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dragon.pl
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dragon.pl
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use strict;
use warnings FATAL => qw ( all );
use Data::Dumper;
use Encode;
use utf8;
#Copyright 2020 Cody Geary and Ebbe S. Andersen
#Permission is hereby granted, free of charge, to any person obtaining
#a copy of this software and associated documentation files (the
#"Software"), \ to deal in the Software without restriction, including
#without limitation the rights to use, copy, modify, merge, publish,
#distribute, sublicense, \ and/or sell copies of the Software, and to
#permit persons to whom the Software is furnished to do so, subject to
#the following conditions:
#The above copyright notice and this permission notice shall be
#included in all copies or substantial portions of the Software.
#THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND,
# EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF
# MERCHANTABILITY,\ FITNESS FOR A PARTICULAR PURPOSE AND
# NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS
# BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER\ LIABILITY, WHETHER IN AN
# ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN
# CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALIN\ GS IN THE
# SOFTWARE.
##########
### User-Configurable Global Vars
##########
my $init_setup = 5; #Number of cycles of the initial setup
my $site_mutation_rate = 45; #Rate of mutation in regions that are mispaired. Only mispaired nts are mutated.
#Mutations that improve the fold are retained, and become the next parent sequence.
my $mutate_loop = 2; #1-100, Rate at which mispaired loop sequence are mutated.
my $drift_period = 3; #Controls frequency of "drift", which allows mutatitions to be tested in properly folded areas.
#5 = drift every 5th round. Targeted mutations do not occur during drift.
my $my_drift_rate = 2; #Rate for random point-mutations to the entire sequence.
my $gc_mut_ratio = 15; #How much GCs are preferred over AUs.
my $double_mutant = 99; #Percent of mutants that target base-pairs rather than single positions.
my $misfold_tolerance = 1.06; #factor of increase in distance that will be allowed during the mutation stage
my $output_threshold = 0; #1=verbose, 0=hide output for neutral mutations
my $max_GC=65; #set desired GC content of initial design
my $max_GC_opt=65; #set the final GC content after optimization, must be greater than $max_GC
my $KL_GC=30; #set GC for Kissing Loops
my $target_GU = 1; #Minimum GU, turned off since we are enforcing GU with the pattern
my $longrange = 250; #define the min distance for pairs to require AUC-limited alphabet in initilization.
my $duplicate_window = 10; ### Length of Duplicates regions that Pattern Search looks for (marked "D", default=10)
my $complement_window = 10; #window-size to search for complement sequences - for GU placer
my $complement_zones=0;
my $duplication_zones=0;
my $max_failed_tries = 100; #sets the number of times it will try to mutate the surrounding nts when the only remaining misfolds are in sequence-locked regions
my $generate_previews=0; #runs trace to preview.txt every new round. This is only helpful for debugging giant designs.
my $KL_min_delta_G = -6; #minimum threshold for non-cognate KL interactions.
##Scoring function for KLs
#my $MinKL = -7.2; ##Default -7.2
#my $MaxKL = -10.8; ##Default -10.8
my $MinKL = -10; ##Default -7.2
my $MaxKL = -12; ##Default -10.8
my $GC_check = 100;
my $fav_rad_level=15; #'radiation' level, ~number of mutants per round, this scales up/down based on success.
my $start_time = time; #start a timer
#my $timeout_length = 7200; #length of timeout in seconds - default is set to 2h
#dragon version has timeout disabled
my $target_ED = 1;
my $lowest_ED = 100000000;
my $keep_ED = 0;
##########
###Define Variables used in subroutines
##########
my $drift_rate = $my_drift_rate;
my $rad_level = $fav_rad_level;
#Initizalize global vars used in subroutines
my $fold = "";
my $distance = 0;
my @map = ();
my $randomletter = "A";
my $name = ""; #the name of the design is extracted from target.txt line 1 and stored here
###These store the folds of all the unique sequences tested
my $unique_sequence_id = 0;
my @sequence_catalog = ();
my @fold_catalog = ();
my $previously_tried=0;
my @duplex_catalog = ();
my @duplex_out_catalog = ();
my @duplex_parens_catalog = ();
my $unique_duplex_id = 0;
#for the KL analysis sub
my $KL_A = "GCGCGC";
my $KL_B = "GUGCGU";
my $KLenergy = "";
my $parens = "";
my $numKL=0;
my @Alist = (); #array of KLA
my @Blist = (); #array of KLB
my @Elist = (); #array of Energies
my @NTlist = (); #store the nt position of each KL
my $KL_score = 0;
my $report_KL = 0; #allows to toggle printed output from the KL analysis sub
my $full_KL_output =0;
my @KL_opt_target = (); #array of KL sites to target for optimization
my $KL_opt_target_text = ""; #the join of the array
my @parent_KL_opt_target = ();
my $numKL_sites=0; #total number of targets in @KL_opt_target (sites may be multiply targeted)
my $best_KL_score=1000;
my @KL_type = (); ##Store the type of KL each site is: (A)=180KL or (B)=120KL
my @KL_specified = (); ##Keep track if any KLs are user-specified, and ignore them for computing the energy calculation penalty
my $KL_notcounted = 0; ##Keep track if any KLs are user-specified, and ignore them for computing the energy calculation penalty
my $non_cognates = "";
my $failed_initial_design = 0; #variable to track if the program needed to change the target structure to solve it
my $failed_pattern_test = 0; #variable to track if the conserved pattern breaks some pattern detection rules
##########
###Define Subroutines for Calculation Functions
##########
my @lines;
sub read_file
{
my ( $file ) = @_;
my ( @lines, $content );
if ( not open FILE, $file ) {
die "Not readable: $file";
}
{
# Reads the whole file by temporarily undefining the
# record separator,
local $/ = undef;
$content = <FILE>;
# Make Unix line ends from either Mac or DOS,
$content =~ s/\r\n/\n/g;
$content =~ s/\r/\n/g;
}
close FILE;
@lines = split "\n", $content;
return wantarray ? @lines : \@lines;
}
sub fold { #outputs to $fold
###These var names store the folds of all the unique sequences tested
# $unique_sequence_id = 0;
# @sequence_catalog = ();
# @fold_catalog = ();
my $dont_fold = 0;
$previously_tried=0;
my $sequence_num_counter = 0;
my $counter = 0;
foreach(@sequence_catalog){
if(defined $sequence_catalog[$sequence_num_counter]){
if($_[0]eq$sequence_catalog[$sequence_num_counter]){
$fold=$fold_catalog[$sequence_num_counter];
$dont_fold=1;
&printer("<found #$sequence_num_counter>\n");
$previously_tried=1;
}
}
$sequence_num_counter ++;
}
$sequence_num_counter = scalar(@sequence_catalog);
if($dont_fold==0){
qx(echo $_[0] > seq.txt);
my $outputs = qx(rnafold --noPS < seq.txt);
my @results = split(' ', $outputs);
$counter = 0; $fold = "";
foreach(@results){
if($counter == 1){ $fold = "$_\n"; }
$counter ++;
}
$sequence_catalog[$unique_sequence_id]=$_[0];
$fold_catalog[$unique_sequence_id]=$fold;
$unique_sequence_id ++;
}
}
sub distance { #outputs to $distance
open(my $din, '>', 'dist.txt');
print $din "$_[0]";
print $din "$_[1]";
close $din;
my $outputs = qx(rnadistance < dist.txt);
my @results = split(' ', $outputs);
my $counter = 0; $distance = "";
foreach(@results){
if($counter == 1){ $distance = "$_\n"; }
$counter ++;
}
}
sub map {
@map = ();
my @secondary = split('', $_[0]);
my @last_bracket;
my @last_brace;
my @last_curlybrace;
my @last_a_brace;
my @last_b_brace;
my @last_c_brace;
my @last_d_brace;
my @last_e_brace;
my @last_f_brace;
my @last_g_brace;
my @last_h_brace;
my @last_i_brace;
my @last_j_brace;
my $counter = 0;
foreach(@secondary){
if ($_ eq "."){
$map[$counter] = $counter; ##Single strands map to themselves
}
elsif ($_ eq "("){
push @last_bracket, $counter;
}
elsif ($_ eq ")"){
my $partner = pop @last_bracket;
$map[$counter]=$partner;
$map[$partner]=$counter;
}
elsif ($_ eq "["){
push @last_brace, $counter;
}
elsif ($_ eq "]"){
my $buddy = pop @last_brace;
$map[$counter]=$buddy;
$map[$buddy]=$counter;
}
elsif ($_ eq "{"){
push @last_curlybrace, $counter;
}
elsif ($_ eq "}"){
my $pal = pop @last_curlybrace;
$map[$counter]=$pal;
$map[$pal]=$counter;
}
elsif ($_ eq "A"){ ##Outwards pointing KLs map as A-1 B-2 C-3.... I-9 J-0
push @last_a_brace, $counter;
}
elsif ($_ eq "1"){
my $pal = pop @last_a_brace;
$map[$counter]=$pal;
$map[$pal]=$counter;
}
elsif ($_ eq "B"){
push @last_b_brace, $counter;
}
elsif ($_ eq "2"){
my $pal = pop @last_b_brace;
$map[$counter]=$pal;
$map[$pal]=$counter;
}
elsif ($_ eq "C"){
push @last_c_brace, $counter;
}
elsif ($_ eq "3"){
my $pal = pop @last_c_brace;
$map[$counter]=$pal;
$map[$pal]=$counter;
}
elsif ($_ eq "D"){
push @last_d_brace, $counter;
}
elsif ($_ eq "4"){
my $pal = pop @last_d_brace;
$map[$counter]=$pal;
$map[$pal]=$counter;
}
elsif ($_ eq "E"){
push @last_e_brace, $counter;
}
elsif ($_ eq "5"){
my $pal = pop @last_e_brace;
$map[$counter]=$pal;
$map[$pal]=$counter;
}
elsif ($_ eq "F"){
push @last_f_brace, $counter;
}
elsif ($_ eq "6"){
my $pal = pop @last_f_brace;
$map[$counter]=$pal;
$map[$pal]=$counter;
}
elsif ($_ eq "G"){
push @last_g_brace, $counter;
}
elsif ($_ eq "7"){
my $pal = pop @last_g_brace;
$map[$counter]=$pal;
$map[$pal]=$counter;
}
elsif ($_ eq "H"){
push @last_h_brace, $counter;
}
elsif ($_ eq "8"){
my $pal = pop @last_h_brace;
$map[$counter]=$pal;
$map[$pal]=$counter;
}
elsif ($_ eq "I"){
push @last_i_brace, $counter;
}
elsif ($_ eq "9"){
my $pal = pop @last_i_brace;
$map[$counter]=$pal;
$map[$pal]=$counter;
}
elsif ($_ eq "J"){
push @last_j_brace, $counter;
}
elsif ($_ eq "0"){
my $pal = pop @last_j_brace;
$map[$counter]=$pal;
$map[$pal]=$counter;
}
$counter ++;
}
}
sub randseq { #returns $randomletter
my $randompick = int(rand(4));
if ($randompick == 0){$randomletter = "A";}
if ($randompick == 1){$randomletter = "U";}
if ($randompick == 2){$randomletter = "C";}
if ($randompick == 3){$randomletter = "G";}
}
#arrays to track patterns and track which 150nt windows to target
my @pattern_zones = ();
##vars for the GC counter subroutine at the end
my $pairs=0;
my $GC_pairs=0;
my $AU_pairs=0;
my $GU_pairs=0;
my $KL_GC_cont=0;
my $KL_pairs=0;
my $KL_GC_pairs=0;
my $KL_repeats = 0;
##########
###Begin Program
##########
my $outputs = "target.txt";
my @results = &read_file( $outputs );
my $counter = 0;
my @target = ();
my @init_target = ();
my @constraint = ();
my @start_seq = ();
my @parent_fold = ();
my $targetstring = "";
my $targetstringpk = "";
my $generate_sequence = 'true';
foreach(@results){
if($counter == 0){ $name = "$_";}
if($counter == 1){ @init_target = split(//,"$_"); @target = split(//,"$_"); $targetstringpk = "$_";}
if($counter == 2){ @constraint = split(//,"$_"); }
if($counter == 3){ @start_seq = split(//,"$_"); $generate_sequence='false'; }
$counter ++;
}
my $output_file_path = "";
( $output_file_path ) = @ARGV;
if ( !defined $output_file_path ) {
qx(mkdir $name);
$output_file_path=$name;
}
$output_file_path = $output_file_path."\/";
###############################
#Setup Output Spool
#################################
open(my $output_spool, '>', "$output_file_path$name\_spool.txt"); ##Output buffer for the screen/file outputs. Spool will hold all screen outputs and send them to a file at the end.
&printer("Output path is $output_file_path \n");
&welcome;
if($generate_sequence eq 'false' ){&printer("I found a sequence!\n");}
&map($targetstringpk);
#######
## Parse the Target string into 2 arrays, 1 with PK and 1 without
#######
my $targetscrubbed = "";
foreach(@target){
if ($_ eq "[" || $_ eq "]" || $_ eq "{" || $_ eq "}" ||
$_ eq "A" || $_ eq "B" || $_ eq "C" || $_ eq "D" || $_ eq "E" || $_ eq "F" || $_ eq "G" || $_ eq "H" || $_ eq "I" || $_ eq "J" ||
$_ eq "1" || $_ eq "2" || $_ eq "3" || $_ eq "4" || $_ eq "5" || $_ eq "6" || $_ eq "7" || $_ eq "8" || $_ eq "9" || $_ eq "0") {
$targetstring=$targetstring.".";
} ## $targetstring has the PKs as .
else {$targetstring=$targetstring.$_;}
if ($_ eq "{" || $_ eq "A" || $_ eq "B" || $_ eq "C" || $_ eq "D" || $_ eq "E" || $_ eq "F" || $_ eq "G" || $_ eq "H" || $_ eq "I" || $_ eq "J" ) { $targetscrubbed=$targetscrubbed."[";} ## $targetscrubbed has the { as [
elsif ($_ eq "}" || $_ eq "1" || $_ eq "2" || $_ eq "3" || $_ eq "4" || $_ eq "5" || $_ eq "6" || $_ eq "7" || $_ eq "8" || $_ eq "9" || $_ eq "0") { $targetscrubbed=$targetscrubbed."]";}
else {$targetscrubbed=$targetscrubbed.$_;}
}
@target = split(//,$targetscrubbed); # @target has (.) and [.] but not {
my @targetnopk = split(//,$targetstring); # @targetnopk has all the [ replaced with .
&printer("Designing $name:\n$targetstringpk\n");
my $strand_length = length $targetstring;
if (scalar(@constraint) != scalar(@target)){
&printer("\nInvalid Constraint, using all N instead.\n");
@constraint = ();
for(my $i=0; $i<$strand_length; $i++){$constraint[$i]="N";}
}
&printer("The design is $strand_length nts long\n\n");
###BEGIN Design
my @best_sol = ();
my @mut_map = ();
my @defect_map = ();
my $mut_map_text = "";
my $best_dist = 1000;
my $i=0;
if($generate_sequence eq 'true'){
##Set LOOP positions match the constraint
for ( $i=0; $i<$strand_length; $i++){
$best_sol[$i]="X";
if ($target[$i] eq "."){
if ($constraint[$i] =~ /[AUCG]/){$best_sol[$i]=$constraint[$i];}
elsif ($constraint[$i] eq "R"){
if(int(rand(2))==0){$best_sol[$i]="G";} else{$best_sol[$i]="A";}
}
elsif ($constraint[$i] eq "N"){
&randseq;
$best_sol[$i]=$randomletter;
}
elsif ($constraint[$i] eq "Y"){
if(int(rand(2))==0){$best_sol[$i]="C";} else{$best_sol[$i]="U";}
}
elsif ($constraint[$i] eq "S"){
if(int(rand(2))==0){$best_sol[$i]="C";} else{$best_sol[$i]="G";}
}
elsif ($constraint[$i] eq "W"){
if(int(rand(2))==0){$best_sol[$i]="A";} else{$best_sol[$i]="U";}
}
else {$best_sol[$i]="A";} #in the odd case just use A, although this should never happen...
}
}
my $GC_cont=100;
my $bp_type=0;
my $bp_direction=0;
##Fill in the stems to match the constraints
for ( $i=0; $i<$strand_length; $i++){
if (($target[$i]eq("(") or $target[$i]eq("[")) && ($constraint[$i] eq "A")){
$best_sol[$i]="A";
$best_sol[$map[$i]]="U";
}
elsif (($target[$i]eq("(") or $target[$i]eq("[")) && ($constraint[$i]eq"C")){
$best_sol[$i]="C";
$best_sol[$map[$i]]="G";
}
elsif (($target[$i]eq("(") or $target[$i]eq("[")) && ($constraint[$i]eq"U")){
$best_sol[$i]="U";
if ($constraint[$map[$i]]eq"A" || $constraint[$map[$i]]eq"N"){
$best_sol[$map[$i]]="A";
}
elsif ($constraint[$map[$i]]eq"G" || $constraint[$map[$i]]eq"K"){
$best_sol[$map[$i]]="G";
}
}
elsif (($target[$i]eq("(") or $target[$i]eq("[")) && ($constraint[$i]eq"G")){
$best_sol[$i]="G";
if ($constraint[$map[$i]]eq"C" || $constraint[$map[$i]]eq"N" || $constraint[$map[$i]]eq"Y"){
$best_sol[$map[$i]]="C";
}
elsif ($constraint[$map[$i]]eq"U" || $constraint[$map[$i]]eq"K"){
$best_sol[$map[$i]]="U";
}
}
elsif (($target[$i]eq("(") or $target[$i]eq("[")) && ($constraint[$i]eq"K")){
if ($constraint[$map[$i]]eq"G" || $constraint[$map[$i]]eq"R" || $constraint[$map[$i]]eq"N"){
$best_sol[$map[$i]]="U";
}
elsif ($constraint[$map[$i]]eq"U"){
$best_sol[$map[$i]]="G";
}
if ($constraint[$map[$i]]eq"K"){
$bp_direction=int(rand(2));
if( ($map[$i]-$i)>$longrange ){$bp_direction = 1;}
if( $bp_direction==0 || $best_sol[$i]eq"G" || $best_sol[$map[$i]]eq"U"){
$best_sol[$i]="G";
$best_sol[$map[$i]]="U";
}
else {
$best_sol[$i]="U";
$best_sol[$map[$i]]="G";
}
}
}
elsif (($target[$i]eq("(") or $target[$i]eq("[")) && ($constraint[$i]eq"S")){
if ($constraint[$map[$i]]eq"G"){
$best_sol[$map[$i]]="C";
}
elsif ($constraint[$map[$i]]eq"C"){
$best_sol[$map[$i]]="G";
}
if ($constraint[$map[$i]]eq"S"){
$bp_direction=int(rand(2));
if( ($map[$i]-$i)>$longrange ){$bp_direction = 1;}
if( $bp_direction==0 || $best_sol[$i]eq"G" || $best_sol[$map[$i]]eq"C"){
$best_sol[$i]="G";
$best_sol[$map[$i]]="C";
}
else {
$best_sol[$i]="C";
$best_sol[$map[$i]]="G";
}
}
}
elsif (($target[$i]eq("(") or $target[$i]eq("[")) && ($constraint[$i]eq"R")){
if ($constraint[$map[$i]]eq"U"){
$best_sol[$map[$i]]="A";
}
elsif ($constraint[$map[$i]]eq"C"){
$best_sol[$map[$i]]="G";
}
if ($constraint[$map[$i]]eq"Y" || $constraint[$map[$i]]eq"N"){
$bp_direction=int(rand(2));
if( $bp_direction==0 || $best_sol[$i]eq"G" || $best_sol[$map[$i]]eq"C"){
$best_sol[$i]="G";
$best_sol[$map[$i]]="C";
}
else {
$best_sol[$i]="A";
$best_sol[$map[$i]]="U";
}
}
}
elsif (($target[$i]eq("(") or $target[$i]eq("[")) && ($constraint[$i]eq"Y")){
if ($constraint[$map[$i]]eq"G"){
$best_sol[$map[$i]]="C";
}
elsif ($constraint[$map[$i]]eq"A"){
$best_sol[$map[$i]]="U";
}
if ($constraint[$map[$i]]eq"R" || $constraint[$map[$i]]eq"N"){
$bp_direction=int(rand(2));
if( $bp_direction==0 || $best_sol[$i]eq"U" || $best_sol[$map[$i]]eq"A"){
$best_sol[$i]="C";
$best_sol[$map[$i]]="G";
}
else {
$best_sol[$i]="U";
$best_sol[$map[$i]]="A";
}
}
}
if (($target[$i]eq("(")) && ($constraint[$i]eq"N")){
if ($constraint[$map[$i]]eq"A"){
$best_sol[$i]="U";
$best_sol[$map[$i]]="A";
}
elsif ($constraint[$map[$i]]eq"U"){
$best_sol[$i]="A";
$best_sol[$map[$i]]="U";
}
elsif ($constraint[$map[$i]]eq"C"){
$best_sol[$i]="G";
$best_sol[$map[$i]]="C";
}
elsif ($constraint[$map[$i]]eq"G"){
$best_sol[$i]="C";
$best_sol[$map[$i]]="G";
}
elsif ($constraint[$map[$i]]eq"N" && $best_sol[$i]eq"X" && $best_sol[$map[$i]]eq"X" ){
$bp_direction= int(rand(2));
$bp_type=(rand(100));
if ($bp_type<(100-$max_GC-$target_GU/2)) {
if ($bp_direction==0) {
$best_sol[$i] = "A";
$best_sol[$map[$i]] = "U";
}
else {
$best_sol[$i] = "U";
$best_sol[$map[$i]] = "A";
}
}
else {
if ($bp_direction==0) {
$best_sol[$i] = "G";
$best_sol[$map[$i]] = "C";
}
else {
$best_sol[$i] = "C";
$best_sol[$map[$i]] = "G";
}
}
}
}
if (($target[$i]eq("[")) && ($constraint[$i]eq"N")){
if ($constraint[$map[$i]]eq"A"){
$best_sol[$i]="U";
$best_sol[$map[$i]]="A";
}
elsif ($constraint[$map[$i]]eq"U"){
$best_sol[$i]="A";
$best_sol[$map[$i]]="U";
}
elsif ($constraint[$map[$i]]eq"C"){
$best_sol[$i]="G";
$best_sol[$map[$i]]="C";
}
elsif ($constraint[$map[$i]]eq"G"){
$best_sol[$i]="C";
$best_sol[$map[$i]]="G";
}
elsif ($constraint[$map[$i]]eq"N" && $best_sol[$i]eq"X" && $best_sol[$map[$i]]eq"X" ){
$bp_direction= int(rand(2));
$bp_type=(rand(100));
if ($bp_type<$KL_GC) {
if ($bp_direction==0) {
$best_sol[$i] = "G";
$best_sol[$map[$i]] = "C";
}
else {
$best_sol[$i] = "C";
$best_sol[$map[$i]] = "G";
}
}
else {
if ($bp_direction==0) {
$best_sol[$i] = "U";
$best_sol[$map[$i]] = "A";
}
else {
$best_sol[$i] = "A";
$best_sol[$map[$i]] = "U";
}
}
}
}
if ($target[$i]eq("(") && ($constraint[$i]eq"N") && ($constraint[$map[$i]]eq"N") && (($map[$i]-$i)>$longrange)){ ####find long-range pairs and make them A,U,C limited.
$bp_type=int(rand(3));
if($bp_type==0){
$best_sol[$i] = "A";
$best_sol[$map[$i]] = "U";
}
if($bp_type==1){
$best_sol[$i] = "C"; ##This puts AUC on the 5' most strand. Flip to have a AUG alphabet instead.
$best_sol[$map[$i]] = "G";
}
if($bp_type==2){
$best_sol[$i] = "U";
$best_sol[$map[$i]] = "A";
}
}
if (($init_target[$i]eq("{")) && ($constraint[$i]eq"N")){ ##apply 100%GC to the dovetails specified with '{'
if ($constraint[$map[$i]]eq"A"){
$best_sol[$i]="U";
$best_sol[$map[$i]]="A";
}
elsif ($constraint[$map[$i]]eq"U"){
$best_sol[$i]="A";
$best_sol[$map[$i]]="U";
}
elsif ($constraint[$map[$i]]eq"C"){
$best_sol[$i]="G";
$best_sol[$map[$i]]="C";
}
elsif ($constraint[$map[$i]]eq"G"){
$best_sol[$i]="C";
$best_sol[$map[$i]]="G";
}
elsif ($constraint[$map[$i]]eq"N"){
$bp_direction= int(rand(2));
if ($bp_direction==0) {
$best_sol[$i] = "G";
$best_sol[$map[$i]] = "C";
}
else {
$best_sol[$i] = "C";
$best_sol[$map[$i]] = "G";
}
}
}
}
}else{
&printer("Inserting new Sequence!\n");
@best_sol = @start_seq;
}
&printer("Target: \n");
&printer("$targetstring\n");
my $constraintstring = join "",@constraint;
&printer("\nConstraint\n");
&printer("$constraintstring\n\n\n");
my $best_sol_seq = join "", @best_sol;
&fold($best_sol_seq);
@parent_fold = split(//,$fold);
#&printer("parent fold = @parent_fold\n");
my $parent_fold_string = $fold;
&distance($parent_fold_string, $targetstring);
my $parent_dist = $distance;
&printer("Initial Sequence: Distance = $parent_dist");
&countGC;
&printer("$best_sol_seq\n");
&printer($fold);
my @trial_sol = @best_sol;
my $new_trial_seq = $best_sol_seq;
my $bp_direction=0; my $bp_type=0; my $GC_cont=100;
################################################
##BEGIN first optimization loop.
my $attempts = 0;
do{ ####
if(($distance==0) or ($generate_sequence eq 'false')){$init_setup=0;}
$attempts += 1; #Keep track of how many times this loops and fails
my $helixend=0;
for ( $i=0; $i<$strand_length-1; $i++){
if ($constraint[$i]eq"N" && $constraint[$map[$i]]eq"N"){
$bp_direction=int(rand(2)); #roll the dice to pick the direction of the bp
$bp_type=(rand(100));
if( $target[$i]eq"." && $parent_fold[$i]ne"." ){$trial_sol[$i]="A";}
if( ($target[$i]eq"(" || $target[$i]eq"[") && $target[$i+1]ne"(" && $target[$i+1]ne"[" ){ $helixend=1;}
if( ($target[$i]eq")" || $target[$i]eq"]") && $target[$i+1]ne")" && $target[$i+1]ne"]" ){ $helixend=1;}
if($helixend==1){$bp_type=100;}
if (($target[$i]eq"(") && ($parent_fold[$i]ne"(")){
if ($bp_type<(100-$max_GC-$target_GU)){
if ($bp_direction==0) {
$trial_sol[$i] = "A";
$trial_sol[$map[$i]] = "U";
}
else {
$trial_sol[$i] = "U";
$trial_sol[$map[$i]] = "A";
}
}
else {
if ($bp_direction==0) {
$trial_sol[$i] = "G";
$trial_sol[$map[$i]] = "C";
}
else {
$trial_sol[$i] = "C";
$trial_sol[$map[$i]] = "G";
}
}
}
if (($target[$i]eq")") && ($target[$i+1]eq"(") && ($parent_fold[$i]ne")") && ($parent_fold[$i+1]ne"(") ){
$trial_sol[$map[$i]] = "G";
$trial_sol[$i] = "C";
$trial_sol[$map[$i+1]] = "C";
$trial_sol[$i+1] = "G";
}
if (($target[$i]eq"(") && ($target[$i-1]eq")") && ($parent_fold[$i]ne"(") && ($parent_fold[$i-1]ne")")){
$trial_sol[$map[$i]] = "C";
$trial_sol[$i] = "G";
$trial_sol[$map[$i-1]] = "G";
$trial_sol[$i-1] = "C";
}
if (($target[$i]eq"(") && ($target[$i+1]eq"(") && (($map[$i]-$map[$i+1])>1) && ($parent_fold[$i]ne"(") && ($parent_fold[$i+1]ne"(")){
$trial_sol[$map[$i]] = "G";
$trial_sol[$i] = "C";
$trial_sol[$map[$i+1]] = "G";
$trial_sol[$i+1] = "C";
}
if (($target[$i]eq")") && ($target[$i+1]eq")") && (($map[$i]-$map[$i+1])>1) && ($parent_fold[$i]ne")") && ($parent_fold[$i+1]ne")")){
$trial_sol[$map[$i]] = "G";
$trial_sol[$i] = "C";
$trial_sol[$map[$i+1]] = "G";
$trial_sol[$i+1] = "C";
}
} elsif ($constraint[$i]eq"S" && $constraint[$map[$i]]eq"S"){
if (($target[$i]eq")") && ($target[$i+1]eq"(") && ($parent_fold[$i]ne")") && ($parent_fold[$i+1]ne"(") ){
$trial_sol[$map[$i]] = "G";
$trial_sol[$i] = "C";
$trial_sol[$map[$i+1]] = "C";
$trial_sol[$i+1] = "G";
}
if (($target[$i]eq"(") && ($target[$i-1]eq")") && ($parent_fold[$i]ne"(") && ($parent_fold[$i-1]ne")")){
$trial_sol[$map[$i]] = "C";
$trial_sol[$i] = "G";
$trial_sol[$map[$i-1]] = "G";
$trial_sol[$i-1] = "C";
}
if (($target[$i]eq"(") && ($target[$i+1]eq"(") && (($map[$i]-$map[$i+1])>1) && ($parent_fold[$i]ne"(") && ($parent_fold[$i+1]ne"(")){
$trial_sol[$map[$i]] = "G";
$trial_sol[$i] = "C";
$trial_sol[$map[$i+1]] = "G";
$trial_sol[$i+1] = "C";
}
if (($target[$i]eq")") && ($target[$i+1]eq")") && (($map[$i]-$map[$i+1])>1) && ($parent_fold[$i]ne")") && ($parent_fold[$i+1]ne")")){
$trial_sol[$map[$i]] = "G";
$trial_sol[$i] = "C";
$trial_sol[$map[$i+1]] = "G";
$trial_sol[$i+1] = "C";
}
}
}
##Compute the fold and distance
$new_trial_seq = join "",@trial_sol; #Make a string to hold the sequence of the new trial
&fold($new_trial_seq);
my $new_fold = $fold; #The fold of puzzle solution
&distance($new_fold, $targetstring); #Distance the puzzle solution is away from the target
my $trial_dist = $distance;
#Decide if to keep the new sequence
my $keep_sequence = 0;
if ($trial_dist<($parent_dist*$misfold_tolerance)){
$keep_sequence=1;
}
else{
$keep_sequence=0;
}
##Process Ouputs
if ($keep_sequence==1){ #Check if the new mutant is better than the parent
@mut_map = ();
@defect_map = ();
for ( $i=0; $i<$strand_length; $i++){
if (($best_sol[$i]) eq ($trial_sol[$i])) {
$mut_map[$i] = "-";
} else {
$mut_map[$i] = $trial_sol[$i];
}
if ($parent_fold[$i]ne$targetnopk[$i]){
$defect_map[$i] = "X";
} else {
$defect_map[$i] = "_";
}
$best_sol[$i]= $trial_sol[$i]; #move the trial solution to best_sol array
}
my $mut_map_text = join "",@mut_map;
my $defect_map_text = join "",@defect_map;
$best_sol_seq = join "", @trial_sol;
$parent_dist=$trial_dist; #copy the trial distance to be the new parent_dist
my $best_dist=$trial_dist;
$parent_fold_string = $fold;
@parent_fold = split(//, $fold);
&printer("\nIteration# $attempts, Distance = $trial_dist"); #Report outputs
&countGC;
if ($attempts>1){&printer("$mut_map_text\n");}
&printer("$best_sol_seq\n");
&printer("$parent_fold_string\n");
&printer("$defect_map_text\n");
}
else {
&printer("( $attempts )");
for ( $i=0; $i<$strand_length; $i++){
$trial_sol[$i]=$best_sol[$i]; #reset the trial solution
}
}
}while($attempts < $init_setup);
################################################
&printer("\n__________________________________________________________\n");
&printer("Begin Directed Mutation Cycle\n");
my $drift_event_count = 0; #initialize the counter for mutation events
my $improved_parent_count = 0;
my $constraint_check = 0;
my $failed_tries =0;
do{
my $keep_sequence=0;
$attempts += 1;
$improved_parent_count += 1;
$drift_event_count += 1;
my @new_fold_array = ();
my $trial_dist=10000;
my $mutate_now=0;
my @mutation_sites = ();
my $coin_flip = 0;
my $num_mutation_spots =0;
&printer("($attempts)");
if($drift_event_count==$drift_period){&printer("drift->");}
if($drift_event_count!=$drift_period){&printer("target->");}
if($improved_parent_count>1 && $drift_event_count!=$drift_period){&printer("expanded->");}
for ( $i=2; $i<$strand_length-2; $i++){
$mutate_now=0;
$coin_flip=int(rand(2));
if (($drift_event_count==$drift_period) && (rand(100)<$drift_rate) && ($constraint[$i]eq"N" || $constraint[$i]eq"K" || $constraint[$i]eq"S")) { #random drift events
$mutate_now=1;
}
if (($parent_fold[$i]ne$targetnopk[$i]) && ($drift_event_count!=$drift_period) && (rand(100)<$site_mutation_rate) && ($constraint[$i]eq"N" || $constraint[$i]eq"K" || $constraint[$i]eq"S") ) { #targeted mutations
$mutate_now=1;
}
if ($improved_parent_count>1) {
if ( (($parent_fold[$i+1] ne $targetnopk[$i+1]) || ($parent_fold[$i-1] ne $targetnopk[$i-1]) || ($parent_fold[$i+2] ne $targetnopk[$i+2]) || ($parent_fold[$i-2] ne $targetnopk[$i-2]) ) &&
($drift_event_count!=$drift_period) && (rand(100)<$site_mutation_rate) && $constraint[$i]eq"N") { #expand mutational window by 2nts
$mutate_now=1;
}
if($parent_fold[$i]ne$target[$i] && $target[$i]eq")" && $target[$i+1]eq"("){
$mutation_sites[$i]=1; $mutation_sites[$map[$i]]=1;
$mutation_sites[$i+1]=1; $mutation_sites[$map[$i+1]]=1;
$mutation_sites[$map[$i]-1]=1;
$mutation_sites[$map[$map[$i]-1]]=1;
$mutate_now=1;
}
if($parent_fold[$map[$i]]ne$target[$map[$i]] || $parent_fold[$map[$i+1]]ne$target[$map[$i+1]] || $parent_fold[$map[$i-1]]ne$target[$map[$i-1]]){
$mutation_sites[$map[$i]]=1; $mutation_sites[$map[$i+1]]=1; $mutation_sites[$map[$i-1]]=1;
$mutate_now=1;
}
}
if ($mutate_now==1){
$mutation_sites[$i]=1; $num_mutation_spots++;}
else{$mutation_sites[$i]=0;}
}
my $mut_scale = $rad_level/($num_mutation_spots+.001); ##scale back the odds of mutation so that there are always ~$radlevel mutations.