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rf-fold
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rf-fold
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#!/usr/bin/env perl
##
# RF Fold
# RNA Framework [http://www.rnaframework.com]
#
# Author: Danny Incarnato (dincarnato[at]rnaframework.com)
# Summary: Produces secondary structures for analyzed transcripts using structural
# probing data to guide folding
#
# This program is free software, and can be redistribute and/or modified
# under the terms of the GNU General Public License as published by
# the Free Software Foundation, either version 3 of the License, or
# any later version.
#
# Please see <http://www.gnu.org/licenses/> for more informations.
##
use strict;
use Config;
use Cwd;
use File::Basename;
use File::Path qw(mkpath);
use File::Spec;
use FindBin qw($Bin);
use Getopt::Long qw(:config no_ignore_case);
use POSIX qw(floor);
use lib $Bin . "/lib";
use Core::Mathematics qw(:all);
use Core::Statistics;
use Core::Process::Queue;
use Core::Utils;
use Data::IO;
use Data::IO::Sequence;
use Data::Sequence;
use Data::Sequence::Structure;
use Data::Sequence::Utils;
use Graphics::Container;
use Graphics::Object::Barplot;
use Graphics::Object::Path;
use Graphics::Object::RNAarcs;
use Graphics::Object::Ruler;
use RNA::Utils;
use RF::Data::IO::XML;
use Term::Constants qw(:screen);
use Term::Progress;
use constant MINWINLENGTH => 50; # Minimum window length for windowed folding
$|++;
my ($output, $ct, $method,
$threads, $img, $t, $noshapevrfoldparams,
$cutoff, $nlp, $ngu, $rnastructure,
$viennarna, $data, $slope, $intercept,
$maxdist, $installed,
$overwrite, $error, $help, $tmpdir,
$ignore, $hc, $progressBar, $processManager,
$windowed, $cwd, $partition, $probplot,
$partwin, $partstep, $wintrim, $foldwin,
$foldstep, $dp, $sh, $pk, $suboptDeltaEnergy,
$pkwin, $pkstep, $p1, $p2, $dotplotOnly,
$pkcutoff, $rnasubopt, $pkmax, $pksubopt,
$shapeknots, $pkmethod, $keeptmp, $constraintDir,
$pkpercentmfe, $nozuker, $zukersubopt, $pkslope,
$pkintercept, $plotMedianShape, $plotMedianShannon,
$ignoreSeq, $onlyCommon, @skparams, @vrfoldparams,
@rsfoldparams, @slope, @intercept, @samples, %xml,
%lambda, %constraints, %results);
%results = ( folded => 0,
parseErr => 0,
foldErr => 0,
pkErr => 0,
diffSeq => 0,
diffConst => 0 );
%lambda = ( "2" => 0,
"3" => 24,
"4" => 24,
"5" => 34,
"6" => 24,
"7" => 35,
"8" => 58,
"9" => 82,
"10" => 65,
"11" => 527,
"12" => 2447,
"13" => 4199,
"14" => 6564,
"15" => 12540 );
do {
local $SIG{__WARN__} = sub { };
GetOptions( "h|help" => \$help,
"o|output-dir=s" => \$output,
"ow|overwrite" => \$overwrite,
"ct|connectivity-table" => \$ct,
"m|folding-method=i" => \$method,
"p|processors=i" => \$threads,
"g|img" => \$img,
"c|constraints=s" => \$constraintDir,
"t|temperature=s" => \$t,
"sl|slope=s" => \$slope,
"in|intercept=s" => \$intercept,
"md|maximum-distance=i" => \$maxdist,
"vrf|vienna-rnafold=s" => \$viennarna,
"f|cutoff=s" => \$cutoff,
"nlp|no-lonely-pairs" => \$nlp,
"ngu|no-closing-gu" => \$ngu,
"rs|rnastructure=s" => \$rnastructure,
"d|data-path=s" => \$data,
"i|ignore-reactivity" => \$ignore,
"is|ignore-sequence" => \$ignoreSeq,
"oc|only-common" => \$onlyCommon,
"hc|hard-constraint" => \$hc,
"w|windowed" => \$windowed,
"fw|fold-window=i" => \$foldwin,
"fo|fold-offset=i" => \$foldstep,
"wt|window-trim=i" => \$wintrim,
"pw|partition-window=i" => \$partwin,
"po|partition-offset=i" => \$partstep,
"dp|dotplot" => \$dp,
"dpo|dotplotOnly" => \$dotplotOnly,
"KT|keep-tmp" => \$keeptmp,
"kt|pseudoknot-tollerance=s" => \$pkpercentmfe,
"sh|shannon-entropy" => \$sh,
"pk|pseudoknots" => \$pk,
"pt|partition=s" => \$partition,
"pp|probabilityplot=s" => \$probplot,
"vrs|vienna-rnasubopt=s" => \$rnasubopt,
"kw|pseudoknot-window=i" => \$pkwin,
"ko|pseudoknot-offset=i" => \$pkstep,
"ksl|pseudoknot-slope=s" => \$pkslope,
"kin|pseudoknot-intercept=s" => \$pkintercept,
"kp1|pseudoknot-penalty1=s" => \$p1,
"kp2|pseudoknot-penalty2=s" => \$p2,
"kc|pseudoknot-cutoff=s" => \$pkcutoff,
"ks|pseudoknot-suboptimal=i" => \$pksubopt,
"kh|pseudoknot-helices=i" => \$pkmax,
"km|pseudoknot-method=i" => \$pkmethod,
"sk|shapeknots=s" => \$shapeknots,
"nz|no-zuker" => \$nozuker,
"zs|zuker-suboptimal=i" => \$zukersubopt,
"ke|subopt-delta-energy=s" => \$suboptDeltaEnergy,
"pmr|plot-median-react" => \$plotMedianShape,
"pms|plot-median-shannon" => \$plotMedianShannon ) or help(1);
@samples = uniq(@ARGV);
};
help() if ($help);
# Default
$cwd = cwd();
$output ||= "rf_fold/";
$method ||= 1;
$threads ||= 1;
$t //= 37.0;
$cutoff //= 0.7;
$slope //= 1.8;
$intercept //= -0.6;
$pkslope //= $slope;
$pkintercept //= $intercept;
$maxdist //= 0;
$partwin ||= 600;
$partstep ||= 200;
$wintrim //= 100;
$foldwin ||= 600;
$foldstep ||= 200;
$pkwin ||= 600;
$pkstep ||= 200;
$p1 //= 0.35;
$p2 //= 0.65;
$pkcutoff //= 0.5;
$pkmax ||= 100;
$pksubopt ||= 1000;
$zukersubopt ||= 1000;
$pkmethod ||= 1;
$pkpercentmfe ||= 0.5;
$suboptDeltaEnergy ||= 1;
$viennarna = $viennarna || which("RNAfold");
$rnasubopt = $rnasubopt || which("RNAsubopt");
$rnastructure = $rnastructure || which("Fold-smp") || which("Fold");
$shapeknots = $shapeknots || which("ShapeKnots-smp") || which("ShapeKnots");
$partition = $partition || which("partition-smp") || which("partition");
$probplot = $probplot || which("ProbabilityPlot");
$data ||= $ENV{DATAPATH};
$installed = eval { require RNA; 1; };
$output =~ s/\/?$/\//;
$tmpdir = $output . "/tmp/";
$constraintDir =~ s/\/?$/\// if (defined $constraintDir);
for ($viennarna, $rnasubopt, $rnastructure,
$shapeknots, $partition, $probplot) { $_ = File::Spec->rel2abs($_) if (defined $_); }
##
# Input validation
##
die "\n [!] Error: No output directory specified\n\n" unless(defined $output);
die "\n [!] Error: No XML directory\/file specified\n\n" if (!@samples);
die "\n [!] Error: Provided constraint directory doesn't exist\n\n" if (defined $constraintDir && !-d $constraintDir);
die "\n [!] Error: Number of processors must be an integer greater than 0\n\n" if ($threads < 1);
die "\n [!] Error: Invalid folding method\n\n" unless($method =~ m/^[12]$/);
die "\n [!] Error: Temperature must be a positive value\n\n" unless(ispositive($t));
die "\n [!] Error: Temperature must be comprised between 0 and 100 degree Celsius\n\n" if ($t > 100);
die "\n [!] Error: Maximum distance value must be a positive integer\n\n" if (!ispositive($maxdist));
die "\n [!] Error: No closing GU parameter requires folding method 1 (ViennaRNA)\n\n" if ($ngu && $method != 1);
die "\n [!] Error: Reactivity cutoff must be a value >0 and <= 1" if (!inrange($cutoff, [0, 1]));
if (!$ignore) {
@slope = split /,/, $slope;
@intercept = split /,/, $intercept;
@slope = ($slope[0]) x scalar(@samples) if (@slope == 1 && @samples > 1);
@intercept = ($intercept[0]) x scalar(@samples) if (@intercept == 1 && @samples > 1);
die "\n [!] Error: Number of provided slope values differs from number of samples\n\n" if (@slope != @samples);
die "\n [!] Error: Number of provided intercept values differs from number of samples\n\n" if (@intercept != @samples);
for (@slope) { die "\n [!] Error: Slope must be numeric\n\n" if (!isnumeric($_)); }
for (@intercept) { die "\n [!] Error: Intercept must be numeric\n\n" if (!isnumeric($_)); }
}
if ($windowed) {
die "\n [!] Error: Folding window's size must be an integer >= 50\n\n" unless($foldwin >= MINWINLENGTH);
die "\n [!] Error: Folding window's sliding offset cannot be greater than window's size\n\n" if ($foldstep > $foldwin);
die "\n [!] Error: Parition function window's size must be an integer >= 50\n\n" unless($partwin >= MINWINLENGTH);
die "\n [!] Error: Partition function window's sliding offset cannot be greater than window's size\n\n" if ($partstep > $partwin);
die "\n [!] Error: Partition function window's trimming value cannot exceed half of" .
"\n the difference between window's size and sliding offset size\n\n" if ($wintrim > int(($partwin - $partstep) / 2));
}
if ($pk) {
die "\n [!] Error: Invalid pseudoknots prediction method\n\n" unless($pkmethod =~ m/^[12]$/);
die "\n [!] Error: Pseudoknot window's size must be an integer >= 50\n\n" unless($pkwin >= MINWINLENGTH);
die "\n [!] Error: Pseudoknot window's sliding offset cannot be greater than window's size\n\n" if ($pkstep > $pkwin);
die "\n [!] Error: Pseudoknot penalty 1 must be numeric\n\n" unless(isnumeric($p1));
die "\n [!] Error: Pseudoknot penalty 2 must be numeric\n\n" unless(isnumeric($p2));
die "\n [!] Error: Pseudoknot reactivity cutoff must be > 0 and <= 1\n\n" if (!inrange($pkcutoff, [0, 1]));
die "\n [!] Error: Pseudoknot suboptimal structures energy tollerance must be > 0 and <= 1\n\n" if (!isnumeric($pkpercentmfe) || !inrange($pkpercentmfe, [0, 1]));
die "\n [!] Error: Pseudoknot suboptimal structure energy delta must be >= 0\n\n" if (!ispositive($suboptDeltaEnergy));
}
die "\n [!] Error: ViennaRNA package's Perl module RNA.pm is required." .
"\n Please ensure that ViennaRNA package v2.2.0 (or greater) is installed and try again\n\n" unless($installed);
print "\n[+] Checking method's requirements...";
if ($method == 1) { # ViennaRNA
my $ret = `$viennarna --version`;
if ($ret =~ m/RNAfold (\d+)\.(\d+)/) {
my ($v1, $v2) = ($1, $2);
die "\n\n [!] Error: RF Fold requires ViennaRNA package v2.2.0 or greater (Detected: v" . $v1 . "." . $v2 . ")\n\n" if ($v1 < 2 ||
$v2 < 2);
}
else { warn "\n\n [!] Warning: Unable to detect ViennaRNA package version\n"; }
}
else { # RNAstructure
if (!defined $data) { die "\n\n [!] Error: Environment variable DATAPATH is not set\n\n"; }
elsif (!-d $data) { die "\n\n [!] Error: Provided DATAPATH directory doesn't exist\n\n"; }
if (!defined $rnastructure) { die "\n\n [!] Error: RNAstructure Fold is not in PATH\n\n"; }
elsif (!-e $rnastructure) { die "\n\n [!] Error: RNAstructure Fold doesn't exist\n\n"; }
elsif (!-x $rnastructure) { die "\n\n [!] Error: RNAstructure Fold is not executable\n\n"; }
if (!defined $partition) { die "\n\n [!] Error: RNAstructure partition is not in PATH\n\n"; }
elsif (!-e $partition) { die "\n\n [!] Error: RNAstructure partition doesn't exist\n\n"; }
elsif (!-x $partition) { die "\n\n [!] Error: RNAstructure partition is not executable\n\n"; }
if (!defined $probplot) { die "\n\n [!] Error: RNAstructure ProbabilityPlot is not in PATH\n\n"; }
elsif (!-e $probplot) { die "\n\n [!] Error: RNAstructure ProbabilityPlot doesn't exist\n\n"; }
elsif (!-x $probplot) { die "\n\n [!] Error: RNAstructure ProbabilityPlot is not executable\n\n"; }
$ENV{DATAPATH} = $data;
}
if ($pk) {
if ($pkmethod == 1) {
if (!defined $rnasubopt) { die "\n\n [!] Error: RNAsubopt is not in PATH\n\n"; }
elsif (!-e $rnasubopt) { die "\n\n [!] Error: RNAsubopt doesn't exist\n\n"; }
elsif (!-x $rnasubopt) { die "\n\n [!] Error: RNAsubopt is not executable\n\n"; }
}
else {
if (!defined $shapeknots) { die "\n\n [!] Error: ShapeKnots is not in PATH\n\n"; }
elsif (!-e $shapeknots) { die "\n\n [!] Error: ShapeKnots doesn't exist\n\n"; }
elsif (!-x $shapeknots) { die "\n\n [!] Error: ShapeKnots is not executable\n\n"; }
}
}
# Setting folding parameters
if ($method == 1 || $pkmethod == 1) { # ViennaRNA
my $vrfoldparams = " -T " . $t;
$vrfoldparams .= " --noLP" if ($nlp);
$vrfoldparams .= " --noClosingGU" if ($ngu);
$vrfoldparams .= " --maxBPspan=" . $maxdist if ($maxdist);
$noshapevrfoldparams = $vrfoldparams;
for my $i (0 .. $#samples) {
my ($tmpParams);
$tmpParams = " --shapeMethod=Dm" . $slope[$i] . "b" . $intercept[$i] if (!$ignore);
push(@vrfoldparams, $vrfoldparams . $tmpParams);
}
}
if ($method == 2 || $pkmethod == 2) { # RNAstructure
my $rsfoldparams = " -t " . ($t + 273.15);
$rsfoldparams .= " -md " . $maxdist if ($maxdist);
for my $i (0 .. $#samples) {
my ($tmpParams);
$tmpParams = " -si " . $intercept[$i] . " -sm " . $slope[$i] if (!$ignore);
push(@rsfoldparams, $rsfoldparams . $tmpParams);
push(@skparams, $tmpParams);
}
}
$RNA::temperature = $t;
# Output directory tree
print "\n[+] Making output directory tree...";
if (-e $output) {
if ($overwrite) {
my $error = rmtree($output);
die "\n\n [!] Error: " . $error . "\n\n" if ($error);
}
else { die "\n\n [!] Error: Output directory already exists." .
"\n Please use -ow (or --overwrite) to overwrite output directory\n\n"; }
}
mkpath($tmpdir, { mode => 0755,
error => \$error });
if (!$dotplotOnly) {
mkpath($output . "structures", { mode => 0755,
error => \$error });
mkpath($output . "images", { mode => 0755,
error => \$error }) if ($img);
mkpath($output . "shannon", { mode => 0755,
error => \$error }) if ($sh);
}
mkpath($output . "dotplot", { mode => 0755,
error => \$error }) if ($dp);
die "\n\n [!] Error: Unable to create output directory (" . $error->[0]->{each(%{$error->[0]})} . ")\n\n" if (@{$error});
# Sets all paths to absolute, before changing working directory to temp dir
$output = File::Spec->rel2abs($output) . "/";
$tmpdir = File::Spec->rel2abs($tmpdir) . "/";
print "\n[+] Importing XML file(s)...";
foreach my $sample (@samples) {
die "\n\n [!] Error: Sample XML file/directory \"$sample\" does not exist\n\n" if (!-e $sample);
if (-d $sample) {
opendir(my $dh, $sample) or die "\n\n [!] Error: Unable to read XML files from directory \"$sample\"\n\n";
while(my $file = readdir($dh)) {
next if (substr($file, -4) ne ".xml");
push(@{$xml{(fileparse($file, ".xml"))[0]}}, File::Spec->rel2abs($sample . "/" . $file));
}
closedir($dh);
}
else { # Single xml file
die "\n\n [!] Error: Provided file \"$sample\" lacks XML extension\n\n" if (substr($sample, -4) ne ".xml");
push(@{$xml{(fileparse($sample, ".xml"))[0]}}, File::Spec->rel2abs($sample));
}
}
%xml = map { $_ => $xml{$_} } grep { @{$xml{$_}} == @samples } keys %xml if ($onlyCommon);
print " " . scalar(keys %xml) . " imported (of which " . scalar(grep { @{$xml{$_}} == @samples } keys %xml) . " common to all replicates)";
die "\n\n [!] Error: Specified folder doesn't contain any XML file\n\n" unless (keys %xml);
warn "\n\n [i] Ignoring XML reactivity data files. Predicting MFE unconstrained structures...\n" if ($ignore);
if ($constraintDir) {
my $err = 0;
print "\n[+] Importing constraint file(s)...";
opendir(my $dh, $constraintDir) or die "\n\n [!] Error: Unable to read constraint files from directory\n\n";
while(my $file = readdir($dh)) {
next if ($file =~ m/^\./);
my $eval = do {
local $@;
eval {
my ($structIO, $entry, $structure);
$structIO = Data::IO::Sequence->new( file => $constraintDir . $file,
lonelypairs => 1 );
$entry = $structIO->read();
$structure = $entry->structure();
$constraints{$entry->id()} = $structure;
};
$@;
};
$err++ if ($eval);
}
closedir($dh);
print " " . scalar(keys %constraints) . " imported";
print ", $err discarded" if ($err);
}
chdir($tmpdir); # Change the working directory before starting threads
$processManager = Core::Process::Queue->new( processors => $threads,
stderr => $output . "error.out",
tmpDir => $tmpdir,
verbosity => 1 );
$processManager->enqueue( command => \&fold,
arguments => $xml{$_},
id => $_ ) for (keys %xml);
$progressBar = Term::Progress->new( max => $processManager->queueSize(),
colored => 1 );
print "\n[+] Folding RNA structures\n\n";
$progressBar->init();
$processManager->parentOnExit(sub {
my ($file, $pid, $ret);
($file, $pid) = @_[0,1];
$ret = ($processManager->dequeue($pid)->exitcode())[0];
$progressBar->update(1);
if (ref($ret) eq "HASH") {
my $err = (keys %$ret)[0];
$results{$err} += $ret->{$err};
}
else { $results{"folded"}++; }
});
$processManager->start();
$processManager->waitall();
chdir($cwd); # Reset to original working directory
my $otherErr = scalar(keys %xml);
$otherErr -= $_ for (values %results);
print "\n\n[+] Folding statistics:\n" .
"\n [*] Folded transcripts: " . $results{"folded"} .
"\n [*] Discarded transcripts: " . (scalar(keys %xml) - $results{"folded"}) . " total" .
"\n " . $results{"parseErr"} . " XML parsing failed" .
"\n " . $results{"foldErr"} . " folding failed";
print "\n " . $results{"diffConst"} . " wrong constraint length" if (keys %constraints);
print "\n " . $results{"diffSeq"} . " different sequence between replicates" if (@samples > 1);
print "\n " . $otherErr . " other" if ($otherErr);
print "\n " . $results{"pkErr"} . " pseudoknotted folding failed" if ($pk);
rmtree($tmpdir) if (!$keeptmp);
if (!-s $output . "error.out") { unlink($output . "error.out"); }
else { print "\n\n [!] Warning: Execution completed with error(s)/warning(s). Please check the \"$output/error.out\" file"; }
print "\n\n[+] All done.\n\n";
sub fold {
my @xml = @_;
my ($sequence, $id, $structure, $cmd,
$ret, $entry, $length, $nwins, $file, $io, $energy,
$constraint, @foldwins, @windows, @constraint,
@pkpairs, @keptpairs, @dotplot, @shannon,
@reactivity, @meanReact, %pairprobs, %npairs, %seen);
foreach my $xml (@xml) {
my ($xmlref);
do {
local $@;
eval { $xmlref = RF::Data::IO::XML->new(file => $xml); };
if ($@) { return({ id => $xml,
parseErr => 1 }); }
};
if (!defined $id) {
$id = $xmlref->id();
$sequence = $xmlref->sequence();
$length = $xmlref->length();
}
else {
return({ id => $xml,
diffSeq => 1 }) if (($sequence ne $xmlref->sequence() && !$ignoreSeq) ||
($length != $xmlref->length() && $ignoreSeq));
}
push(@reactivity, [ $xmlref->reactivity() ]);
}
@meanReact = map { my $i = $_; [ map { $_->[$i] } @reactivity ] } 0 .. $#{$reactivity[0]};
@meanReact = map { isnumeric(@$_) ? mean(@$_) : "NaN" } @meanReact;
$constraint = exists $constraints{$id} ? $constraints{$id} : "." x $length;
return({ id => $id,
diffConst => 1 }) if (length($constraint) != $length);
######### Partition function block ##########
# We enter here only if we need to do windowed analysis, or if
# the user requests the bp dotplot or the shannon entropy
if ($windowed || $sh || $dp) {
$nwins = $windowed ? max(1, floor(($length - $partwin) / $partstep) + 1) : 1;
# Define windows to be analyzed
# Windows are defined as: [start, end, 5'-trim, 3'-trim]
if ($nwins > 1) {
for (50, 100) {
my $end = $partwin - $_ - 1;
last if ($end < MINWINLENGTH - 1);
push(@foldwins, [0, $end, 0, $wintrim]);
}
for (0 .. $nwins - 1) {
my ($start, $end);
$start = $partstep * $_;
$end = $_ == $nwins - 1 ? $length - 1 : $start + $partwin - 1;
push(@foldwins, [$start, $end, $wintrim, $wintrim]);
}
for (50, 100) {
my $start = $length - $partwin + $_;
last if ($start > $length - MINWINLENGTH);
push(@foldwins, [$start, $length - 1, $wintrim, 0]);
}
}
else { push(@foldwins, [0, $length - 1, 0, 0]); }
# This fixes the cases in which the first or last windows are < MINWINLENGTH
$foldwins[0]->[2] = 0;
$foldwins[-1]->[3] = 0;
for my $i (0 .. $#reactivity) {
# Calculate window partition function
foreach my $win (@foldwins) {
my ($start, $end, $trim5, $trim3,
$winconst, $subseq, $tmpfile, @winreact);
($start, $end, $trim5, $trim3) = @{$win};
$subseq = substr($sequence, $start, $end - $start + 1);
$winconst = fixdotbracket(substr($constraint, $start, $end - $start + 1));
@winreact = @{$reactivity[$i]}[$start .. $end];
$tmpfile = "$id\_$start-$end\_$$\_$i";
my ($win, $shannon) = partition( file => $tmpfile,
sample => $i,
sequence => $subseq,
reactivity => \@winreact,
pos => $start,
trim5 => $trim5,
trim3 => $trim3,
constraint => $winconst );
if (!$win) {
unlink(glob($tmpfile . "*")) unless($keeptmp);
return({ id => $id,
foldErr => 1 });
}
push(@{$pairprobs{$_}->{pairs}}, $win->{$_}) for (keys %{$win});
push(@{$shannon[$_]}, $shannon->{$_}) for (keys %{$shannon});
push(@windows, [$start + $trim5, $end - $trim3]);
}
}
@shannon = map { mean(@{$_}) } @shannon; # Shannon entropy vector
# Just do this for windowed analysis, otherwise just fold
if ($windowed) {
# Build constraint with pairs > 0.99 probability
foreach my $pair (sort keys %pairprobs) {
my ($i, $j, $prob);
($i, $j) = split(/-/, $pair);
next if (exists $seen{$i} || exists $seen{$j});
for (@windows) { $pairprobs{$pair}->{n}++ if (inrange($i, $_) && inrange($j, $_)); }
$prob = sum(@{$pairprobs{$pair}->{pairs}}) / $pairprobs{$pair}->{n};
# If average p(i,j) is >= 0.99
if ($prob >= 0.99) {
push(@constraint, [$i, $j, $prob]);
$seen{$i} = 1;
$seen{$j} = 1;
}
}
}
undef($nwins);
undef(@foldwins);
undef(@windows);
# We can take advantage of the rmpseudoknots() function to discard incompatible pairs
# The rmpseudoknots() function takes the sequence, an array ref of base-pairs, and
# optionally a scoring function to be applied (in this case the average of bp probs)
$constraint = (rmpseudoknots($sequence, \@constraint, \&Core::Mathematics::mean))[0];
}
######### End of partition function block ##########
# Build dotplot of pairing probabilities (only if necessary)
if ($windowed || $dp || $sh) {
foreach my $pair (keys %pairprobs) {
my ($i, $j) = split(/-/, $pair);
push(@dotplot, [$i, $j, mean(@{$pairprobs{$pair}->{pairs}})]);
}
@dotplot = sort {$a->[0] <=> $b->[0]} @dotplot;
}
if ($dp) {
open(my $wh, ">" . $output . "dotplot/" . $id . ".dp") or next;
select((select($wh), $|=1)[0]);
print $wh length($sequence) . "\ni\tj\t-log10(Probability)\n";
print $wh join("\t", $_->[0] + 1, $_->[1] + 1, -logarithm($_->[2], 10)) . "\n" for (@dotplot);
close($wh);
}
return if ($dotplotOnly);
######### MFE folding block ##########
$nwins = $windowed ? max(1, floor(($length - $foldwin) / $foldstep) + 1) : 1;
# Define windows to be analyzed
# Windows are defined as: [start, end]
if ($windowed && $nwins > 1) {
for (100, 50, -50, -100) {
my $end = $foldwin - $_ - 1;
$end = $length - 1 if ($end > $length - 1);
next if ($end < MINWINLENGTH - 1);
push(@foldwins, [0, $end]);
last if ($end == $length - 1);
}
for (0 .. $nwins - 1) {
my ($start, $end);
$start = $foldstep * $_;
$end = $_ == $nwins - 1 ? $length - 1 : $start + $foldwin - 1;
push(@foldwins, [$start, $end]);
}
for (100, 50, -50, -100) {
my $start = $length - $foldwin + $_;
$start = 0 if (!ispositive($start));
next if ($start > $length - MINWINLENGTH);
push(@foldwins, [$start, $length - 1]);
last if (!$start);
}
}
else { push(@foldwins, [0, $length - 1]); }
for my $i (0 .. $#reactivity) {
# Calculate MFE folding in window
foreach my $win (@foldwins) {
my ($start, $end, $subseq, $winconst,
$tmpfile, @winreact);
($start, $end) = @{$win};
$subseq = substr($sequence, $start, $end - $start + 1);
@winreact = @{$reactivity[$i]}[$start .. $end];
$winconst = rmlonelypairs(fixdotbracket(substr($constraint, $start, $end - $start + 1)));
$tmpfile = "$id\_$start-$end\_$$\_$i";
my $win = winfold( file => $tmpfile,
sample => $i,
sequence => $subseq,
reactivity => \@winreact,
pos => $start,
constraint => $winconst );
if (!$win) {
unlink(glob($tmpfile . "*")) unless($keeptmp);
return({ id => $id,
foldErr => 1 });
}
$npairs{$_}->{pairs}++ for (keys %{$win});
push(@windows, [$start, $end]);
}
}
# Build structure with pairs in > 0.50 of windows
foreach my $pair (sort {(split(/-/, $a))[0] <=> (split(/-/, $b))[0]} keys %npairs) {
my ($i, $j, $n1, $n2);
($i, $j) = split(/-/, $pair);
for (@windows) {
$n1++ if (inrange($i, $_));
$n2++ if (inrange($j, $_));
}
# If pair(i,j) is present in > 0.50 windows
if ($npairs{$pair}->{pairs} / $n1 > 0.5 &&
$npairs{$pair}->{pairs} / $n2 > 0.5) { push(@keptpairs, [$i, $j]); }
}
$entry = Data::Sequence::Structure->new( id => $id,
sequence => $sequence,
basepairs => \@keptpairs,
lonelypairs => $nlp ? 0 : 1 );
undef(@foldwins);
######### End of MFE folding block ##########
######### Pseudoknots detection block ##########
if ($pk) { # Pseudoknot detection
my (@keptpkpairs, %pkpairs, %basepairs, %pkbases);
$nwins = $windowed ? max(1, floor(($length - $pkwin) / $pkstep) + 1) : 1;
# Define windows to be analyzed
# Windows are defined as: [start, end]
if ($windowed &&
$nwins > 1) {
for (50, 100) {
my $end = $pkwin - $_ - 1;
last if ($end < MINWINLENGTH - 1);
push(@foldwins, [0, $end]);
}
for (0 .. $nwins - 1) {
my ($start, $end);
$start = $pkstep * $_;
$end = $_ == $nwins - 1 ? $length - 1 : $start + $pkwin - 1;
push(@foldwins, [$start, $end]);
}
for (50, 100) {
my $start = $length - $pkwin + $_;
last if ($start > $length - MINWINLENGTH);
push(@foldwins, [$start, $length - 1]);
}
}
else { push(@foldwins, [0, $length - 1]); }
for my $i (0 .. $#reactivity) {
# Predict pseudoknot in window
foreach my $win (@foldwins) {
my ($start, $end, $subseq, $tmpfile,
$winconst, @winreact);
($start, $end) = @{$win};
$subseq = substr($sequence, $start, $end - $start + 1);
$winconst = fixdotbracket(substr($entry->structure(), $start, $end - $start + 1));
@winreact = @{$reactivity[$i]}[$start .. $end];
$tmpfile = "$id\_$start-$end\_$$\_$i";
my ($winpk, $winbp) = pkdetect( file => $tmpfile,
sample => $i,
sequence => $subseq,
reactivity => \@winreact,
pos => $start,
constraint => $winconst );
if (!$winpk) {
unlink(glob($tmpfile . "*")) unless($keeptmp);
return({ id => $id,
pkErr => 1 });
}
$pkpairs{$_}++ for (keys %{$winpk});
$basepairs{$_}++ for (keys %{$winbp});
push(@windows, [$start, $end]);
}
}
# Discard pseudoknotted pairs in < 0.50 of windows
foreach my $pair (keys %pkpairs) {
my ($i, $j, $n1, $n2);
($i, $j) = split(/-/, $pair);
for (@windows) {
$n1++ if (inrange($i, $_));
$n2++ if (inrange($j, $_));
}
# We essentially check whether the pseudoknot base-pair exists in other windows,
# even if it has not been marked as a pseudoknot
$pkpairs{$pair} += $basepairs{$pair} if (exists $basepairs{$pair});
# If pseudoknotted pair is present in > 0.50 windows
push(@pkpairs, [$i, $j]) if ($pkpairs{$pair} / $n1 > 0.5 && $pkpairs{$pair} / $n2 > 0.5);
}
# In the pkdetect() function, we have already discarded the pk helices that overlap by more than 50%
# with helices in the MFE structure. Now, we remove the pairs from the MFE structure that still overlap (if any)
%pkbases = map { $_ => 1 } map { @$_ } @pkpairs;
@keptpairs = grep { !exists $pkbases{$_->[0]} && !exists $pkbases{$_->[1]} } @keptpairs;
# Shouldn't be necessary anymore
# In case we have incompatible base-pairs, we let rmpseudoknots() resolve that
#@keptpkpairs = rmpseudoknots($sequence, [ @keptpairs, @pkpairs ]);
#@pkpairs = @{$keptpkpairs[1]} ? @{$keptpkpairs[1]} : listpairs($keptpkpairs[0]);
undef($nwins);
undef(@foldwins);
undef(@windows);
}
######### End of pseudoknots detection block ##########
$entry = Data::Sequence::Structure->new( id => $id,
sequence => $sequence,
basepairs => [ @keptpairs, @pkpairs ],
pseudoknots => 1,
lonelypairs => $nlp ? 0 : 1 );
$energy = RNA::energy_of_struct($sequence, $entry->structure());
$energy += pseudodG($entry->structure(), \@meanReact) if (!$ignore);
$entry->energy(sprintf("%.2f", min(0, $energy)));
$file = $output . "structures/" . $id . "." . ($ct ? "ct" : "db");
# Fix the ID changed by ViennaRNA
$entry->id($id) if ($method == 1);
eval { $io = Data::IO::Sequence->new( file => $file,
mode => "w",
format => $ct ? "CT" : "Vienna" ); };
if ($@) {
return({ id => $id,
foldErr => 1 });
next;
}
$io->write($entry);
$io->close();
if ($dp) {
open(my $wh, ">" . $output . "dotplot/" . $id . ".dp") or next;
select((select($wh), $|=1)[0]);