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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: 2013/7/19
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6 #Modified:
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7 #Description:
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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 RNA;
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13
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14 my %opts;
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15 GetOptions(\%opts,"map=s","g=s","d:i","f:i","o=s","e:f","s=s","h");
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16 if (!(defined $opts{map} and defined $opts{g} and defined $opts{o} and defined $opts{s} ) || defined $opts{h}) { #necessary arguments
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17 &usage;
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18 }
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19
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20 my $checkno=1;
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21 my $filein=$opts{'map'};
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22 my $faout=$opts{'o'};
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23 my $strout=$opts{'s'};
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24 my $genome= $opts{'g'};
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25
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26 my $maxd=defined $opts{'d'} ? $opts{'d'} : 200;
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27 my $flank=defined $opts{'f'}? $opts{'f'} : 10;
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28
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29 my $MAX_ENERGY=-18;
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30 if (defined $opts{'e'}) {$MAX_ENERGY=$opts{'e'};}
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31 my $MAX_UNPAIR=5;
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32 my $MIN_PAIR=15;
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33 my $MAX_SIZEDIFF=4;
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34 my $MAX_BULGE=2;
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35 my $ASYMMETRY=5;
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36 my $MIN_UNPAIR=0;
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37 my $MIN_SPACE=5;
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38 my $MAX_SPACE=$maxd;
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39 my $FLANK=$flank;
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40
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41 ######### load in genome sequences start ########
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42 my %genome;
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43 my %lng;
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44 my $name;
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45 open IN,"<$genome";
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46 while (my $aline=<IN>) {
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47 chomp $aline;
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48 next if($aline=~/^\#/);
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49 if ($aline=~/^>(\S+)/) {
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50 $name=$1;
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51 next;
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52 }
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53 $genome{$name} .=$aline;
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54 }
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55 close IN;
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56 foreach my $key (keys %genome) {
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57 $lng{$key}=length($genome{$key});
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58 }
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59 ####### load in genome sequences end ##########
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60
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61 my %breaks; ### reads number bigger than 3
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62 open IN,"<$filein"; #input file
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63 while (my $aline=<IN>) {
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64 chomp $aline;
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65 my @tmp=split/\t/,$aline;
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66 $tmp[0]=~/_x(\d+)$/;
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67 my $no=$1;
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68 next if($no<3);
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69 #my $trand=&find_strand($tmp[9]);
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70 #my @pos=split/\.\./,$tmp[5];
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71 my $end=$tmp[3]+length($tmp[4])-1;
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72 if($tmp[1] eq "-"){$tmp[4]=revcom($tmp[4]);}
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73 push @{$breaks{$tmp[2]}{$tmp[1]}},[$tmp[3],$end,$no,$tmp[4]]; ### 0 base
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74 }
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75 close IN;
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76
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77 my %cites; ### peaks
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78 foreach my $chr (keys %breaks) {
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79 foreach my $strand (keys %{$breaks{$chr}}) {
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80 my @array=@{$breaks{$chr}{$strand}};
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81 @array=sort{$a->[0]<=>$b->[0]} @array;
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82 for (my $i=0;$i<@array;$i++) {
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83 my $start=$array[$i][0];my $end=$array[$i][1];
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84 my @subarray=();
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85 push @subarray,$array[$i];
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86
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87 for (my $j=$i+1;$j<@array;$j++) {
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88 if ($start<$array[$j][1] && $end>$array[$j][0]) { ###overlap
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89 push @subarray,$array[$j];
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90 ($start,$end)=&newpos($start,$end,$array[$j][0],$array[$j][1]);
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91 }
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92 else{
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93 $i=$j-1;
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94 &find_cites(\@subarray,$chr,$strand);
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95 last;
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96 }
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97 }
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98 }
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99 }
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100 }
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101
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102 my %cluster;
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103 foreach my $chr (keys %cites) {
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104 foreach my $strand (keys %{$cites{$chr}}) {
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105 my @array=@{$sites{$chr}{$strand}};
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106 @array=sort{$a->[0]<=>$b->[0]} @array;
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107 for (my $i=0;$i<@array;$i++) {
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108 my $start=$array[$i][0];my $end=$array[$i][1];
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109 my @subarray=();
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110 push @subarray,$array[$i];
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111
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112 for (my $j=$i+1;$j<@array;$j++) {
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113 if ($end>$array[$j][0]-$maxd) { ###distance less than 200bp
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114 push @subarray,$array[$j];
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115 ($start,$end)=&newpos($start,$end,$array[$j][0],$array[$j][1]);
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116 }
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117 else{
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118 @{$cluster{$chr}{$strand}{$i}}=@subarray;
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119 $i=$j-1;
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120 last;
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121 }
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122 }
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123 }
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124
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125 }
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126 }
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127
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128
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129 open FA,">$faout"; #output file
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130 open STR,">$strout";
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131 foreach my $chr (keys %cluster) {
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132 foreach my $strand (keys %{$cluster{$chr}}) {
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133 foreach my $no (keys %{$cluster{$chr}{$strand}}) {
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134 my @array2=@{$cluster{$chr}{$strand}{$no}};
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135 @array2=sort{$a->[0]<=>$b->[0]} @array2;
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136 &excise(\@array2,$chr,$strand);
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137 }
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138 }
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139 }
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140 close FA;
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141 close STR;
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142 sub oneCiteDn{
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143 my ($array,$a,$chr,$strand)=@_;
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144
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145 my $ss=$$array[$a][0]-$flank;
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146 $ss=0 if($ss<0);
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147 my $ee=$$array[$a][1]+$maxd+$flank;
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148 $ee=$lng{$chr} if($ee>$lng{$chr});
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149
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150 my $seq=substr($genome{$chr},$ss,$ee-$ss+1);
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151 if($strand eq "-"){$seq=revcom($seq);}
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152
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153 my $val=&ffw1($seq,$$array[$a][3],$chr,$strand,$ss,$ee);
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154 return $val;
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155 }
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156 sub oneCiteUp{
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157 my ($array,$a,$chr,$strand)=@_;
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158
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159 my $ss=$$array[$a][0]-$maxd-$flank;
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160 $ss=0 if($ss<0);
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161 my $ee=$$array[$a][1]+$flank;
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162 $ee=$lng{$chr} if($ee>$lng{$chr});
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163
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164 my $seq=substr($genome{$chr},$ss,$ee-$ss+1);
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165 if($strand eq "-"){$seq=revcom($seq);}
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166
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167 my $val=&ffw1($seq,$$array[$a][3],$chr,$strand,$ss,$ee);
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168 return $val;
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169
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170 }
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171
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172 sub twoCites{
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173 my ($array,$a,$b,$chr,$strand)=@_;
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174
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175 my $ss=$$array[$a][0]-$flank;
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176 $ss=0 if($ss<0);
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177 my $ee=$$array[$b][1]+$flank;
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178 $ee=$lng{$chr} if($ee>$lng{$chr});
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179
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180 my $seq=substr($genome{$chr},$ss,$ee-$ss+1);
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181 if($strand eq "-"){$seq=revcom($seq);}
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182
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183 # my( $str,$mfe)=RNA::fold($seq);
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184 # return 0 if($mfe>$MAX_ENERGY); ### minimum mfe
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185 my $val=&ffw2($seq,$$array[$a][3],$$array[$b][3],$chr,$strand,$ss,$ee);
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186
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187 return $val;
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188
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189 }
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190 sub excise{
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191 my ($cluster,$chr,$strand)=@_;
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192
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193 if(@{$cluster}==1){
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194 $ok=&oneCiteDn($cluster,0,$chr,$strand);
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195 $ok=&oneCiteUp($cluster,0,$chr,$strand);
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196 }else{
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197 my $peak_pos=0;
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198
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199 for (my $i=0;$i<@{$cluster};$i++) {
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200 if($$cluster[$i][2]>$$cluster[$peak_pos][2]){$peak_pos=$i;}
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201 }
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202
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203 my $ok=0;
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204 for (my $i=0;$i<@{$cluster};$i++) {
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205 next if($i==$peak_pos);
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206 if($i<$peak_pos){$ok=&twoCites($cluster,$i,$peak_pos,$chr,$strand);}
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207 else{$ok=&twoCites($cluster,$peak_pos,$i,$chr,$strand);}
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208 last if($ok);
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209 }
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210 if (!$ok) {
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211 $ok=&oneCiteDn($cluster,$peak_pos,$chr,$strand);
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212 $ok=&oneCiteUp($cluster,$peak_pos,$chr,$strand);
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213 }
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214
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215 }
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216 }
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217
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218 sub ffw2{
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219 my ($seq,$tag1,$tag2,$chr,$strand,$ss,$ee)=@_;
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220
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221 my $N_count=$seq=~tr/N//; ## precursor sequence has not more than 5 Ns
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222 if ($N_count > 5) {
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223 return 0;
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224 }
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225
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226 my $seq_length=length $seq;
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227 # position tag1 and tag2
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228 my $tag1_beg=index($seq,$tag1,0)+1;
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229 if ($tag1_beg < 1) {
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230 warn "[ffw2] coordinate error.\n";
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231 # $fold->{reason}="coordinate error";
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232 return 0;
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233 }
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234 my $tag2_beg=index($seq,$tag2,0)+1;
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235 if ($tag2_beg < 1) {
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236 warn "[ffw2] coordinate error.\n";
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237 # $fold->{reason}="coordinate error";
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238 return 0;
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239 }
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240 if ($tag2_beg < $tag1_beg) {
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241 # swap tag1 and tag2
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242 ($tag1,$tag2)=($tag2,$tag1);
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243 ($tag1_beg,$tag2_beg)=($tag2_beg,$tag1_beg);
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244 }
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245 my $tag1_end=$tag1_beg+length($tag1)-1;
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246 my $tag2_end=$tag2_beg+length($tag2)-1;
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247 # re-clipping
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248 my $beg=$tag1_beg-$FLANK; $beg=1 if $beg < 1;
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249 my $end=$tag2_end+$FLANK; $end=$seq_length if $end > $seq_length;
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250 $seq=substr($seq,$beg-1,$end-$beg+1);
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251 $seq_length=length $seq;
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252 # re-reposition
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253 $tag1_beg=index($seq,$tag1,0)+1;
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254 if ($tag1_beg < 1) {
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255 warn "[ffw2] coordinate error.\n";
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256 # $fold->{reason}="coordinate error";
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257 return 0;
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258 }
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259
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260 $tag2_beg=index($seq,$tag2,0)+1;
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261 if ($tag2_beg < 1) {
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262 warn "[ffw2] coordinate error.\n";
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263 # $fold->{reason}="coordinate error";
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264 return 0;
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265 }
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266 $tag1_end=$tag1_beg+length($tag1)-1;
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267 $tag2_end=$tag2_beg+length($tag2)-1;
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268
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269 # fold
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270 #my ($struct,$mfe)=RNA::fold($seq);
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271 my $rnafold=`perl -e 'print "$seq"' | RNAfold --noPS`;
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272 my @rawfolds=split/\s+/,$rnafold;
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273 my $struct=$rawfolds[1];
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274 my $mfe=$rawfolds[-1];
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275 $mfe=~s/\(//;
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276 $mfe=~s/\)//;
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277 #$mfe=sprintf "%.2f", $mfe;
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278 if ($mfe > $MAX_ENERGY) {return 0;}
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279
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280 # tag1
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281 my $tag1_length=$tag1_end-$tag1_beg+1;
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282 my $tag1_struct=substr($struct,$tag1_beg-1,$tag1_length);
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283 my $tag1_arm=which_arm($tag1_struct);
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284 my $tag1_unpair=$tag1_struct=~tr/.//;
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285 my $tag1_pair=$tag1_length-$tag1_unpair;
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286 my $tag1_max_bulge=biggest_bulge($tag1_struct);
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287 if ($tag1_arm ne "5p") { return 0;} # tag not in stem
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288 # if ($tag1_unpair > $MAX_UNPAIR) {$fold->{reason}="unpair=$tag1_unpair ($MAX_UNPAIR)"; return $pass}
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289 if ($tag1_pair < $MIN_PAIR) {return 0;}
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290 if ($tag1_max_bulge > $MAX_BULGE) {return 0;}
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291
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292 # tag2
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293 my $tag2_length=$tag2_end-$tag2_beg+1;
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294 my $tag2_struct=substr($struct,$tag2_beg-1,$tag2_length);
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295 my $tag2_arm=which_arm($tag2_struct);
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296 my $tag2_unpair=$tag2_struct=~tr/.//;
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297 my $tag2_pair=$tag2_length-$tag2_unpair;
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298 my $tag2_max_bulge=biggest_bulge($tag2_struct);
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299 if ($tag2_arm ne "3p") {return 0;} # star not in stem
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300 # if ($tag2_unpair > $MAX_UNPAIR) {$fold->{reason}="unpair=$tag2_unpair ($MAX_UNPAIR)"; return $pass}
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301 if ($tag2_pair < $MIN_PAIR) {return 0;}
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302 if ($tag2_max_bulge > $MAX_BULGE) {return 0;}
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303
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304 # space size between miR and miR*
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305 my $space=$tag2_beg-$tag1_end-1;
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306 if ($space < $MIN_SPACE) {return 0;}
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307 if ($space > $MAX_SPACE) {return 0;}
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308
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309 # size diff of miR and miR*
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310 my $size_diff=abs($tag1_length-$tag2_length);
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311 if ($size_diff > $MAX_SIZEDIFF) {return 0;}
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312
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313 # build base pairing table
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314 my %pairtable;
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315 &parse_struct($struct,\%pairtable); # coords count from 1
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316
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317 my $asy1=get_asy(\%pairtable,$tag1_beg,$tag1_end);
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318 my $asy2=get_asy(\%pairtable,$tag2_beg,$tag2_end);
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319 my $asy=($asy1 < $asy2) ? $asy1 : $asy2;
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320 if ($asy > $ASYMMETRY) {return 0}
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321
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322 # duplex fold, determine whether two matures like a miR/miR* ike duplex
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323 my ($like_mir_duplex1,$duplex_pair,$overhang1,$overhang2)=likeMirDuplex1($tag1,$tag2);
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324 # parse hairpin, determine whether two matures form miR/miR* duplex in hairpin context
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325 my ($like_mir_duplex2,$duplex_pair2,$overhang_b,$overhang_t)=likeMirDuplex2(\%pairtable,$tag1_beg,$tag1_end,$tag2_beg,$tag2_end);
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326 if ($like_mir_duplex1==0 && $like_mir_duplex2==0) {
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327 return 0;
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328 }
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329
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330 print FA ">$chr:$strand:$ss..$ee\n$seq\n";
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331 print STR ">$chr:$strand:$ss..$ee\n$seq\n$struct\t($mfe)\n";
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332
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333 return 1;
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334 }
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335
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336 sub ffw1{
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337 my ($seq,$tag,$chr,$strand,$ss,$ee)=@_;
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338 my $pass=0;
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339
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340 my $N_count=$seq=~tr/N//;
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341 if ($N_count > 5) {
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342 return 0;
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343 }
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344
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345 my $seq_length=length $seq;
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346 my $tag_length=length $tag;
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347
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348 # position
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349 my $tag_beg=index($seq,$tag,0)+1;
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350 if ($tag_beg < 1) {
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351 warn "[ffw1] coordinate error.\n";
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352 return $pass;
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353 }
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354 my $tag_end=$tag_beg+length($tag)-1;
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355
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356
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357 # define candidate precursor by hybrid short arm to long arm, not solid enough
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358 my($beg,$end)=define_precursor($seq,$tag);
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359 if (not defined $beg) {
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360 return $pass;
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361 }
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362 if (not defined $end) {
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363 return $pass;
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364 }
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365 $seq=substr($seq,$beg-1,$end-$beg+1);
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366 $seq_length=length $seq;
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367
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368
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369 # fold
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370 #my ($struct,$mfe)=RNA::fold($seq);
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371 my $rnafold=`perl -e 'print "$seq"' | RNAfold --noPS`;
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372 my @rawfolds=split/\s+/,$rnafold;
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373 my $struct=$rawfolds[1];
|
|
|
374 my $mfe=$rawfolds[-1];
|
|
|
375 $mfe=~s/\(//;
|
|
|
376 $mfe=~s/\)//;
|
|
|
377
|
|
|
378 if ($mfe > $MAX_ENERGY) {
|
|
|
379 $pass=0;
|
|
|
380 return $pass;
|
|
|
381 }
|
|
|
382
|
|
|
383 # reposition
|
|
|
384 $tag_beg=index($seq,$tag,0)+1;
|
|
|
385 if ($tag_beg < 1) {
|
|
|
386 warn "[ffw1] coordinate error.\n";
|
|
|
387 return 0;
|
|
|
388 }
|
|
|
389 $tag_end=$tag_beg+length($tag)-1;
|
|
|
390
|
|
|
391 my $tag_struct=substr($struct,$tag_beg-1,$tag_length);
|
|
|
392 my $tag_arm=which_arm($tag_struct);
|
|
|
393 my $tag_unpair=$tag_struct=~tr/.//;
|
|
|
394 my $tag_pair=$tag_length-$tag_unpair;
|
|
|
395 my $tag_max_bulge=biggest_bulge($tag_struct);
|
|
|
396 if ($tag_arm eq "-") { return $pass;}
|
|
|
397 # if ($tag_unpair > $MAX_UNPAIR) {$fold->{reason}="unpair=$tag_unpair ($MAX_UNPAIR)"; return $pass}
|
|
|
398 if ($tag_pair < $MIN_PAIR) { return $pass;}
|
|
|
399 if ($tag_max_bulge > $MAX_BULGE) {return $pass;}
|
|
|
400
|
|
|
401 # build base pairing table
|
|
|
402 my %pairtable;
|
|
|
403 &parse_struct($struct,\%pairtable); # coords count from 1
|
|
|
404
|
|
|
405 # get star
|
|
|
406 my ($star_beg,$star_end)=get_star(\%pairtable,$tag_beg,$tag_end);
|
|
|
407 my $star=substr($seq,$star_beg-1,$star_end-$star_beg+1);
|
|
|
408 my $star_length=$star_end-$star_beg+1;
|
|
|
409 my $star_struct=substr($struct,$star_beg-1,$star_end-$star_beg+1);
|
|
|
410 my $star_arm=which_arm($star_struct);
|
|
|
411 my $star_unpair=$star_struct=~tr/.//;
|
|
|
412 my $star_pair=$star_length-$star_unpair;
|
|
|
413 my $star_max_bulge=biggest_bulge($star_struct);
|
|
|
414 if ($star_arm eq "-") { return $pass;}
|
|
|
415 # if ($star_unpair > $MAX_UNPAIR) {$fold->{reason}="unpair=$star_unpair ($MAX_UNPAIR)"; return $pass}
|
|
|
416 if ($star_pair < $MIN_PAIR) {return $pass;}
|
|
|
417 if ($star_max_bulge > $MAX_BULGE) {return $pass;}
|
|
|
418
|
|
|
419 if ($tag_arm eq $star_arm) {return $pass;}
|
|
|
420
|
|
|
421 # space size between miR and miR*
|
|
|
422 my $space;
|
|
|
423 if ($tag_beg < $star_beg) {
|
|
|
424 $space=$star_beg-$tag_end-1;
|
|
|
425 }
|
|
|
426 else {
|
|
|
427 $space=$tag_beg-$star_end-1;
|
|
|
428 }
|
|
|
429 if ($space < $MIN_SPACE) { return $pass;}
|
|
|
430 if ($space > $MAX_SPACE) { return $pass;}
|
|
|
431
|
|
|
432 # size diff
|
|
|
433 my $size_diff=abs($tag_length-$star_length);
|
|
|
434 if ($size_diff > $MAX_SIZEDIFF) { return $pass;}
|
|
|
435
|
|
|
436 # asymmetry
|
|
|
437 my $asy=get_asy(\%pairtable,$tag_beg,$tag_end);
|
|
|
438 if ($asy > $ASYMMETRY) {return $pass;}
|
|
|
439
|
|
|
440 $pass=1;
|
|
|
441 print FA ">$chr:$strand:$ss..$ee\n$seq\n";
|
|
|
442 print STR ">$chr:$strand:$ss..$ee\n$seq\n$struct\t($mfe)\n";
|
|
|
443 return $pass;
|
|
|
444
|
|
|
445 }
|
|
|
446 sub get_star {
|
|
|
447 my($table,$beg,$end)=@_;
|
|
|
448
|
|
|
449 my ($s1,$e1,$s2,$e2); # s1 pair to s2, e1 pair to e2
|
|
|
450 foreach my $i ($beg..$end) {
|
|
|
451 if (defined $table->{$i}) {
|
|
|
452 my $j=$table->{$i};
|
|
|
453 $s1=$i;
|
|
|
454 $s2=$j;
|
|
|
455 last;
|
|
|
456 }
|
|
|
457 }
|
|
|
458 foreach my $i (reverse ($beg..$end)) {
|
|
|
459 if (defined $table->{$i}) {
|
|
|
460 my $j=$table->{$i};
|
|
|
461 $e1=$i;
|
|
|
462 $e2=$j;
|
|
|
463 last;
|
|
|
464 }
|
|
|
465 }
|
|
|
466 # print "$s1,$e1 $s2,$e2\n";
|
|
|
467
|
|
|
468 # correct terminus
|
|
|
469 my $off1=$s1-$beg;
|
|
|
470 my $off2=$end-$e1;
|
|
|
471 $s2+=$off1;
|
|
|
472 $s2+=2; # 081009
|
|
|
473 $e2-=$off2; $e2=1 if $e2 < 1;
|
|
|
474 $e2+=2; $e2=1 if $e2 < 1; # 081009
|
|
|
475 ($s2,$e2)=($e2,$s2) if ($s2 > $e2);
|
|
|
476 return ($s2,$e2);
|
|
|
477 }
|
|
|
478
|
|
|
479 sub define_precursor {
|
|
|
480 my $seq=shift;
|
|
|
481 my $tag=shift;
|
|
|
482
|
|
|
483 my $seq_length=length $seq;
|
|
|
484 my $tag_length=length $tag;
|
|
|
485 my $tag_beg=index($seq,$tag,0)+1;
|
|
|
486 my $tag_end=$tag_beg+$tag_length-1;
|
|
|
487
|
|
|
488 # split the candidate region into short arm and long arm
|
|
|
489 my $tag_arm;
|
|
|
490 my ($larm,$larm_beg,$larm_end);
|
|
|
491 my ($sarm,$sarm_beg,$sarm_end);
|
|
|
492 if ($tag_beg-1 < $seq_length-$tag_end) { # on 5' arm
|
|
|
493 $sarm=substr($seq,0,$tag_end);
|
|
|
494 $larm=substr($seq,$tag_end);
|
|
|
495 $sarm_beg=1;
|
|
|
496 $sarm_end=$tag_end;
|
|
|
497 $larm_beg=$tag_end+1;
|
|
|
498 $larm_end=$seq_length;
|
|
|
499 $tag_arm="5p";
|
|
|
500 }
|
|
|
501 else {
|
|
|
502 $larm=substr($seq,0,$tag_beg-1); # on 3' arm
|
|
|
503 $sarm=substr($seq,$tag_beg-1);
|
|
|
504 $larm_beg=1;
|
|
|
505 $larm_end=$tag_beg-1;
|
|
|
506 $sarm_beg=$tag_beg;
|
|
|
507 $sarm_end=$seq_length;
|
|
|
508 $tag_arm="3p";
|
|
|
509 }
|
|
|
510
|
|
|
511 # print "$sarm_beg,$sarm_end $sarm\n";
|
|
|
512 # print "$larm_beg,$larm_end $larm\n";
|
|
|
513
|
|
|
514 # clipping short arm
|
|
|
515 if ($tag_arm eq "5p") {
|
|
|
516 $sarm_beg=$tag_beg-$flank; $sarm_beg=1 if $sarm_beg < 1;
|
|
|
517 $sarm=substr($seq,$sarm_beg-1,$sarm_end-$sarm_beg+1);
|
|
|
518 }
|
|
|
519 else {
|
|
|
520 $sarm_end=$tag_end+$flank; $sarm_end=$seq_length if $sarm_end > $seq_length;
|
|
|
521 $sarm=substr($seq,$sarm_beg-1,$sarm_end-$sarm_beg+1);
|
|
|
522 }
|
|
|
523 # print "$sarm_beg,$sarm_end $sarm\n";
|
|
|
524 # print "$larm_beg,$larm_end $larm\n";
|
|
|
525
|
|
|
526 # define the precursor by hybriding short arm to long arm
|
|
|
527 =cut #modify in 2014-10-28
|
|
|
528 my $duplex=RNA::duplexfold($sarm,$larm);
|
|
|
529 my $struct=$duplex->{structure};
|
|
|
530 my $energy=sprintf "%.2f", $duplex->{energy};
|
|
|
531 my ($str1,$str2)=split(/&/,$struct);
|
|
|
532 my $pair=$str1=~tr/(//;
|
|
|
533 # print "pair=$pair\n";
|
|
|
534 my $beg1=$duplex->{i}+1-length($str1);
|
|
|
535 my $end1=$duplex->{i};
|
|
|
536 my $beg2=$duplex->{j};
|
|
|
537 my $end2=$duplex->{j}+length($str2)-1;
|
|
|
538 =cut
|
|
|
539 ###### new codes begin
|
|
|
540 my $duplex=`perl -e 'print "$sarm\n$larm"' | RNAduplex`;
|
|
|
541 #(.(.(((.....(((.&))))))...).). 1,16 : 1,13 (-7.20)
|
|
|
542 my @tmpduplex=split/\s+/,$duplex;
|
|
|
543 my $struct=$tmpduplex[0];
|
|
|
544 $tmpduplex[-1]=~s/[(|)]//g;
|
|
|
545 my $energy=$tmpduplex[-1];
|
|
|
546 my ($str1,$str2)=split(/&/,$struct);
|
|
|
547 my $pair=$str1=~tr/(//;
|
|
|
548 my ($beg1,$end1)=split/,/,$tmpduplex[1];
|
|
|
549 my ($beg2,$end2)=split/,/,$tmpduplex[3];
|
|
|
550 ######## new codes end
|
|
|
551
|
|
|
552 # print "$beg1:$end1 $beg2:$end2\n";
|
|
|
553 # transform coordinates
|
|
|
554 $beg1=$beg1+$sarm_beg-1;
|
|
|
555 $end1=$end1+$sarm_beg-1;
|
|
|
556 $beg2=$beg2+$larm_beg-1;
|
|
|
557 $end2=$end2+$larm_beg-1;
|
|
|
558 # print "$beg1:$end1 $beg2:$end2\n";
|
|
|
559
|
|
|
560 my $off5p=$beg1-$sarm_beg;
|
|
|
561 my $off3p=$sarm_end-$end1;
|
|
|
562 $beg2-=$off3p; $beg2=1 if $beg2 < 1;
|
|
|
563 $end2+=$off5p; $end2=$seq_length if $end2 > $seq_length;
|
|
|
564
|
|
|
565 # print "$beg1:$end1 $beg2:$end2\n";
|
|
|
566
|
|
|
567 my $beg=$sarm_beg < $beg2 ? $sarm_beg : $beg2;
|
|
|
568 my $end=$sarm_end > $end2 ? $sarm_end : $end2;
|
|
|
569
|
|
|
570 return if $pair < $MIN_PAIR;
|
|
|
571 # print "$beg,$end\n";
|
|
|
572 return ($beg,$end);
|
|
|
573 }
|
|
|
574
|
|
|
575
|
|
|
576 # duplex fold, judge whether two short seqs like a miRNA/miRNA* duplex
|
|
|
577 sub likeMirDuplex1 {
|
|
|
578 my $seq1=shift;
|
|
|
579 my $seq2=shift;
|
|
|
580 my $like_mir_duplex=1;
|
|
|
581
|
|
|
582 my $length1=length $seq1;
|
|
|
583 my $length2=length $seq2;
|
|
|
584 =cut
|
|
|
585 my $duplex=RNA::duplexfold($seq1, $seq2);
|
|
|
586 my $duplex_struct=$duplex->{structure};
|
|
|
587 my $duplex_energy=sprintf "%.2f", $duplex->{energy};
|
|
|
588 my ($str1,$str2)=split(/&/,$duplex_struct);
|
|
|
589 my $beg1=$duplex->{i}+1-length($str1);
|
|
|
590 my $end1=$duplex->{i};
|
|
|
591 my $beg2=$duplex->{j};
|
|
|
592 my $end2=$duplex->{j}+length($str2)-1;
|
|
|
593 =cut
|
|
|
594 my $duplex=`perl -e 'print "$seq1\n$seq2"' | RNAduplex`;
|
|
|
595 #(.(.(((.....(((.&))))))...).). 1,16 : 1,13 (-7.20)
|
|
|
596 my @tmpduplex=split/\s+/,$duplex;
|
|
|
597 my $duplex_struct=$tmpduplex[0];
|
|
|
598 $tmpduplex[-1]=~s/[(|)]//g;
|
|
|
599 my $duplex_energy=$tmpduplex[-1];
|
|
|
600 my ($str1,$str2)=split(/&/,$duplex_struct);
|
|
|
601 #my $pair=$str1=~tr/(//;
|
|
|
602 my ($beg1,$end1)=split/,/,$tmpduplex[1];
|
|
|
603 my ($beg2,$end2)=split/,/,$tmpduplex[3];
|
|
|
604
|
|
|
605 # revise beg1, end1, beg2, end2
|
|
|
606 $str1=~/^(\.*)/;
|
|
|
607 $beg1+=length($1);
|
|
|
608 $str1=~/(\.*)$/;
|
|
|
609 $end1-=length($1);
|
|
|
610 $str2=~/^(\.*)/;
|
|
|
611 $beg2+=length($1);
|
|
|
612 $str2=~/(\.*)$/;
|
|
|
613 $end2-=length($1);
|
|
|
614
|
|
|
615 my $pair_num=$str1=~tr/(//;
|
|
|
616 my $overhang1=($length2-$end2)-($beg1-1); # 3' overhang at hairpin bottom
|
|
|
617 my $overhang2=($length1-$end1)-($beg2-1); # 3' overhang at hairpin neck
|
|
|
618 # print $pair_num,"\n";
|
|
|
619 # print $overhang1,"\n";
|
|
|
620 # print $overhang2,"\n";
|
|
|
621 if ($pair_num < 13) {
|
|
|
622 $like_mir_duplex=0;
|
|
|
623 }
|
|
|
624 if ($overhang1 < 0 || $overhang2 < 0 ) {
|
|
|
625 $like_mir_duplex=0;
|
|
|
626 }
|
|
|
627 if ($overhang1 > 4 || $overhang2 > 4) {
|
|
|
628 $like_mir_duplex=0;
|
|
|
629 }
|
|
|
630 return ($like_mir_duplex,$pair_num,$overhang1,$overhang2);
|
|
|
631 }
|
|
|
632
|
|
|
633 # judge whether two matures form miR/miR* duplex, in hairpin context
|
|
|
634 sub likeMirDuplex2 {
|
|
|
635 my ($table,$beg1,$end1,$beg2,$end2)=@_;
|
|
|
636 my $like_mir_duplex=1;
|
|
|
637
|
|
|
638 # s1 e1
|
|
|
639 # 5 ----------------------------3
|
|
|
640 # | | |||| ||| |
|
|
|
641 #3 -------------------------------5
|
|
|
642 # e2 s2
|
|
|
643
|
|
|
644 my $pair_num=0;
|
|
|
645 my $overhang1=0;
|
|
|
646 my $overhang2=0;
|
|
|
647 my ($s1,$e1,$s2,$e2);
|
|
|
648 foreach my $i ($beg1..$end1) {
|
|
|
649 if (defined $table->{$i}) {
|
|
|
650 my $j=$table->{$i};
|
|
|
651 if ($j <= $end2 && $j >= $beg2) {
|
|
|
652 $s1=$i;
|
|
|
653 $e2=$j;
|
|
|
654 last;
|
|
|
655 }
|
|
|
656 }
|
|
|
657 }
|
|
|
658 foreach my $i (reverse ($beg1..$end1)) {
|
|
|
659 if (defined $table->{$i}) {
|
|
|
660 my $j=$table->{$i};
|
|
|
661 if ($j <= $end2 && $j >= $beg2) {
|
|
|
662 $e1=$i;
|
|
|
663 $s2=$j;
|
|
|
664 last;
|
|
|
665 }
|
|
|
666 }
|
|
|
667 }
|
|
|
668
|
|
|
669 # print "$beg1,$end1 $s1,$e1\n";
|
|
|
670 # print "$beg2,$end2 $s2,$e2\n";
|
|
|
671
|
|
|
672 foreach my $i ($beg1..$end1) {
|
|
|
673 if (defined $table->{$i}) {
|
|
|
674 my $j=$table->{$i};
|
|
|
675 if ($j <= $end2 && $j >= $beg2) {
|
|
|
676 ++$pair_num;
|
|
|
677 }
|
|
|
678 }
|
|
|
679 }
|
|
|
680 if (defined $s1 && defined $e2) {
|
|
|
681 $overhang1=($end2-$e2)-($s1-$beg1);
|
|
|
682 }
|
|
|
683 if (defined $e1 && defined $s2) {
|
|
|
684 $overhang2=($end1-$e1)-($s2-$beg2);
|
|
|
685 }
|
|
|
686
|
|
|
687 if ($pair_num < 13) {
|
|
|
688 $like_mir_duplex=0;
|
|
|
689 }
|
|
|
690 if ($overhang1 < 0 && $overhang2 < 0) {
|
|
|
691 $like_mir_duplex=0;
|
|
|
692 }
|
|
|
693 return ($like_mir_duplex,$pair_num,$overhang1,$overhang2);
|
|
|
694 }
|
|
|
695 sub parse_struct {
|
|
|
696 my $struct=shift;
|
|
|
697 my $table=shift;
|
|
|
698
|
|
|
699 my @t=split('',$struct);
|
|
|
700 my @lbs; # left brackets
|
|
|
701 foreach my $k (0..$#t) {
|
|
|
702 if ($t[$k] eq "(") {
|
|
|
703 push @lbs, $k+1;
|
|
|
704 }
|
|
|
705 elsif ($t[$k] eq ")") {
|
|
|
706 my $lb=pop @lbs;
|
|
|
707 my $rb=$k+1;
|
|
|
708 $table->{$lb}=$rb;
|
|
|
709 $table->{$rb}=$lb;
|
|
|
710 }
|
|
|
711 }
|
|
|
712 if (@lbs) {
|
|
|
713 warn "unbalanced RNA struct.\n";
|
|
|
714 }
|
|
|
715 }
|
|
|
716 sub which_arm {
|
|
|
717 my $substruct=shift;
|
|
|
718 my $arm;
|
|
|
719 if ($substruct=~/\(/ && $substruct=~/\)/) {
|
|
|
720 $arm="-";
|
|
|
721 }
|
|
|
722 elsif ($substruct=~/\(/) {
|
|
|
723 $arm="5p";
|
|
|
724 }
|
|
|
725 else {
|
|
|
726 $arm="3p";
|
|
|
727 }
|
|
|
728 return $arm;
|
|
|
729 }
|
|
|
730 sub biggest_bulge {
|
|
|
731 my $struct=shift;
|
|
|
732 my $bulge_size=0;
|
|
|
733 my $max_bulge=0;
|
|
|
734 while ($struct=~/(\.+)/g) {
|
|
|
735 $bulge_size=length $1;
|
|
|
736 if ($bulge_size > $max_bulge) {
|
|
|
737 $max_bulge=$bulge_size;
|
|
|
738 }
|
|
|
739 }
|
|
|
740 return $max_bulge;
|
|
|
741 }
|
|
|
742 sub get_asy {
|
|
|
743 my($table,$a1,$a2)=@_;
|
|
|
744 my ($pre_i,$pre_j);
|
|
|
745 my $asymmetry=0;
|
|
|
746 foreach my $i ($a1..$a2) {
|
|
|
747 if (defined $table->{$i}) {
|
|
|
748 my $j=$table->{$i};
|
|
|
749 if (defined $pre_i && defined $pre_j) {
|
|
|
750 my $diff=($i-$pre_i)+($j-$pre_j);
|
|
|
751 $asymmetry += abs($diff);
|
|
|
752 }
|
|
|
753 $pre_i=$i;
|
|
|
754 $pre_j=$j;
|
|
|
755 }
|
|
|
756 }
|
|
|
757 return $asymmetry;
|
|
|
758 }
|
|
|
759
|
|
|
760 sub peaks{
|
|
|
761 my @cluster=@{$_[0]};
|
|
|
762
|
|
|
763 return if(@cluster<1);
|
|
|
764
|
|
|
765 my $max=0; my $index=-1;
|
|
|
766 for (my $i=0;$i<@cluster;$i++) {
|
|
|
767 if($cluster[$i][2]>$max){
|
|
|
768 $max=$cluster[$i][2];
|
|
|
769 $index=$i;
|
|
|
770 }
|
|
|
771 }
|
|
|
772 # &excise(\@cluster,$index,$_[1],$_[2]);
|
|
|
773 return($index);
|
|
|
774 }
|
|
|
775
|
|
|
776 sub find_cites{
|
|
|
777 my @tmp=@{$_[0]};
|
|
|
778 my $i=&peaks(\@tmp);
|
|
|
779
|
|
|
780 my $start=$tmp[$i][0];
|
|
|
781 my $total=0; my $node5=0;
|
|
|
782 for (my $j=0;$j<@tmp ;$j++) {
|
|
|
783 $total+=$tmp[$j][2];
|
|
|
784 $node5 +=$tmp[$j][2] if($tmp[$j][0]-$start<=2 && $tmp[$j][0]-$start>=-2);
|
|
|
785 }
|
|
|
786 push @{$cites{$_[1]}{$_[2]}},$tmp[$i] if($node5/$total>0.80 && $tmp[$i][2]/$node5>0.5);
|
|
|
787 }
|
|
|
788
|
|
|
789 sub newpos{
|
|
|
790 my ($a,$b,$c,$d)=@_;
|
|
|
791 my $s= $a>$c ? $c : $a;
|
|
|
792 my $e=$b>$d ? $b : $d;
|
|
|
793 return($s,$e);
|
|
|
794 }
|
|
|
795
|
|
|
796 sub rev{
|
|
|
797
|
|
|
798 my($sequence)=@_;
|
|
|
799
|
|
|
800 my $rev=reverse $sequence;
|
|
|
801
|
|
|
802 return $rev;
|
|
|
803 }
|
|
|
804
|
|
|
805 sub com{
|
|
|
806
|
|
|
807 my($sequence)=@_;
|
|
|
808
|
|
|
809 $sequence=~tr/acgtuACGTU/TGCAATGCAA/;
|
|
|
810
|
|
|
811 return $sequence;
|
|
|
812 }
|
|
|
813
|
|
|
814 sub revcom{
|
|
|
815
|
|
|
816 my($sequence)=@_;
|
|
|
817
|
|
|
818 my $revcom=rev(com($sequence));
|
|
|
819
|
|
|
820 return $revcom;
|
|
|
821 }
|
|
|
822
|
|
|
823 sub find_strand{
|
|
|
824
|
|
|
825 #A subroutine to find the strand, parsing different blast formats
|
|
|
826 my($other)=@_;
|
|
|
827
|
|
|
828 my $strand="+";
|
|
|
829
|
|
|
830 if($other=~/-/){
|
|
|
831 $strand="-";
|
|
|
832 }
|
|
|
833
|
|
|
834 if($other=~/minus/i){
|
|
|
835 $strand="-";
|
|
|
836 }
|
|
|
837
|
|
|
838 return($strand);
|
|
|
839 }
|
|
|
840 sub usage{
|
|
|
841 print <<"USAGE";
|
|
|
842 Version $version
|
|
|
843 Usage:
|
|
|
844 $0 -map -g -d -f -o -s -e
|
|
|
845 options:
|
|
|
846 -map input file# align result # bst. format
|
|
|
847 -g input file # genome sequence fasta format
|
|
|
848 -d <int> Maximal space between miRNA and miRNA* (200)
|
|
|
849 -f <int> Flank sequence length of miRNA precursor (10)
|
|
|
850 -o output file# percursor fasta file
|
|
|
851 -s output file# precursor structure file
|
|
|
852 -e <folat> Maximal free energy allowed for a miRNA precursor (-18 kcal/mol)
|
|
|
853
|
|
|
854 -h help
|
|
|
855 USAGE
|
|
|
856 exit(1);
|
|
|
857 }
|
|
|
858
|
