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