--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/precursors.pl	Fri Jul 25 05:22:09 2014 -0400
@@ -0,0 +1,789 @@
+#!/usr/bin/perl -w
+#Filename:
+#Author: Tian Dongmei
+#Email: tiandm@big.ac.cn
+#Date: 2013/7/19
+#Modified:
+#Description: 
+my $version=1.00;
+
+use strict;
+use Getopt::Long;
+use RNA;
+
+my %opts;
+GetOptions(\%opts,"map=s","g=s","d:i","f:i","o=s","e:f","s=s","h");
+if (!(defined $opts{map} and defined $opts{g} and defined $opts{o} and defined $opts{s} ) || defined $opts{h}) { #necessary arguments
+&usage;
+}
+
+my $filein=$opts{'map'};
+my $faout=$opts{'o'};
+my $strout=$opts{'s'};
+my $genome= $opts{'g'};
+
+my $maxd=defined $opts{'d'} ? $opts{'d'} : 200;
+my $flank=defined $opts{'f'}? $opts{'f'} : 10;
+
+my $MAX_ENERGY=-18;
+if (defined $opts{'e'}) {$MAX_ENERGY=$opts{'e'};}
+my $MAX_UNPAIR=5;
+my $MIN_PAIR=15;
+my $MAX_SIZEDIFF=4;
+my $MAX_BULGE=2;
+my $ASYMMETRY=5;
+my $MIN_UNPAIR=0;
+my $MIN_SPACE=5;
+my $MAX_SPACE=$maxd;
+my $FLANK=$flank;
+
+######### load in genome sequences start ########
+my %genome;
+my %lng;
+my $name;
+open IN,"<$genome";
+while (my $aline=<IN>) {
+	chomp $aline;
+	next if($aline=~/^\#/);
+	if ($aline=~/^>(\S+)/) {
+		$name=$1;
+		next;
+	}
+	$genome{$name} .=$aline;
+}
+close IN;
+foreach my $key (keys %genome) {
+	$lng{$key}=length($genome{$key});
+}
+####### load in genome sequences end ##########
+
+my %breaks; ### reads number bigger than 3
+open IN,"<$filein"; #input file  
+while (my $aline=<IN>) {
+	chomp $aline;
+	my @tmp=split/\t/,$aline;
+	$tmp[0]=~/_x(\d+)$/;
+	my $no=$1;
+	next if($no<3);
+	#my $trand=&find_strand($tmp[9]);
+	#my @pos=split/\.\./,$tmp[5];
+	my $end=$tmp[3]+length($tmp[4])-1;
+	if($tmp[1] eq "-"){$tmp[4]=revcom($tmp[4]);}
+	push @{$breaks{$tmp[2]}{$tmp[1]}},[$tmp[3],$end,$no,$tmp[4]]; ### 0 base
+}
+close IN;
+
+my %cites; ### peaks
+foreach my $chr (keys %breaks) {
+	foreach my $strand (keys %{$breaks{$chr}}) {
+		my @array=@{$breaks{$chr}{$strand}};
+		@array=sort{$a->[0]<=>$b->[0]} @array;
+		for (my $i=0;$i<@array;$i++) {
+			my $start=$array[$i][0];my $end=$array[$i][1];
+			my @subarray=();
+			push @subarray,$array[$i];
+
+			for (my $j=$i+1;$j<@array;$j++) {
+				if ($start<$array[$j][1] && $end>$array[$j][0]) {
+					push @subarray,$array[$j];
+					($start,$end)=&newpos($start,$end,$array[$j][0],$array[$j][1]);
+				}
+				else{
+					$i=$j;
+					&find_cites(\@subarray,$chr,$strand);
+					last;
+				}
+			}
+		}
+	}
+}
+
+open FA,">$faout"; #output file 
+open STR,">$strout";
+foreach my $chr (keys %cites) {
+	foreach my $strand (keys %{$cites{$chr}}) {
+
+		my @array2=@{$cites{$chr}{$strand}};
+		@array2=sort{$a->[0]<=>$b->[0]} @array2;
+		&excise(\@array2,$chr,$strand);
+	}
+}
+close FA;
+close STR;
+sub oneCiteDn{
+	my ($array,$a,$chr,$strand)=@_;
+
+	my $ss=$$array[$a][0]-$flank;
+	$ss=0 if($ss<0);
+	my $ee=$$array[$a][1]+$maxd+$flank;
+	$ee=$lng{$chr} if($ee>$lng{$chr});
+	
+	my $seq=substr($genome{$chr},$ss,$ee-$ss+1);
+	if($strand eq "-"){$seq=revcom($seq);}
+
+	my $val=&ffw1($seq,$$array[$a][3],$chr,$strand,$ss,$ee);
+	return $val;
+}
+sub oneCiteUp{
+	my ($array,$a,$chr,$strand)=@_;
+
+	my $ss=$$array[$a][0]-$maxd-$flank;
+	$ss=0 if($ss<0);
+	my $ee=$$array[$a][1]+$flank;
+	$ee=$lng{$chr} if($ee>$lng{$chr});
+	
+	my $seq=substr($genome{$chr},$ss,$ee-$ss+1);
+	if($strand eq "-"){$seq=revcom($seq);}
+
+	my $val=&ffw1($seq,$$array[$a][3],$chr,$strand,$ss,$ee);
+	return $val;
+
+}
+
+sub twoCites{
+	my ($array,$a,$b,$chr,$strand)=@_;
+
+	my $ss=$$array[$a][0]-$flank;
+	$ss=0 if($ss<0);
+	my $ee=$$array[$b][1]+$flank;
+	$ee=$lng{$chr} if($ee>$lng{$chr});
+	
+	my $seq=substr($genome{$chr},$ss,$ee-$ss+1);
+	if($strand eq "-"){$seq=revcom($seq);}
+
+#	my( $str,$mfe)=RNA::fold($seq);
+#	return 0 if($mfe>$MAX_ENERGY); ### minimum mfe
+	my $val=&ffw2($seq,$$array[$a][3],$$array[$b][3],$chr,$strand,$ss,$ee);
+	
+	return $val;
+
+}
+sub excise{
+	my ($cluster,$chr,$strand)=@_;
+
+	my $last_pos=0;
+	for (my $i=0;$i<@{$cluster};$i++) {
+		next if($$cluster[$i][0]<$last_pos);
+		my $ok=0;
+		for (my $j=$i+1;$j<@{$cluster} ;$j++) {
+			if($$cluster[$j][0]-$$cluster[$i][1]>$maxd){
+				$i=$j;
+				last;
+			}else{
+				$ok=&twoCites($cluster,$i,$j,$chr,$strand);
+				if($ok){ $last_pos=$$cluster[$j][1]+$flank; $i=$j; last;}
+			}
+		}
+		next if($ok);
+
+		$ok=&oneCiteDn($cluster,$i,$chr,$strand);
+		if($ok){$last_pos=$$cluster[$i][1]+$maxd+$flank; next;}
+		$ok=&oneCiteUp($cluster,$i,$chr,$strand);
+		if($ok){$last_pos=$$cluster[$i][1]+$flank;next;}
+	}
+
+
+}
+
+sub ffw2{
+	my ($seq,$tag1,$tag2,$chr,$strand,$ss,$ee)=@_;
+	
+	my $N_count=$seq=~tr/N//;  ## precursor sequence has not more than 5 Ns
+	if ($N_count > 5) {
+		return 0;
+	}
+
+	my $seq_length=length $seq;
+	# position tag1 and tag2
+	my $tag1_beg=index($seq,$tag1,0)+1;
+	if ($tag1_beg < 1) {
+		warn "[ffw2] coordinate error.\n";
+#		$fold->{reason}="coordinate error";
+		return 0;
+	}
+	my $tag2_beg=index($seq,$tag2,0)+1;
+	if ($tag2_beg < 1) {
+		warn "[ffw2] coordinate error.\n";
+#		$fold->{reason}="coordinate error";
+		return 0;
+	}
+	if ($tag2_beg < $tag1_beg) {
+		# swap tag1 and tag2
+		($tag1,$tag2)=($tag2,$tag1);
+		($tag1_beg,$tag2_beg)=($tag2_beg,$tag1_beg);
+	}
+	my $tag1_end=$tag1_beg+length($tag1)-1;
+	my $tag2_end=$tag2_beg+length($tag2)-1;
+	# re-clipping
+	my $beg=$tag1_beg-$FLANK; $beg=1 if $beg < 1;
+	my $end=$tag2_end+$FLANK; $end=$seq_length if $end > $seq_length;
+	$seq=substr($seq,$beg-1,$end-$beg+1);
+	$seq_length=length $seq;
+	# re-reposition
+	$tag1_beg=index($seq,$tag1,0)+1;
+	if ($tag1_beg < 1) {
+		warn "[ffw2] coordinate error.\n";
+#		$fold->{reason}="coordinate error";
+		return 0;
+	}
+	
+	$tag2_beg=index($seq,$tag2,0)+1;
+	if ($tag2_beg < 1) {
+		warn "[ffw2] coordinate error.\n";
+#		$fold->{reason}="coordinate error";
+		return 0;
+	}
+	$tag1_end=$tag1_beg+length($tag1)-1;
+	$tag2_end=$tag2_beg+length($tag2)-1;
+
+	# fold
+	my ($struct,$mfe)=RNA::fold($seq);
+	$mfe=sprintf "%.2f", $mfe;
+	if ($mfe > $MAX_ENERGY) {return 0;}
+
+	# tag1
+	my $tag1_length=$tag1_end-$tag1_beg+1;
+	my $tag1_struct=substr($struct,$tag1_beg-1,$tag1_length);
+	my $tag1_arm=which_arm($tag1_struct);
+	my $tag1_unpair=$tag1_struct=~tr/.//;
+	my $tag1_pair=$tag1_length-$tag1_unpair;
+	my $tag1_max_bulge=biggest_bulge($tag1_struct);
+	if ($tag1_arm ne "5p") { return 0;} # tag not in stem
+#	if ($tag1_unpair > $MAX_UNPAIR) {$fold->{reason}="unpair=$tag1_unpair ($MAX_UNPAIR)"; return $pass}
+	if ($tag1_pair < $MIN_PAIR) {return 0;}
+	if ($tag1_max_bulge > $MAX_BULGE) {return 0;}
+
+	# tag2
+	my $tag2_length=$tag2_end-$tag2_beg+1;
+	my $tag2_struct=substr($struct,$tag2_beg-1,$tag2_length);
+	my $tag2_arm=which_arm($tag2_struct);
+	my $tag2_unpair=$tag2_struct=~tr/.//;
+	my $tag2_pair=$tag2_length-$tag2_unpair;
+	my $tag2_max_bulge=biggest_bulge($tag2_struct);
+	if ($tag2_arm ne "3p") {return 0;} # star not in stem
+#	if ($tag2_unpair > $MAX_UNPAIR) {$fold->{reason}="unpair=$tag2_unpair ($MAX_UNPAIR)"; return $pass}
+	if ($tag2_pair < $MIN_PAIR) {return 0;}
+	if ($tag2_max_bulge > $MAX_BULGE) {return 0;}
+
+	# space size between miR and miR*
+	my $space=$tag2_beg-$tag1_end-1;
+	if ($space < $MIN_SPACE) {return 0;}
+	if ($space > $MAX_SPACE) {return 0;}
+	
+	# size diff of miR and miR*
+	my $size_diff=abs($tag1_length-$tag2_length);
+	if ($size_diff > $MAX_SIZEDIFF) {return 0;}
+
+	# build base pairing table
+	my %pairtable;
+	&parse_struct($struct,\%pairtable); # coords count from 1
+
+	my $asy1=get_asy(\%pairtable,$tag1_beg,$tag1_end);
+	my $asy2=get_asy(\%pairtable,$tag2_beg,$tag2_end);
+	my $asy=($asy1 < $asy2) ? $asy1 : $asy2;
+	if ($asy > $ASYMMETRY) {return 0}
+
+	# duplex fold, determine whether two matures like a miR/miR* ike duplex
+	my ($like_mir_duplex1,$duplex_pair,$overhang1,$overhang2)=likeMirDuplex1($tag1,$tag2);
+	# parse hairpin, determine whether two matures form miR/miR* duplex in hairpin context
+	my ($like_mir_duplex2,$duplex_pair2,$overhang_b,$overhang_t)=likeMirDuplex2(\%pairtable,$tag1_beg,$tag1_end,$tag2_beg,$tag2_end);
+	if ($like_mir_duplex1==0 && $like_mir_duplex2==0) {
+		return 0;
+	}
+
+	print FA ">$chr:$strand:$ss..$ee\n$seq\n";
+	print STR ">$chr:$strand:$ss..$ee\n$seq\n$struct\t($mfe)\n";
+	
+	return 1;	
+}
+
+sub ffw1{
+	my ($seq,$tag,$chr,$strand,$ss,$ee)=@_;
+	my $pass=0;
+
+	my $N_count=$seq=~tr/N//;
+	if ($N_count > 5) {
+		return 0;
+	}
+
+	my $seq_length=length $seq;
+	my $tag_length=length $tag;
+
+	# position
+	my $tag_beg=index($seq,$tag,0)+1;
+	if ($tag_beg < 1) {
+		 warn "[ffw1] coordinate error.\n";
+		 return $pass;
+	}
+	my $tag_end=$tag_beg+length($tag)-1;
+	
+	
+	# define candidate precursor by hybrid short arm to long arm, not solid enough
+	my($beg,$end)=define_precursor($seq,$tag);
+	if (not defined $beg) {
+		return $pass;
+	}
+	if (not defined $end) {
+		return $pass;
+	}
+	$seq=substr($seq,$beg-1,$end-$beg+1);
+	$seq_length=length $seq;
+	
+	
+	# fold
+	my ($struct,$mfe)=RNA::fold($seq);
+	$mfe=sprintf "%.2f",$mfe;
+	if ($mfe > $MAX_ENERGY) {
+		$pass=0;
+		return $pass;
+	}
+
+	# reposition
+	$tag_beg=index($seq,$tag,0)+1;
+	if ($tag_beg < 1) {
+		 warn "[ffw1] coordinate error.\n";
+		 return 0;
+	}
+	$tag_end=$tag_beg+length($tag)-1;
+
+	my $tag_struct=substr($struct,$tag_beg-1,$tag_length);
+	my $tag_arm=which_arm($tag_struct);
+	my $tag_unpair=$tag_struct=~tr/.//;
+	my $tag_pair=$tag_length-$tag_unpair;
+	my $tag_max_bulge=biggest_bulge($tag_struct);
+	if ($tag_arm eq "-") { return $pass;}
+#	if ($tag_unpair > $MAX_UNPAIR) {$fold->{reason}="unpair=$tag_unpair ($MAX_UNPAIR)"; return $pass}
+	if ($tag_pair < $MIN_PAIR) { return $pass;}
+	if ($tag_max_bulge > $MAX_BULGE) {return $pass;}
+
+	# build base pairing table
+	my %pairtable;
+	&parse_struct($struct,\%pairtable); # coords count from 1
+	
+	# get star
+	my ($star_beg,$star_end)=get_star(\%pairtable,$tag_beg,$tag_end);
+	my $star=substr($seq,$star_beg-1,$star_end-$star_beg+1);
+	my $star_length=$star_end-$star_beg+1;
+	my $star_struct=substr($struct,$star_beg-1,$star_end-$star_beg+1);
+	my $star_arm=which_arm($star_struct);
+	my $star_unpair=$star_struct=~tr/.//;
+	my $star_pair=$star_length-$star_unpair;
+	my $star_max_bulge=biggest_bulge($star_struct);
+	if ($star_arm eq "-") { return $pass;}
+#	if ($star_unpair > $MAX_UNPAIR) {$fold->{reason}="unpair=$star_unpair ($MAX_UNPAIR)"; return $pass}
+	if ($star_pair < $MIN_PAIR) {return $pass;}
+	if ($star_max_bulge > $MAX_BULGE) {return $pass;}
+
+	if ($tag_arm eq $star_arm) {return $pass;}
+	
+	# space size between miR and miR*
+	my $space;
+	if ($tag_beg < $star_beg) {
+		$space=$star_beg-$tag_end-1;
+	}
+	else {
+		$space=$tag_beg-$star_end-1;
+	}
+	if ($space < $MIN_SPACE) { return $pass;}
+	if ($space > $MAX_SPACE) { return $pass;}
+
+	# size diff
+	my $size_diff=abs($tag_length-$star_length);
+	if ($size_diff > $MAX_SIZEDIFF) { return $pass;}
+	
+	# asymmetry
+	my $asy=get_asy(\%pairtable,$tag_beg,$tag_end);
+	if ($asy > $ASYMMETRY) {return $pass;}
+	
+	$pass=1;
+	print FA ">$chr:$strand:$ss..$ee\n$seq\n";
+	print STR ">$chr:$strand:$ss..$ee\n$seq\n$struct\t($mfe)\n";
+	return $pass;
+
+}
+sub get_star {
+	my($table,$beg,$end)=@_;
+	
+	my ($s1,$e1,$s2,$e2); # s1 pair to s2, e1 pair to e2
+	foreach my $i ($beg..$end) {
+		if (defined $table->{$i}) {
+			my $j=$table->{$i};
+			$s1=$i;
+			$s2=$j;
+			last;
+		}
+	}
+	foreach my $i (reverse ($beg..$end)) {
+		if (defined $table->{$i}) {
+			my $j=$table->{$i};
+			$e1=$i;
+			$e2=$j;
+			last;
+		}
+	}
+#	print "$s1,$e1 $s2,$e2\n";
+	
+	# correct terminus
+	my $off1=$s1-$beg;
+	my $off2=$end-$e1;
+	$s2+=$off1;
+	$s2+=2; # 081009
+	$e2-=$off2; $e2=1 if $e2 < 1;
+	$e2+=2; $e2=1 if $e2 < 1; # 081009
+	($s2,$e2)=($e2,$s2) if ($s2 > $e2);
+	return ($s2,$e2);
+}
+
+sub define_precursor {
+	my $seq=shift;
+	my $tag=shift;
+
+	my $seq_length=length $seq;
+	my $tag_length=length $tag;
+	my $tag_beg=index($seq,$tag,0)+1;
+	my $tag_end=$tag_beg+$tag_length-1;
+
+	# split the candidate region into short arm and long arm
+	my $tag_arm;
+        my ($larm,$larm_beg,$larm_end);
+	my ($sarm,$sarm_beg,$sarm_end);
+        if ($tag_beg-1 < $seq_length-$tag_end) { # on 5' arm
+                $sarm=substr($seq,0,$tag_end);
+		$larm=substr($seq,$tag_end);
+		$sarm_beg=1;
+		$sarm_end=$tag_end;
+		$larm_beg=$tag_end+1;
+		$larm_end=$seq_length;
+		$tag_arm="5p";
+        }
+        else {
+            $larm=substr($seq,0,$tag_beg-1); # on 3' arm
+			$sarm=substr($seq,$tag_beg-1);
+			$larm_beg=1;
+			$larm_end=$tag_beg-1;
+			$sarm_beg=$tag_beg;
+			$sarm_end=$seq_length;
+			$tag_arm="3p";
+        }
+	
+#	print "$sarm_beg,$sarm_end $sarm\n";
+#	print "$larm_beg,$larm_end $larm\n";
+
+	# clipping short arm
+	if ($tag_arm eq "5p") {
+		$sarm_beg=$tag_beg-$flank; $sarm_beg=1 if $sarm_beg < 1;
+		$sarm=substr($seq,$sarm_beg-1,$sarm_end-$sarm_beg+1);
+	}
+	else {
+		$sarm_end=$tag_end+$flank; $sarm_end=$seq_length if $sarm_end > $seq_length;
+		$sarm=substr($seq,$sarm_beg-1,$sarm_end-$sarm_beg+1);
+	}
+#	print "$sarm_beg,$sarm_end $sarm\n";
+#	print "$larm_beg,$larm_end $larm\n";
+
+	# define the precursor by hybriding short arm to long arm
+	my $duplex=RNA::duplexfold($sarm,$larm);
+	my $struct=$duplex->{structure};
+	my $energy=sprintf "%.2f", $duplex->{energy};
+	my ($str1,$str2)=split(/&/,$struct);
+	my $pair=$str1=~tr/(//;
+#	print "pair=$pair\n";
+	my $beg1=$duplex->{i}+1-length($str1);
+	my $end1=$duplex->{i};
+	my $beg2=$duplex->{j};
+	my $end2=$duplex->{j}+length($str2)-1;
+#	print "$beg1:$end1 $beg2:$end2\n";
+	# transform coordinates
+	$beg1=$beg1+$sarm_beg-1;
+	$end1=$end1+$sarm_beg-1;
+	$beg2=$beg2+$larm_beg-1;
+	$end2=$end2+$larm_beg-1;
+#	print "$beg1:$end1 $beg2:$end2\n";
+	
+	my $off5p=$beg1-$sarm_beg;
+	my $off3p=$sarm_end-$end1;
+	$beg2-=$off3p; $beg2=1 if $beg2 < 1;
+	$end2+=$off5p; $end2=$seq_length if $end2 > $seq_length;
+
+#	print "$beg1:$end1 $beg2:$end2\n";
+	
+	my $beg=$sarm_beg < $beg2 ? $sarm_beg : $beg2;
+	my $end=$sarm_end > $end2 ? $sarm_end : $end2;
+	
+	return if $pair < $MIN_PAIR;
+#	print "$beg,$end\n";
+	return ($beg,$end);
+}
+
+
+# duplex fold, judge whether two short seqs like a miRNA/miRNA* duplex
+sub likeMirDuplex1 {
+	my $seq1=shift;
+	my $seq2=shift;
+	my $like_mir_duplex=1;
+	
+	my $length1=length $seq1;
+	my $length2=length $seq2;
+	my $duplex=RNA::duplexfold($seq1, $seq2);
+	my $duplex_struct=$duplex->{structure};
+	my $duplex_energy=sprintf "%.2f", $duplex->{energy};
+	my ($str1,$str2)=split(/&/,$duplex_struct);
+	my $beg1=$duplex->{i}+1-length($str1);
+	my $end1=$duplex->{i};
+	my $beg2=$duplex->{j};
+	my $end2=$duplex->{j}+length($str2)-1;
+	
+	# revise beg1, end1, beg2, end2
+	$str1=~/^(\.*)/;
+	$beg1+=length($1);
+	$str1=~/(\.*)$/;
+	$end1-=length($1);
+	$str2=~/^(\.*)/;
+	$beg2+=length($1);
+	$str2=~/(\.*)$/;
+	$end2-=length($1);
+
+	my $pair_num=$str1=~tr/(//;
+	my $overhang1=($length2-$end2)-($beg1-1); # 3' overhang at hairpin bottom
+	my $overhang2=($length1-$end1)-($beg2-1); # 3' overhang at hairpin neck
+#	print $pair_num,"\n";
+#	print $overhang1,"\n";
+#	print $overhang2,"\n";
+	if ($pair_num < 13) {
+		$like_mir_duplex=0;
+	}
+	if ($overhang1 < 0 || $overhang2 < 0 ) {
+		$like_mir_duplex=0;
+	}
+	if ($overhang1 > 4 || $overhang2 > 4) {
+		$like_mir_duplex=0;
+	}
+	return ($like_mir_duplex,$pair_num,$overhang1,$overhang2);
+}
+
+# judge whether two matures form miR/miR* duplex, in hairpin context
+sub likeMirDuplex2 {
+	my ($table,$beg1,$end1,$beg2,$end2)=@_;
+	my $like_mir_duplex=1;
+
+#	   s1         e1
+#   5 ----------------------------3	
+#      | | |||| |||               |
+#3 -------------------------------5
+#      e2         s2
+
+	my $pair_num=0;
+	my $overhang1=0;
+	my $overhang2=0;
+	my ($s1,$e1,$s2,$e2);
+	foreach my $i ($beg1..$end1) {
+		if (defined $table->{$i}) {
+			my $j=$table->{$i};
+			if ($j <= $end2 && $j >= $beg2) {
+				$s1=$i;
+				$e2=$j;
+				last;
+			}
+		}
+	}
+	foreach my $i (reverse ($beg1..$end1)) {
+		if (defined $table->{$i}) {
+			my $j=$table->{$i};
+			if ($j <= $end2 && $j >= $beg2) {
+				$e1=$i;
+				$s2=$j;
+				last;
+			}
+		}
+	}
+
+#	print "$beg1,$end1 $s1,$e1\n";
+#	print "$beg2,$end2 $s2,$e2\n";
+
+	foreach my $i ($beg1..$end1) {
+		if (defined $table->{$i}) {
+			my $j=$table->{$i};
+			if ($j <= $end2 && $j >= $beg2) {
+				++$pair_num;
+			}
+		}
+	}
+	if (defined $s1 && defined $e2) {
+		$overhang1=($end2-$e2)-($s1-$beg1);
+	}
+	if (defined $e1 && defined $s2) {
+		$overhang2=($end1-$e1)-($s2-$beg2);
+	}
+	
+	if ($pair_num < 13) {
+		$like_mir_duplex=0;
+	}
+	if ($overhang1 < 0 && $overhang2 < 0) {
+		$like_mir_duplex=0;
+	}
+	return ($like_mir_duplex,$pair_num,$overhang1,$overhang2);
+}
+sub parse_struct {
+	my $struct=shift;
+	my $table=shift;
+
+	my @t=split('',$struct);
+	my @lbs; # left brackets
+	foreach my $k (0..$#t) {
+		if ($t[$k] eq "(") {
+			push @lbs, $k+1;
+		}
+		elsif ($t[$k] eq ")") {
+			my $lb=pop @lbs;
+			my $rb=$k+1;
+			$table->{$lb}=$rb;
+			$table->{$rb}=$lb;
+		}
+	}
+	if (@lbs) {
+		warn "unbalanced RNA struct.\n";
+	}
+}
+sub which_arm {
+	my $substruct=shift;
+	my $arm;
+	if ($substruct=~/\(/ && $substruct=~/\)/) {
+		$arm="-";
+	}
+	elsif ($substruct=~/\(/) {
+		$arm="5p";
+	}
+	else {
+		$arm="3p";
+	}
+	return $arm;
+}
+sub biggest_bulge {
+	my $struct=shift;
+	my $bulge_size=0;
+	my $max_bulge=0;
+	while ($struct=~/(\.+)/g) {
+		$bulge_size=length $1;
+		if ($bulge_size > $max_bulge) {
+			$max_bulge=$bulge_size;
+		}
+	}
+	return $max_bulge;
+}
+sub get_asy {
+	my($table,$a1,$a2)=@_;
+	my ($pre_i,$pre_j);
+	my $asymmetry=0;
+	foreach my $i ($a1..$a2) {
+		if (defined $table->{$i}) {
+			my $j=$table->{$i};
+			if (defined $pre_i && defined $pre_j) {
+				my $diff=($i-$pre_i)+($j-$pre_j);
+				$asymmetry += abs($diff);
+			}
+			$pre_i=$i;
+			$pre_j=$j;
+		}
+	}
+	return $asymmetry;
+}
+
+sub peaks{
+	my @cluster=@{$_[0]};
+
+	return if(@cluster<1);
+
+	my $max=0; my $index=-1;
+	for (my $i=0;$i<@cluster;$i++) {
+		if($cluster[$i][2]>$max){
+			$max=$cluster[$i][2];
+			$index=$i;
+		}
+	}
+#	&excise(\@cluster,$index,$_[1],$_[2]);
+	return($index);
+}
+
+sub find_cites{
+	my @tmp=@{$_[0]};
+	my $i=&peaks(\@tmp);
+	
+	my $start=$tmp[$i][0];
+	my $total=0; my $node5=0;
+	for (my $j=0;$j<@tmp ;$j++) {
+		$total+=$tmp[$j][2];
+		$node5 +=$tmp[$j][2] if($tmp[$j][0]-$start<=2 && $tmp[$j][0]-$start>=-2);
+	}
+	push @{$cites{$_[1]}{$_[2]}},$tmp[$i] if($node5/$total>0.80 && $tmp[$i][2]/$node5>0.5);
+}
+
+sub newpos{
+	my ($a,$b,$c,$d)=@_;
+	my $s= $a>$c ? $c : $a;
+	my $e=$b>$d ? $b : $d;
+	return($s,$e);
+}
+
+sub rev{
+
+    my($sequence)=@_;
+
+    my $rev=reverse $sequence;   
+
+    return $rev;
+}
+
+sub com{
+
+    my($sequence)=@_;
+
+    $sequence=~tr/acgtuACGTU/TGCAATGCAA/;   
+ 
+    return $sequence;
+}
+
+sub revcom{
+
+    my($sequence)=@_;
+
+    my $revcom=rev(com($sequence));
+
+    return $revcom;
+}
+
+sub find_strand{
+
+    #A subroutine to find the strand, parsing different blast formats
+    my($other)=@_;
+
+    my $strand="+";
+
+    if($other=~/-/){
+	$strand="-";
+    }
+
+    if($other=~/minus/i){
+	$strand="-";
+    }
+
+    return($strand);
+}
+sub usage{
+print <<"USAGE";
+Version $version
+Usage:
+$0 -map -g -d -f -o -s -e
+options:
+  -map input file# align result # bst. format
+  -g input file # genome sequence fasta format
+  -d <int>   Maximal space between miRNA and miRNA* (200)
+  -f <int>   Flank sequence length of miRNA precursor (10)
+  -o output file# percursor fasta file
+  -s output file# precursor structure file
+  -e <folat> Maximal free energy allowed for a miRNA precursor (-18 kcal/mol)
+
+  -h help
+USAGE
+exit(1);
+}
+