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49
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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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13 #use threads::shared;
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14 use File::Path;
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15 use File::Basename;
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16 #use RNA;
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17 #use Term::ANSIColor;
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18
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19 my %opts;
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50
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20 GetOptions(\%opts,"i:s@","tag:s@","phred:i","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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49
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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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31 #&printErr();
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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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50
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36 if (defined $opts{'phred'}) {
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37 $phred_qv=$opts{'phred'};
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38 }
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49
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39
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40 my @inputfiles=@{$opts{'i'}};
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41 my @inputtags=@{$opts{'tag'}};
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42
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43 my $mypath=`pwd`;
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44 chomp $mypath;
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45
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46 my $dir=defined $opts{'o'} ? $opts{'o'} : "$mypath/miRPlant_out/";
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47
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48
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49 unless ($dir=~/\/$/) {$dir.="/";}
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50 if (not -d $dir) {
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51 mkdir $dir;
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52 }
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53 my $config=$dir."/input_config";
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54 open CONFIG,">$config";
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55 for (my $i=0;$i<@inputfiles;$i++) {
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56 print CONFIG $inputfiles[$i],"\t",$inputtags[$i],"\n";
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57 }
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58 close CONFIG;
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59
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60 my $scipt_path=defined $opts{'path'} ? $opts{'path'} : "/Users/big/galaxy-dist/tools/myTools/";
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61
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62 my $a="ATCTCGTATG"; #adapter
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63 if (defined $opts{'a'}) {$a=$opts{'a'};}
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64
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65 my $m=6; #adapter minimum mapped nt
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66 if (defined $opts{'M'}) {$m=$opts{'M'};}
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67
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68 my $t=1; #threads number
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69 if (defined $opts{'t'}) {$t=$opts{'t'};}
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70
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71 my $min_nt=19; # minimum reads length
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72 if (defined $opts{'min'}) {$min_nt=$opts{'min'};}
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73
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74 my $max_nt=28; #maximum reads length
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75 if (defined $opts{'max'}) {$max_nt=$opts{'max'};}
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76
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77 my $mis=0; #mismatch number for microRNA
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78 if (defined $opts{'mis'}) {$mis=$opts{'mis'};}
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79
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80 my $mis_rfam=0;# mismatch number for rfam
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81 if (defined $opts{'v'}) {$mis_rfam=$opts{'v'};}
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82
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83 my $hit=25; # maximum reads mapping hits in genome
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84 if (defined $opts{'r'}) {$hit=$opts{'r'};}
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85
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86 my $upstream = 2; # microRNA 5' extension
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87 $upstream = $opts{'e'} if(defined $opts{'e'});
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88
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89 my $downstream = 5;# microRNA 3' extension
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90 $downstream = $opts{'f'} if(defined $opts{'f'});
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91
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92 my $maxd=defined $opts{'dis'} ? $opts{'dis'} : 200;
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93 my $flank=defined $opts{'flank'} ? $opts{'flank'} :10;
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94 my $mfe=defined $opts{'mfe'} ? $opts{'mfe'} : -20;
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95
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96 $time=&Time();
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97 print "$time, Checking input file!\n";
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98
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99 my (@filein,@mark,@clean);
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100 #&read_config();
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101 @filein=@inputfiles;
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102 @mark=@inputtags;
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103
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104 &checkfa($opts{pre});
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105 &checkfa($opts{mat});
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106 &checkfa($opts{gfa});
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107
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108
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109 ##### clip adpter --> clean data start
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110 $time=&Time();
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111 print "$time, Preprocess:\n trim adapter, reads collapse and filter reads by length.\n";
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112
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113 $time=~s/:/-/g;
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114 $time=~s/ /-/g;
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115 my $preprocess=$dir."preProcess_${time}/";
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116 mkdir $preprocess;
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117 my $can_use_threads = eval 'use threads; 1';
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118 if ($can_use_threads) {
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119 # Do processing using threads
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120 print "Do processing using threads\n";
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121 my @filein1=@filein; my @mark1=@mark;
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122 while (@filein1>0) {
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123 my @thrs; my @res;
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124 for (my $i=0;$i<$t ;$i++) {
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125 last if(@filein1==0);
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126 my $in=shift @filein1;
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127 my $out=shift @mark1;
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128 push @clean,$preprocess.$out."_clips_adapter.fq";
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129 $thrs[$i]=threads->create(\&clips,$in,$out);
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130 }
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131 for (my $i=0;$i<@thrs;$i++) {
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132 $res[$i]=$thrs[$i]->join();
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133 }
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134 }
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135 } else {
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136 # Do not processing using threads
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137 print "Do not processing using threads\n";
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138 for (my $i=0;$i<@filein ;$i++) {
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139 my $in=$filein[$i];
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140 my $out=$mark[$i];
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141 push @clean,$preprocess.$out."_clips_adapter.fq";
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142 &clips($in,$out);
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143 }
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144 }
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145
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146 ##### clip adpter --> clean data end
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147
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148 my $collapsed=$preprocess."collapse_reads.fa";
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149 my $data=$preprocess."collapse_reads_${min_nt}_${max_nt}.fa"; ## raw clean data
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150 my $data2; ### mirbase not mapped reads
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151 my $data3; ### rfam not mapped reads
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152 &collapse(\@clean,$collapsed); #collapse reads to tags
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153
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154 &filterbylength(); # filter <$min_nt && >$max_nt
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155
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156 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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157
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158 $time=Time();
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159 print "$time: known microRNA quantify!\n\n";
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160
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161 chdir $dir;
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162
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163 $time=~s/:/-/g;
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164 $time=~s/ /-/g;
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165 my $known_result=$dir."miRNA_Express_${time}/";
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166 &quantify(); ### known microRAN quantify
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167
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168
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169 #my $miR_exp_dir=&search($known_result,"miRNA_Express_");
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170 $data2=$known_result."/mirbase_not_mapped.fa";
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171
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172 my $pathfile="$dir/path.txt";
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173 open PA,">$pathfile";
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174 print PA "$config\n";
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175 print PA "$preprocess\n";
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176 print PA "$known_result\n";
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177
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178 if (defined $opts{'rfam'}) { #rfam mapping and analysis
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179 $time=Time();
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180 print "$time: RNA annotate!\n\n";
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181 $time=~s/:/-/g;
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182 $time=~s/ /-/g;
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183 my $rfam_exp_dir=$dir."rfam_match_${time}";
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184 &rfam();
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185 #my $rfam_exp_dir=&search($dir,"rfam_match_");
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186 $data3=$rfam_exp_dir."/rfam_not_mapped.fa";
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187 print PA "$rfam_exp_dir\n";
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188
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189 my $tag=join "\\;" ,@mark;
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190 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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191 }
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192
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193 my $data4=$data;
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194 if (defined $opts{'D'}) { #genome mapping
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195 $data4=$data3;
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196 }else{
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197 $data4=$data2;
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198 }
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199
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200 $time=Time();
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201 print "$time: Genome alignment!\n\n";
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202 $time=~s/:/-/g;
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203 $time=~s/ /-/g;
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204 my $genome_map=$dir."genome_match_${time}";
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205 &genome($data4);
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206 print PA "$genome_map\n";
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207 #my $genome_map=&search($dir,"genome_match_");
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208 my $mapfile=$genome_map."/genome_mapped.bwt";
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209 my $mapfa=$genome_map."/genome_mapped.fa";
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210 my $unmap=$genome_map."/genome_not_mapped.fa";
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211
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212 #$time=Time();
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213 #print "$time: Novel microRNA prediction!\n\n";
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214
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215 &predict($mapfa);
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216
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217 close PA;
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218 system("perl $scipt_path/html.pl -i $pathfile -format $format -o $dir/result.html");
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219
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220 $time=Time();
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221 print "$time: Program end!!\n";
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222
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223 ############################## sub programs ###################################
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224 sub predict{
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225 my ($file)=@_;
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226 $time=&Time();
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227 print "$time: Novel microRNA prediction!\n\n";
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228 $time=~s/:/-/g;
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229 $time=~s/ /-/g;
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230 my $predict=$dir."miRNA_predict_${time}";
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231 print PA "$predict\n";
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232 mkdir $predict;
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233 chdir $predict;
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234 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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235 # 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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236
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237 system("bowtie-build -f excised_precursor.fa excised_precursor");
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238 # print "\nbowtie-build -f excised_precursor.fa excised_precursor\n";
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239
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240 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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241 # print "\nbowtie -v $mis -f -p $t -m $hit -a --best --strata excised_precursor $file > precursor_mapped.bwt\n";
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242
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243 system("perl $scipt_path/convert_bowtie_to_blast.pl precursor_mapped.bwt $file excised_precursor.fa > precursor_mapped.bst");
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244 # print "\nconvert_bowtie_to_blast.pl precursor_mapped.bwt $file excised_precursor.fa > precursor_mapped.bst\n";
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245
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246 system("sort -k 4 precursor_mapped.bst > signatures.bst");
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247 # print "\nsort +3 -25 precursor_mapped.bst > ../signatures.bst\n";
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248
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249 chdir $dir;
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250 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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251 # print "\nmiRDeep_plant.pl $dir/signatures.bst $predict/excised_precursor_struc.txt tmp_dir -y > microRNA_prediction.txt\n";
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252 #system("rm novel_tmp_dir -rf");
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253 my $tag=join "," ,@mark;
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254 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 $tag");
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255 }
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256
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257 sub genome{
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258 my ($file)=@_;
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259 if(defined $opts{'idx'}){
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260 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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261 # 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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262 }else{
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263 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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264 # print "\nmatching.pl -i $file -g $opts{gfa} -v $mis -p $t -r $hit -o $dir -time $time\n";
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265 }
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266 }
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267 sub rfam{
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268 if (defined $opts{'idx2'}) {
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269 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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270 # 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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271 }else{
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272 system("perl $scipt_path/rfam.pl -i $data2 -ref $opts{rfam} -v $mis_rfam -p $t -o $dir -time $time");
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273 # print "\nrfam.pl -i $data2 -ref $opts{rfam} -v $mis_rfam -p $t -o $dir -time $time\n";
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274 }
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275 }
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276 sub quantify{
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277 my $tag=join "\\;" ,@mark;
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278 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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279 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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280 }
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281 sub filterbylength{
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282 my $tmpmark=join ",", @mark;
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283 system("perl $scipt_path/filterReadsByLength.pl -i $collapsed -o $data -min $min_nt -max $max_nt -mark $tmpmark");
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284 system("perl $scipt_path/Length_Distibution.pl -i $preprocess/reads_length_distribution.txt -o $preprocess/length.html");
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285 # print "\nfilterReadsByLength.pl -i $collapsed -o $data -min $min_nt -max $max_nt -mark $tmpmark\n";
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286
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287 }
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288 sub collapse{
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289 my ($ins,$data)=@_;
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290 my $str="";
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291 for (my $i=0;$i<@{$ins};$i++) {
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292 $str .="-i $$ins[$i] ";
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293 }
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294 system ("perl $scipt_path/collapseReads2Tags.pl $str -mark seq -o $data -format $format");
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295 # print "\ncollapseReads2Tags.pl $str -mark seq -o $data -format $format\n";
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296 }
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297
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298 sub clips{
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299 my ($in,$out)=@_;
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300 my $adapter=$preprocess.$out."_clips_adapter.fq";
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301 if($format eq "fq" || $format eq "fastq"){
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302 system("fastx_clipper -a $a -M $m -Q $phred_qv -i $in -o $adapter") ;
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303 print "\nfastx_clipper -a $a -M $m -Q $phred_qv -i $in -o $adapter\n";
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304 }
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305 if($format eq "fa" || $format eq "fasta"){
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306 system("fastx_clipper -a $a -M $m -i $in -o $adapter") ;
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307 # print "\nfastx_clipper -a $a -M $m -i $in -o $adapter\n";
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308 }
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309 #my $clean=$preprocess.$out."_clean.fq";
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310 #system("filterReadsByLength.pl -i $adapter -o $clean -min $min_nt -max $max_nt ");
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311
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312 return;
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313 }
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314
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315 sub read_config{
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316 open CON,"<$config";
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317 while (my $aline=<CON>) {
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318 chomp $aline;
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319 my @tmp=split/\t/,$aline;
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320 push @filein,$tmp[0];
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321 push @mark,$tmp[1];
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322 &check_rawdata($tmp[0]);
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323 }
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324 close CON;
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325 if (@filein != @mark) {
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326 #&printErr();
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327 die "Maybe config file have some wrong!!!\n";
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328 }
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329 }
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330 sub check_rawdata{
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331 my ($fileforcheck)=@_;
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332 if (!(-s $fileforcheck)) {
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333 #&printErr();
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334 die "Can not find $fileforcheck, or file is empty!!!\n";
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335 }
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336 if ($format eq "fasta" || $format eq "fa") {
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337 &checkfa($fileforcheck);
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338 }
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339 if ($format eq "fastq" || $format eq "fq") {
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340 &checkfq($fileforcheck);
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341 }
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342 }
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343 sub checkfa{
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344 my ($file_reads)=@_;
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345 open N,"<$file_reads";
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346 my $line=<N>;
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347 chomp $line;
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348 if($line !~ /^>\S+/){
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349 #printErr();
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350 die "The first line of file $file_reads does not start with '>identifier'
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351 Reads file $file_reads is not a valid fasta file\n\n";
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352 }
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353 if(<N> !~ /^[ACGTNacgtn]*$/){
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354 #printErr();
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355 die "File $file_reads contains not allowed characters in sequences
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356 Allowed characters are ACGTN
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357 Reads file $file_reads is not a fasta file\n\n";
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358 }
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359 close N;
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360 }
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361 sub checkfq{
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362 my ($file_reads)=@_;
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363
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364 open N,"<$file_reads";
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365 for (my $i=0;$i<10;$i++) {
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366 my $a=<N>;
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367 my $b=<N>;
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368 my $c=<N>;
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369 my $d=<N>;
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370 chomp $a;
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371 chomp $b;
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372 chomp $c;
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373 chomp $d;
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374 if($a!~/^\@/){
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375 #&printErr();
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376 die "$file_reads is not a fastq file\n\n";
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377 }
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378 if($b!~ /^[ACGTNacgtn]*$/){
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379 #&printErr();
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380 die "File $file_reads contains not allowed characters in sequences
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381 Allowed characters are ACGTN
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382 Reads file $file_reads is not a fasta file\n\n";
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383 }
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384 if ($c!~/^\@/ && $c!~/^\+/) {
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385 #&printErr();
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386 die "$file_reads is not a fastq file\n\n";
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387 }
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388 if ((length $b) != (length $d)) {
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389 #&printErr();
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390 die "$file_reads is not a fastq file\n\n";
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391 }
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392 my @qv=split //,$d;
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393 for (my $j=0;$j<@qv ;$j++) {
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394 my $q=ord($qv[$j])-64;
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395 if($q<0){$phred_qv=33;}
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396 }
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|
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397 }
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|
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398 close N;
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|
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399 }
|
|
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400
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|
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401 sub search{
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402 my ($dir,$str)=@_;
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403 opendir I,$dir;
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|
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404 my @ret;
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|
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405 while (my $file=readdir I) {
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|
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406 if ($file=~/$str/) {
|
|
|
407 push @ret, $file;
|
|
|
408 }
|
|
|
409 }
|
|
|
410 closedir I;
|
|
|
411 if (@ret != 1) {
|
|
|
412 #&printErr();
|
|
|
413
|
|
|
414 die "Can not find directory or file which name has string: $str !!!\n";
|
|
|
415 }
|
|
|
416 return $ret[0];
|
|
|
417 }
|
|
|
418
|
|
|
419 =cut
|
|
|
420
|
|
|
421 sub printErr{
|
|
|
422 print STDERR color 'bold red';
|
|
|
423 print STDERR "Error: ";
|
|
|
424 print STDERR color 'reset';
|
|
|
425 }
|
|
|
426 sub Time{
|
|
|
427 my $time=time();
|
|
|
428 my ($sec,$min,$hour,$day,$month,$year) = (localtime($time))[0,1,2,3,4,5,6];
|
|
|
429 $month++;
|
|
|
430 $year+=1900;
|
|
|
431 if (length($sec) == 1) {$sec = "0"."$sec";}
|
|
|
432 if (length($min) == 1) {$min = "0"."$min";}
|
|
|
433 if (length($hour) == 1) {$hour = "0"."$hour";}
|
|
|
434 if (length($day) == 1) {$day = "0"."$day";}
|
|
|
435 if (length($month) == 1) {$month = "0"."$month";}
|
|
|
436 #print "$year-$month-$day $hour:$min:$sec\n";
|
|
|
437 return("$year-$month-$day-$hour-$min-$sec");
|
|
|
438 }
|
|
|
439 =cut
|
|
|
440 sub Time{
|
|
|
441 my $time=time();
|
|
|
442 my ($sec,$min,$hour,$day,$month,$year) = (localtime($time))[0,1,2,3,4,5,6];
|
|
|
443 $month++;
|
|
|
444 $year+=1900;
|
|
|
445 if (length($sec) == 1) {$sec = "0"."$sec";}
|
|
|
446 if (length($min) == 1) {$min = "0"."$min";}
|
|
|
447 if (length($hour) == 1) {$hour = "0"."$hour";}
|
|
|
448 if (length($day) == 1) {$day = "0"."$day";}
|
|
|
449 if (length($month) == 1) {$month = "0"."$month";}
|
|
|
450 #print "$year-$month-$day $hour:$min:$sec\n";
|
|
|
451 return("$year-$month-$day $hour:$min:$sec");
|
|
|
452 }
|
|
|
453
|
|
|
454
|
|
|
455 sub usage{
|
|
|
456 print <<"USAGE";
|
|
|
457 Version $version
|
|
|
458 Usage:
|
|
|
459
|
|
|
460 $0 -i -format -gfa -index -pre -mat -rfam -D -a -M -min -max -mis -e -f -v -t -o -path
|
|
|
461 options:
|
|
|
462 -i input files, # raw data file, can be multipe eg. -i xxx.fq -i xxx .fq ...
|
|
|
463 -tag string # raw data file names, -tag xxx -tag xxx
|
|
|
464
|
|
|
465 -format string,#specific input rawdata file format : fastq|fq|fasta|fa
|
|
|
466
|
|
|
467 -path scirpt path
|
|
|
468
|
|
|
469 -gfa string, input file # genome fasta. sequence file
|
|
|
470 -idx string, genome file index, file-prefix #(must be indexed by bowtie-build) The parameter
|
|
|
471 string must be the prefix of the bowtie index. For instance, if
|
|
|
472 the first indexed file is called 'h_sapiens_37_asm.1.ebwt' then
|
|
|
473 the prefix is 'h_sapiens_37_asm'.##can be null
|
|
|
474
|
|
|
475 -pre string, input file #species specific microRNA precursor sequences
|
|
|
476 -mat string, input file #species specific microRNA mature sequences
|
|
|
477
|
|
|
478 -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.
|
|
|
479 -idx2 string, rfam file index, file-prefix #(must be indexed by bowtie-build) The parameter
|
|
|
480 string must be the prefix of the bowtie index. For instance, if
|
|
|
481 the first indexed file is called 'h_sapiens_37_asm.1.ebwt' then
|
|
|
482 the prefix is 'h_sapiens_37_asm'.##can be null
|
|
|
483
|
|
|
484 -D If [-D] is specified,will discard rfam mapped reads(nead -rfam).
|
|
|
485
|
|
|
486 -a string, ADAPTER string. default is ATCTCGTATG.
|
|
|
487 -M int, require minimum adapter alignment length of N. If less than N nucleotides aligned with the adapter - don't clip it.
|
|
|
488 -min int, reads min length,default is 19.
|
|
|
489 -max int, reads max length,default is 28.
|
|
|
490
|
|
|
491 -mis [int] number of allowed mismatches when mapping reads to precursors, default 0
|
|
|
492 -e [int] number of nucleotides upstream of the mature sequence to consider, default 2
|
|
|
493 -f [int] number of nucleotides downstream of the mature sequence to consider, default 5
|
|
|
494 -v <int> report end-to-end hits w/ <=v mismatches; ignore qualities,default 0; used in rfam alignment
|
|
|
495 -r int a read is allowed to map up to this number of positions in the genome,default is 25
|
|
|
496
|
|
|
497 -dis <int> Maximal space between miRNA and miRNA* (200)
|
|
|
498 -flank <int> Flank sequence length of miRNA precursor (10)
|
|
|
499 -mfe <folat> Maximal free energy allowed for a miRNA precursor (-20)
|
|
|
500
|
|
|
501 -t int, number of threads [1]
|
|
|
502
|
|
|
503 -o output directory# absolute path
|
|
|
504 -h help
|
|
|
505 USAGE
|
|
|
506 exit(1);
|
|
|
507 }
|
|
|
508
|
