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14
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1 #!/usr/bin/perl -w
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2 #Filename:
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3 #Author: Tian Dongmei
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4 #Email: tiandm@big.ac.cn
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5 #Date: 2014-4-22
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6 #Modified:
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7 #Description: plant microRNA prediction
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8 my $version=1.00;
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9
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10 use strict;
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11 use Getopt::Long;
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12 use threads;
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46
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13 #use threads::shared;
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14
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14 use File::Path;
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15 use File::Basename;
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46
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16 #use RNA;
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17 #use Term::ANSIColor;
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14
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18
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19 my %opts;
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20 GetOptions(\%opts,"i:s@","tag:s@","format=s","gfa=s","pre=s","mat=s","rfam:s","dis:i","flank:i","mfe:f","idx:s","idx2:s","mis:i","r:i","v:i","e:i","f:i","a:s","M:i","t:i","min:i","max:i","o:s","path:s","D","h");
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21 if (!(defined $opts{i} and defined $opts{format} and defined $opts{gfa} and defined $opts{pre} and defined $opts{mat}) || defined $opts{h}) { #necessary arguments
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22 &usage;
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23 }
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24
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25 my $time=&Time();
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26 print "miPlant program start:\n The time is $time!\n";
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27 print "Command line:\n $0 @ARGV\n";
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28
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29 my $format=$opts{'format'};
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30 if ($format ne "fastq" && $format ne "fq" && $format ne "fasta" && $format ne "fa") {
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46
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31 #&printErr();
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14
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32 die "Parameter \"-format\" is error! Parameter is fastq, fq, fasta or fa\n";
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33 }
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34
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35 my $phred_qv=64;
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36
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37
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38 my @inputfiles=@{$opts{'i'}};
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39 my @inputtags=@{$opts{'tag'}};
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40
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41 my $mypath=`pwd`;
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42 chomp $mypath;
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43
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44 my $dir=defined $opts{'o'} ? $opts{'o'} : "$mypath/miRPlant_out/";
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45
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46
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47 unless ($dir=~/\/$/) {$dir.="/";}
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48 if (not -d $dir) {
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49 mkdir $dir;
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50 }
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51 my $config=$dir."/input_config";
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52 open CONFIG,">$config";
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53 for (my $i=0;$i<@inputfiles;$i++) {
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54 print CONFIG $inputfiles[$i],"\t",$inputtags[$i],"\n";
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55 }
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56 close CONFIG;
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57
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58 my $scipt_path=defined $opts{'path'} ? $opts{'path'} : "/Users/big/galaxy-dist/tools/myTools/";
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59
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60 my $a="ATCTCGTATG"; #adapter
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61 if (defined $opts{'a'}) {$a=$opts{'a'};}
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62
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63 my $m=6; #adapter minimum mapped nt
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64 if (defined $opts{'M'}) {$m=$opts{'M'};}
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65
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66 my $t=1; #threads number
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67 if (defined $opts{'t'}) {$t=$opts{'t'};}
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68
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69 my $min_nt=19; # minimum reads length
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70 if (defined $opts{'min'}) {$min_nt=$opts{'min'};}
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71
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72 my $max_nt=28; #maximum reads length
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73 if (defined $opts{'max'}) {$max_nt=$opts{'max'};}
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74
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75 my $mis=0; #mismatch number for microRNA
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76 if (defined $opts{'mis'}) {$mis=$opts{'mis'};}
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77
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78 my $mis_rfam=0;# mismatch number for rfam
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79 if (defined $opts{'v'}) {$mis_rfam=$opts{'v'};}
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80
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81 my $hit=25; # maximum reads mapping hits in genome
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82 if (defined $opts{'r'}) {$hit=$opts{'r'};}
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83
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84 my $upstream = 2; # microRNA 5' extension
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85 $upstream = $opts{'e'} if(defined $opts{'e'});
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86
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87 my $downstream = 5;# microRNA 3' extension
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88 $downstream = $opts{'f'} if(defined $opts{'f'});
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89
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90 my $maxd=defined $opts{'dis'} ? $opts{'dis'} : 200;
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91 my $flank=defined $opts{'flank'} ? $opts{'flank'} :10;
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92 my $mfe=defined $opts{'mfe'} ? $opts{'mfe'} : -20;
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93
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94 $time=&Time();
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95 print "$time, Checking input file!\n";
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96
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97 my (@filein,@mark,@clean);
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98 #&read_config();
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99 @filein=@inputfiles;
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100 @mark=@inputtags;
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101
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102 &checkfa($opts{pre});
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103 &checkfa($opts{mat});
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104 &checkfa($opts{gfa});
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105
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106
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107 ##### clip adpter --> clean data start
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108 $time=&Time();
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109 print "$time, Preprocess:\n trim adapter, reads collapse and filter reads by length.\n";
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110
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111 $time=~s/:/-/g;
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112 $time=~s/ /-/g;
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113 my $preprocess=$dir."preProcess_${time}/";
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114 mkdir $preprocess;
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115 my $can_use_threads = eval 'use threads; 1';
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116 if ($can_use_threads) {
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117 # Do processing using threads
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118 print "Do processing using threads\n";
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119 my @filein1=@filein; my @mark1=@mark;
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120 while (@filein1>0) {
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121 my @thrs; my @res;
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122 for (my $i=0;$i<$t ;$i++) {
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123 last if(@filein1==0);
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124 my $in=shift @filein1;
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125 my $out=shift @mark1;
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126 push @clean,$preprocess.$out."_clips_adapter.fq";
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127 $thrs[$i]=threads->create(\&clips,$in,$out);
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128 }
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129 for (my $i=0;$i<@thrs;$i++) {
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130 $res[$i]=$thrs[$i]->join();
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131 }
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132 }
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133 } else {
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134 # Do not processing using threads
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135 print "Do not processing using threads\n";
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136 for (my $i=0;$i<@filein ;$i++) {
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137 my $in=$filein[$i];
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138 my $out=$mark[$i];
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139 push @clean,$preprocess.$out."_clips_adapter.fq";
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140 &clips($in,$out);
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141 }
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142 }
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143
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144 ##### clip adpter --> clean data end
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145
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146 my $collapsed=$preprocess."collapse_reads.fa";
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147 my $data=$preprocess."collapse_reads_${min_nt}_${max_nt}.fa"; ## raw clean data
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148 my $data2; ### mirbase not mapped reads
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149 my $data3; ### rfam not mapped reads
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150 &collapse(\@clean,$collapsed); #collapse reads to tags
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151
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152 &filterbylength(); # filter <$min_nt && >$max_nt
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153
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154 print "The final clean data file is $data, only contains reads which length is among $min_nt\~$max_nt\n\n";
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155
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156 $time=Time();
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157 print "$time: known microRNA quantify!\n\n";
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158
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159 chdir $dir;
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160
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161 $time=~s/:/-/g;
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162 $time=~s/ /-/g;
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163 my $known_result=$dir."miRNA_Express_${time}/";
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164 &quantify(); ### known microRAN quantify
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165
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166
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167 #my $miR_exp_dir=&search($known_result,"miRNA_Express_");
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168 $data2=$known_result."/mirbase_not_mapped.fa";
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169
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170 my $pathfile="$dir/path.txt";
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171 open PA,">$pathfile";
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172 print PA "$config\n";
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173 print PA "$preprocess\n";
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174 print PA "$known_result\n";
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175
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176 if (defined $opts{'rfam'}) { #rfam mapping and analysis
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177 $time=Time();
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178 print "$time: RNA annotate!\n\n";
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179 $time=~s/:/-/g;
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180 $time=~s/ /-/g;
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181 my $rfam_exp_dir=$dir."rfam_match_${time}";
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182 &rfam();
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183 #my $rfam_exp_dir=&search($dir,"rfam_match_");
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184 $data3=$rfam_exp_dir."/rfam_not_mapped.fa";
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185 print PA "$rfam_exp_dir\n";
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186
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187 my $tag=join "\\;" ,@mark;
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188 system("perl $scipt_path/count_rfam_express.pl -i $rfam_exp_dir/rfam_mapped.bwt -tag $tag -o rfam_non-miRNA_annotation.txt");
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189 }
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190
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191 my $data4=$data;
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192 if (defined $opts{'D'}) { #genome mapping
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193 $data4=$data3;
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194 }else{
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195 $data4=$data2;
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196 }
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197
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198 $time=Time();
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199 print "$time: Genome alignment!\n\n";
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200 $time=~s/:/-/g;
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201 $time=~s/ /-/g;
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202 my $genome_map=$dir."genome_match_${time}";
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203 &genome($data4);
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204 print PA "$genome_map\n";
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205 #my $genome_map=&search($dir,"genome_match_");
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206 my $mapfile=$genome_map."/genome_mapped.bwt";
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207 my $mapfa=$genome_map."/genome_mapped.fa";
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208 my $unmap=$genome_map."/genome_not_mapped.fa";
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209
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210 #$time=Time();
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211 #print "$time: Novel microRNA prediction!\n\n";
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212
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213 &predict($mapfa);
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214
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215 close PA;
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216 system("perl $scipt_path/html.pl -i $pathfile -format $format -o $dir/result.html");
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217
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218 $time=Time();
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219 print "$time: Program end!!\n";
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220
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221 ############################## sub programs ###################################
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222 sub predict{
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223 my ($file)=@_;
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224 $time=&Time();
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225 print "$time: Novel microRNA prediction!\n\n";
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226 $time=~s/:/-/g;
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227 $time=~s/ /-/g;
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228 my $predict=$dir."miRNA_predict_${time}";
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229 print PA "$predict\n";
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230 mkdir $predict;
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231 chdir $predict;
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232 system("perl $scipt_path/precursors.pl -map $mapfile -g $opts{gfa} -d $maxd -f $flank -o $predict/excised_precursor.fa -s $predict/excised_precursor_struc.txt -e $mfe");
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233 # print "\nprecursors.pl -map $mapfile -g $opts{gfa} -d $maxd -f $flank -o $predict/excised_precursor.fa -s $predict/excised_precursor_struc.txt -e $mfe\n";
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234
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235 system("bowtie-build -f excised_precursor.fa excised_precursor");
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236 # print "\nbowtie-build -f excised_precursor.fa excised_precursor\n";
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237
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44
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238 system("bowtie -v $mis -f -p $t -m $hit -a --best --strata excised_precursor $file > precursor_mapped.bwt 2> run.log");
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239 # print "\nbowtie -v $mis -f -p $t -m $hit -a --best --strata excised_precursor $file > precursor_mapped.bwt\n";
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240
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241 system("perl $scipt_path/convert_bowtie_to_blast.pl precursor_mapped.bwt $file excised_precursor.fa > precursor_mapped.bst");
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242 # print "\nconvert_bowtie_to_blast.pl precursor_mapped.bwt $file excised_precursor.fa > precursor_mapped.bst\n";
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243
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44
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244 system("sort -k 4 precursor_mapped.bst > signatures.bst");
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245 # print "\nsort +3 -25 precursor_mapped.bst > ../signatures.bst\n";
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246
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247 chdir $dir;
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248 system("perl $scipt_path/miRDeep_plant.pl $predict/signatures.bst $predict/excised_precursor_struc.txt novel_tmp_dir -y > microRNA_prediction.mrd");
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249 # print "\nmiRDeep_plant.pl $dir/signatures.bst $predict/excised_precursor_struc.txt tmp_dir -y > microRNA_prediction.txt\n";
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250 #system("rm novel_tmp_dir -rf");
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251 my $tag=join "," ,@mark;
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252 system("perl $scipt_path/miRNA_Express_and_sequence.pl -i microRNA_prediction.mrd -list novel_microRNA_express.txt -fa novel_microRNA_mature.fa -pre novel_microRNA_precursor.fa -tag $tag");
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253 }
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254
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255 sub genome{
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256 my ($file)=@_;
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257 if(defined $opts{'idx'}){
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258 system("perl $scipt_path/matching.pl -i $file -g $opts{gfa} -v $mis -p $t -r $hit -o $dir -index $opts{idx} -time $time") ;
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259 # print "\nmatching.pl -i $file -g $opts{gfa} -v $mis -p $t -r $hit -o $dir -index $opts{idx} -time $time\n";
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260 }else{
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261 system("perl $scipt_path/matching.pl -i $file -g $opts{gfa} -v $mis -p $t -r $hit -o $dir -time $time") ;
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262 # print "\nmatching.pl -i $file -g $opts{gfa} -v $mis -p $t -r $hit -o $dir -time $time\n";
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263 }
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264 }
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265 sub rfam{
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266 if (defined $opts{'idx2'}) {
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267 system("perl $scipt_path/rfam.pl -i $data2 -ref $opts{rfam} -v $mis_rfam -p $t -o $dir -index $opts{idx2} -time $time");
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268 # print "\nrfam.pl -i $data2 -ref $opts{rfam} -v $mis_rfam -p $t -o $dir -index $opts{idx2} -time $time\n";
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269 }else{
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270 system("perl $scipt_path/rfam.pl -i $data2 -ref $opts{rfam} -v $mis_rfam -p $t -o $dir -time $time");
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271 # print "\nrfam.pl -i $data2 -ref $opts{rfam} -v $mis_rfam -p $t -o $dir -time $time\n";
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272 }
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273 }
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274 sub quantify{
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275 my $tag=join "\\;" ,@mark;
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276 system("perl $scipt_path/quantify.pl -p $opts{pre} -m $opts{mat} -r $data -o $dir -time $time -mis $mis -t $t -e $upstream -f $downstream -tag $tag");
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277 print "\nquantify.pl -p $opts{pre} -m $opts{mat} -r $data -o $dir -time $time -mis $mis -t $t -e $upstream -f $downstream -tag $tag\n";
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278 }
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279 sub filterbylength{
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280 my $tmpmark=join ",", @mark;
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281 system("perl $scipt_path/filterReadsByLength.pl -i $collapsed -o $data -min $min_nt -max $max_nt -mark $tmpmark");
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44
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282 system("perl $scipt_path/Length_Distibution.pl -i $preprocess/reads_length_distribution.txt -o $preprocess/length.html");
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14
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283 # print "\nfilterReadsByLength.pl -i $collapsed -o $data -min $min_nt -max $max_nt -mark $tmpmark\n";
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284
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285 }
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286 sub collapse{
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287 my ($ins,$data)=@_;
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288 my $str="";
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289 for (my $i=0;$i<@{$ins};$i++) {
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290 $str .="-i $$ins[$i] ";
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291 }
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292 system ("perl $scipt_path/collapseReads2Tags.pl $str -mark seq -o $data -format $format");
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293 # print "\ncollapseReads2Tags.pl $str -mark seq -o $data -format $format\n";
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294 }
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295
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296 sub clips{
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297 my ($in,$out)=@_;
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298 my $adapter=$preprocess.$out."_clips_adapter.fq";
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299 if($format eq "fq" || $format eq "fastq"){
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300 system("fastx_clipper -a $a -M $m -Q $phred_qv -i $in -o $adapter") ;
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301 print "\nfastx_clipper -a $a -M $m -Q $phred_qv -i $in -o $adapter\n";
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302 }
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303 if($format eq "fa" || $format eq "fasta"){
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304 system("fastx_clipper -a $a -M $m -i $in -o $adapter") ;
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305 # print "\nfastx_clipper -a $a -M $m -i $in -o $adapter\n";
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306 }
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307 #my $clean=$preprocess.$out."_clean.fq";
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308 #system("filterReadsByLength.pl -i $adapter -o $clean -min $min_nt -max $max_nt ");
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309
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310 return;
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311 }
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312
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313 sub read_config{
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314 open CON,"<$config";
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315 while (my $aline=<CON>) {
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316 chomp $aline;
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317 my @tmp=split/\t/,$aline;
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318 push @filein,$tmp[0];
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319 push @mark,$tmp[1];
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320 &check_rawdata($tmp[0]);
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321 }
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322 close CON;
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323 if (@filein != @mark) {
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46
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324 #&printErr();
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14
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325 die "Maybe config file have some wrong!!!\n";
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326 }
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327 }
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328 sub check_rawdata{
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329 my ($fileforcheck)=@_;
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330 if (!(-s $fileforcheck)) {
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46
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331 #&printErr();
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14
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332 die "Can not find $fileforcheck, or file is empty!!!\n";
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333 }
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334 if ($format eq "fasta" || $format eq "fa") {
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335 &checkfa($fileforcheck);
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336 }
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337 if ($format eq "fastq" || $format eq "fq") {
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338 &checkfq($fileforcheck);
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339 }
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340 }
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341 sub checkfa{
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342 my ($file_reads)=@_;
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343 open N,"<$file_reads";
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344 my $line=<N>;
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345 chomp $line;
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346 if($line !~ /^>\S+/){
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46
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347 #printErr();
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14
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348 die "The first line of file $file_reads does not start with '>identifier'
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349 Reads file $file_reads is not a valid fasta file\n\n";
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350 }
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351 if(<N> !~ /^[ACGTNacgtn]*$/){
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46
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352 #printErr();
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14
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353 die "File $file_reads contains not allowed characters in sequences
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354 Allowed characters are ACGTN
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355 Reads file $file_reads is not a fasta file\n\n";
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356 }
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357 close N;
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358 }
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359 sub checkfq{
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360 my ($file_reads)=@_;
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361
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362 open N,"<$file_reads";
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363 for (my $i=0;$i<10;$i++) {
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364 my $a=<N>;
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365 my $b=<N>;
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366 my $c=<N>;
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367 my $d=<N>;
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368 chomp $a;
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369 chomp $b;
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370 chomp $c;
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371 chomp $d;
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372 if($a!~/^\@/){
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46
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373 #&printErr();
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14
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374 die "$file_reads is not a fastq file\n\n";
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375 }
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376 if($b!~ /^[ACGTNacgtn]*$/){
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46
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377 #&printErr();
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14
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378 die "File $file_reads contains not allowed characters in sequences
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379 Allowed characters are ACGTN
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380 Reads file $file_reads is not a fasta file\n\n";
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381 }
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382 if ($c!~/^\@/ && $c!~/^\+/) {
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46
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383 #&printErr();
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14
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384 die "$file_reads is not a fastq file\n\n";
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385 }
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386 if ((length $b) != (length $d)) {
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46
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387 #&printErr();
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14
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388 die "$file_reads is not a fastq file\n\n";
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389 }
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390 my @qv=split //,$d;
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391 for (my $j=0;$j<@qv ;$j++) {
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392 my $q=ord($qv[$j])-64;
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393 if($q<0){$phred_qv=33;}
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394 }
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|
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395 }
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|
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396 close N;
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|
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397 }
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|
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398
|
|
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399 sub search{
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400 my ($dir,$str)=@_;
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401 opendir I,$dir;
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402 my @ret;
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|
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403 while (my $file=readdir I) {
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|
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404 if ($file=~/$str/) {
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|
|
405 push @ret, $file;
|
|
|
406 }
|
|
|
407 }
|
|
|
408 closedir I;
|
|
|
409 if (@ret != 1) {
|
|
46
|
410 #&printErr();
|
|
14
|
411
|
|
|
412 die "Can not find directory or file which name has string: $str !!!\n";
|
|
|
413 }
|
|
|
414 return $ret[0];
|
|
|
415 }
|
|
|
416
|
|
46
|
417 =cut
|
|
|
418
|
|
14
|
419 sub printErr{
|
|
|
420 print STDERR color 'bold red';
|
|
|
421 print STDERR "Error: ";
|
|
|
422 print STDERR color 'reset';
|
|
|
423 }
|
|
|
424 sub Time{
|
|
|
425 my $time=time();
|
|
|
426 my ($sec,$min,$hour,$day,$month,$year) = (localtime($time))[0,1,2,3,4,5,6];
|
|
|
427 $month++;
|
|
|
428 $year+=1900;
|
|
|
429 if (length($sec) == 1) {$sec = "0"."$sec";}
|
|
|
430 if (length($min) == 1) {$min = "0"."$min";}
|
|
|
431 if (length($hour) == 1) {$hour = "0"."$hour";}
|
|
|
432 if (length($day) == 1) {$day = "0"."$day";}
|
|
|
433 if (length($month) == 1) {$month = "0"."$month";}
|
|
|
434 #print "$year-$month-$day $hour:$min:$sec\n";
|
|
|
435 return("$year-$month-$day-$hour-$min-$sec");
|
|
|
436 }
|
|
|
437 =cut
|
|
|
438 sub Time{
|
|
|
439 my $time=time();
|
|
|
440 my ($sec,$min,$hour,$day,$month,$year) = (localtime($time))[0,1,2,3,4,5,6];
|
|
|
441 $month++;
|
|
|
442 $year+=1900;
|
|
|
443 if (length($sec) == 1) {$sec = "0"."$sec";}
|
|
|
444 if (length($min) == 1) {$min = "0"."$min";}
|
|
|
445 if (length($hour) == 1) {$hour = "0"."$hour";}
|
|
|
446 if (length($day) == 1) {$day = "0"."$day";}
|
|
|
447 if (length($month) == 1) {$month = "0"."$month";}
|
|
|
448 #print "$year-$month-$day $hour:$min:$sec\n";
|
|
|
449 return("$year-$month-$day $hour:$min:$sec");
|
|
|
450 }
|
|
|
451
|
|
|
452
|
|
|
453 sub usage{
|
|
|
454 print <<"USAGE";
|
|
|
455 Version $version
|
|
|
456 Usage:
|
|
46
|
457
|
|
14
|
458 $0 -i -format -gfa -index -pre -mat -rfam -D -a -M -min -max -mis -e -f -v -t -o -path
|
|
|
459 options:
|
|
46
|
460 -i input files, # raw data file, can be multipe eg. -i xxx.fq -i xxx .fq ...
|
|
|
461 -tag string # raw data file names, -tag xxx -tag xxx
|
|
14
|
462
|
|
|
463 -format string,#specific input rawdata file format : fastq|fq|fasta|fa
|
|
|
464
|
|
46
|
465 -path scirpt path
|
|
|
466
|
|
14
|
467 -gfa string, input file # genome fasta. sequence file
|
|
|
468 -idx string, genome file index, file-prefix #(must be indexed by bowtie-build) The parameter
|
|
|
469 string must be the prefix of the bowtie index. For instance, if
|
|
|
470 the first indexed file is called 'h_sapiens_37_asm.1.ebwt' then
|
|
|
471 the prefix is 'h_sapiens_37_asm'.##can be null
|
|
|
472
|
|
|
473 -pre string, input file #species specific microRNA precursor sequences
|
|
|
474 -mat string, input file #species specific microRNA mature sequences
|
|
|
475
|
|
|
476 -rfam string, input file# rfam database file, microRNAs must not be contained in this file## if not define, rfam small RNA will not be count.
|
|
|
477 -idx2 string, rfam file index, file-prefix #(must be indexed by bowtie-build) The parameter
|
|
|
478 string must be the prefix of the bowtie index. For instance, if
|
|
|
479 the first indexed file is called 'h_sapiens_37_asm.1.ebwt' then
|
|
|
480 the prefix is 'h_sapiens_37_asm'.##can be null
|
|
|
481
|
|
|
482 -D If [-D] is specified,will discard rfam mapped reads(nead -rfam).
|
|
|
483
|
|
|
484 -a string, ADAPTER string. default is ATCTCGTATG.
|
|
|
485 -M int, require minimum adapter alignment length of N. If less than N nucleotides aligned with the adapter - don't clip it.
|
|
|
486 -min int, reads min length,default is 19.
|
|
|
487 -max int, reads max length,default is 28.
|
|
|
488
|
|
|
489 -mis [int] number of allowed mismatches when mapping reads to precursors, default 0
|
|
|
490 -e [int] number of nucleotides upstream of the mature sequence to consider, default 2
|
|
|
491 -f [int] number of nucleotides downstream of the mature sequence to consider, default 5
|
|
|
492 -v <int> report end-to-end hits w/ <=v mismatches; ignore qualities,default 0; used in rfam alignment
|
|
|
493 -r int a read is allowed to map up to this number of positions in the genome,default is 25
|
|
|
494
|
|
|
495 -dis <int> Maximal space between miRNA and miRNA* (200)
|
|
|
496 -flank <int> Flank sequence length of miRNA precursor (10)
|
|
|
497 -mfe <folat> Maximal free energy allowed for a miRNA precursor (-20)
|
|
|
498
|
|
|
499 -t int, number of threads [1]
|
|
|
500
|
|
|
501 -o output directory# absolute path
|
|
|
502 -h help
|
|
|
503 USAGE
|
|
|
504 exit(1);
|
|
|
505 }
|
|
|
506
|
