
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106
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107 my $length_seq1 = length($ligne2_r1);
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108 my $length_qual1 =length($ligne4_r1);
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109 my $seq1;
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110 my $qual1;
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111
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112 my $length_seq2 = length($ligne2_r2);
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113 my $length_qual2 =length($ligne4_r2);
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114 my $seq2;
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115 my $qual2;
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116 my $header1="";
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117 my $header2="";
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118 my $repheader1="";
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119 my $repheader2="";
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120
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121
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122 if ($ligne1_r1 =~/^\@(.*?)\#/){
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123 $header1 = $1;
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124 }
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125
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126 if ($ligne3_r1 =~/^\+(.*?)\#/){
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127 $repheader1 = $1;
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128 }
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129
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130 if ($ligne1_r2 =~/^\@(.*?)\#/){
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131 $header2 = $1;
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132 }
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133
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134 if ($ligne3_r2 =~/^\+(.*?)\#/){
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135 $repheader2 = $1;
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136 }
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137 #@ 2 sec
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138
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139 ### Verification de la coherence sequence /qualité @ 1 sec
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140 if (($TYPE eq "illumina")&&((!$header1)||(!$header2)||(!$repheader1)||(!$repheader2))){
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141 if ($VERBOSE eq "ON"){
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142 print "Error in header : empty\n";
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143 print $ligne1_r1;
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144 print $ligne2_r1;
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145 print $ligne3_r1;
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146 print $ligne4_r1;
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147 print $ligne1_r2;
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148 print $ligne2_r2;
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149 print $ligne3_r2;
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150 print $ligne4_r2;
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151 print "\n";
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152 }
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153 $error3++;
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154 }
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155 elsif (($TYPE eq "sanger")&&((!$header1)||(!$header2))){
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156 if ($VERBOSE eq "ON"){
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157 print "Error in header refgsd : empty\n";
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158 print $ligne1_r1;
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159 print $ligne2_r1;
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160 print $ligne3_r1;
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161 print $ligne4_r1;
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162 print $ligne1_r2;
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163 print $ligne2_r2;
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164 print $ligne3_r2;
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165 print $ligne4_r2;
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166 print "\n";
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167 }
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168 $error3++;
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169 }
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170 elsif (($TYPE eq "illumina")&&(($header1 ne $repheader1)||($header2 ne $repheader2)||($header1 ne $header2))){
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171 if ($VERBOSE eq "ON"){
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172 print "Error in header : different\n";
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173 print $ligne1_r1;
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174 print $ligne2_r1;
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175 print $ligne3_r1;
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176 print $ligne4_r1;
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177 print $ligne1_r2;
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178 print $ligne2_r2;
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179 print $ligne3_r2;
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180 print $ligne4_r2;
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181 print "\n";
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182 }
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183 $error4++;
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184 }
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185 elsif (($TYPE eq "sanger")&&($header1 ne $header2)){
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186 if ($VERBOSE eq "ON"){
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187 print "Error in header : different\n";
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188 print $ligne1_r1;
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189 print $ligne2_r1;
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190 print $ligne3_r1;
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191 print $ligne4_r1;
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192 print $ligne1_r2;
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193 print $ligne2_r2;
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194 print $ligne3_r2;
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195 print $ligne4_r2;
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196 print "\n";
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197 }
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198 $error4++;
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199 }
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200 elsif (($length_seq1 != $length_qual1)||($length_seq2 != $length_qual2)){
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201 if ($VERBOSE eq "ON"){
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202 print "Error in seq/qual length\n";
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203 print $ligne1_r1;
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204 print $ligne2_r1;
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205 print $ligne3_r1;
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206 print $ligne4_r1;
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207 print $ligne1_r2;
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208 print $ligne2_r2;
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209 print $ligne3_r2;
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210 print $ligne4_r2;
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211 print "\n";
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212 }
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213 $error5++;
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214 }
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215 #@ 1 - 2 sec
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216 else {
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217 ### Parsing sequence & qualité
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218 if ($ligne2_r1 =~ /^([ATGCNX]+)\s*$/i){
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219 $seq1 = $1;
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220 $nb_base_read1 += length($seq1);
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221 }
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222 if ($ligne2_r2 =~ /^([ATGCNX]+)\s*$/i){
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223 $seq2 = $1;
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224 $nb_base_read2 += length($seq2);
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225 }
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226 if ($ligne4_r1 =~ /^(.*)\s*$/i){
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227 $qual1 = $1;
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228 }
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229 if ($ligne4_r2 =~ /^(.*)\s*$/i){
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230 $qual2 = $1;
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231 }
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232 #@ 2 sec
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233 ### Verification du parsing et de la coherence sequence /qualité (n°2)
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234 if ((!$seq1)||(!$seq2)||(!$qual1)||(!$qual2)){
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235 if ($VERBOSE eq "ON"){
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236 print "Error parsing seq / quality \n";
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237 print $ligne1_r1;
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238 print $ligne2_r1;
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239 print $ligne3_r1;
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240 print $ligne4_r1;
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241 print $ligne1_r2;
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242 print $ligne2_r2;
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243 print $ligne3_r2;
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244 print $ligne4_r2;
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245 print "\n";
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246 }
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247 $error6++;
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248 }
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249 elsif ((length($seq1) != length($qual1))||(length($seq2) != length($qual2))){
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250 if ($VERBOSE eq "ON"){
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251 print "Error in seq/qual length after parsing\n";
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252 print $ligne1_r1;
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253 print $ligne2_r1;
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254 print $ligne3_r1;
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255 print $ligne4_r1;
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256 print $ligne1_r2;
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257 print $ligne2_r2;
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258 print $ligne3_r2;
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259 print $ligne4_r2;
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260 print "\n";
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261 }
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262 $error7++;
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263 }
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264 #@ <1 sec
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265 else {
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266 my $fastq_lines_r1="";
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267 my $fastq_lines_r2="";
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268 my $nb_base_current_read1_t = 0;
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269 my $nb_base_current_read2_t = 0;
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270
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271 $fastq_lines_r1 = &grooming_and_trimming($ligne1_r1,$seq1,$qual1);
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272 $nb_base_current_read1_t = $nb_base_current_t;
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273 if ($fastq_lines_r1){
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274 $fastq_lines_r2 = &grooming_and_trimming($ligne1_r2,$seq2,$qual2);
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275 $nb_base_current_read2_t = $nb_base_current_t;
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276 }
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277 if ($fastq_lines_r2){
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278 print OUT1 $fastq_lines_r1;
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279 print OUT2 $fastq_lines_r2;
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280
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281 $nb_read1_t++;
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282 $nb_read2_t++;
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283 $nb_base_read1_t += $nb_base_current_read1_t;
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284 $nb_base_read2_t += $nb_base_current_read2_t;
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285
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286
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287 }
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288 }
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289 }
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290
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291
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292 #@ 7 sec
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293 }
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294 }
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295
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296 close (READ1);
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297 close (READ2);
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298 close (OUT1);
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299 close (OUT2);
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300
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301 open (LF,">$log_file") or die("Can't open $log_file\n");
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302 print LF "\n####\t Fastq preparation \n";
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303 print LF "## Before preparation\n";
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304 print LF "#Read1 :\t$nb_read1\t#Base :\t$nb_base_read1\n";
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305 print LF "#Read2 :\t$nb_read2\t#Base :\t$nb_base_read2\n";
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306 print LF "## After preparation\n";
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307 print LF "#Read1 :\t$nb_read1_t\t#Base :\t$nb_base_read1_t\n";
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308 print LF "#Read2 :\t$nb_read2_t\t#Base :\t$nb_base_read2_t\n";
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309 close (LF);
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310
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311
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312 sub grooming_and_trimming{
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313 my $header = shift;
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314 my $seq = shift;
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315 my $quality = shift;
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316 my $quality_converted="";
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317 my $quality_ori=$quality;
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318
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319 my $lengthseq = length($seq);
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320 my $startTrim = 0;
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321 my $stopTrim = length($quality)-1;
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322 my $startnoN = $startTrim;
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323 my $stopnoN = $stopTrim;
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324
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325
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326 my $chercheN = $seq;
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327 my @bad_position_N;
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328 my @bad_position_Q;
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329 my $current_index = index($chercheN,"N");
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330 my $abs_index = $current_index;
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331 while ($current_index >=0){
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332 push (@bad_position_N,$abs_index);
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333
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334 if ($current_index<length($seq)){
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335 $chercheN = substr($chercheN,$current_index+1);
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336 $current_index = index($chercheN,"N");
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337 $abs_index = $current_index + $bad_position_N[$#bad_position_N]+1;
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338 }
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339 else {
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340 last;
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341 }
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342 }
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343
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344 my @q = split(//,$quality);
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345 for (my $i=0;$i<=$#q;$i++){
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346 my $chr = $q[$i];
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347 my $num = ord($q[$i]);
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348 if ($TYPE eq "illumina"){
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349 $num = $num - 31; # 31 comme la difference entre la plage sanger (33-> 93 / 0->60) et illumina (64->104 / 0->40)
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350 $quality_converted .= chr($num);
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351 }
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352
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353 if ($num < $MIN_QUALITY + 33){ #33 comme le départ de la plage sanger
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354 push(@bad_position_Q,$i);
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355 }
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356 }
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357 if ($quality_converted){$quality = $quality_converted;}
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358
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359 my @bad_position = (@bad_position_N, @bad_position_Q);
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360
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361 if ($#bad_position>=0){
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362 @bad_position = sort {$a <=> $b} @bad_position;
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363 my %coord=%{&extract_longer_string_coordinates_from_bad_position(0,$stopTrim,\@bad_position)};
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364 $startTrim = $coord{"start"};
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365 $stopTrim = $coord{"stop"};
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366 #print "$startTrim .. $stopTrim\n";
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367
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368 }
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369 my $lengthTrim = $stopTrim - $startTrim +1;
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370
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371 #if ($stats_length{$lengthTrim}){
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372 # $stats_length{$lengthTrim} = 1;
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373 #}
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374 #else {
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375 # $stats_length{$lengthTrim}++;
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376 #}
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377 my $fastq_lines="";
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378
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379 # if ($header =~ /GA8\-EAS671_0005\:3\:1\:1043\:4432/){
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380 # print "HEAD:\t$header";
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381 # print "SEQ:\n$seq\n";
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382 # print "$quality_ori\n";
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383 # print "$quality\n";
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384 # for (my $i=0;$i<=$#bad_position;$i++){
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385 # print $bad_position[$i]."(".$q[$bad_position[$i]]." : ".ord($q[$bad_position[$i]]).")"."\t";
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386 # }
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387 # print "\n";
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388 # print "$startTrim .. $stopTrim / $lengthTrim \n";
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389 # print $fastq_lines;
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390 # print "\n";
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391 # }
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392
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393 #for (my $i=$startTrim;$i<=$stopTrim;$i++){
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394 # if ($stats_quality{ord($q{$i])}){
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395 # $stats_quality{ord($q{$i])}=1;
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396 # }
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397 # else {
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398 # $stats_quality{ord($q{$i])}++;
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399 # }
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400 #}
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401
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402 if ($lengthTrim >= $MIN_LENGTH){
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403 $fastq_lines .= $header;
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404 my $new_seq = substr($seq,$startTrim,$lengthTrim);
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405 # $nb_base_current_t = length($new_seq);
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406 $fastq_lines .= $new_seq."\n";
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407 $fastq_lines .= "+\n";
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408 my $new_q = substr($quality,$startTrim,$lengthTrim);
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409 $fastq_lines .= $new_q."\n";
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410 return $fastq_lines;
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411
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412 }
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413 else {
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414 #print "Insufficient length after trimming\n";
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415 return "";
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416 }
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417 }
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418
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419 sub extract_longer_string_coordinates_from_bad_position{
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420 my $start=shift;
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421 my $stop =shift;
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422 my $refbad = shift;
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423 my @bad_position = @$refbad;
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424 my %coord;
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425
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426 my $current_start = $start;
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427 my $current_stop = $bad_position[0]-1;
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428 if ($current_stop < $start){$current_stop = $start;}
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429
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430
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431 #debut -> premier N
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432 my $current_length = $current_stop - $current_start +1;
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433 my $test_length;
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434
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435 #entre les N
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436 for (my $i=1;$i<=$#bad_position;$i++){
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437 $test_length = $bad_position[$i]+1-$bad_position[$i-1]-1;
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438 if ( $test_length > $current_length){
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439 $current_start = $bad_position[$i-1]+1;
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440 $current_stop = $bad_position[$i]-1;
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441 $current_length = $current_stop - $current_start +1;
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442 }
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443 }
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444
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445 #dernier N -> fin
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446 $test_length = $stop-$bad_position[$#bad_position]+1;
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447 if ( $test_length > $current_length){
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448 $current_start = $bad_position[$#bad_position]+1;
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449 if ($current_start > $stop){$current_start=$stop;}
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450 $current_stop = $stop;
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451 }
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452 $coord{"start"}=$current_start;
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453 $coord{"stop"}= $current_stop;
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454 $coord{"lenght"}=$current_stop-$current_start+1;
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455
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456 return \%coord;
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457 }
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