# $Id: Mutator.pm 16123 2009-09-17 12:57:27Z cjfields $
#
# bioperl module for Bio::LiveSeq::Mutator
#
# Please direct questions and support issues to <bioperl-l@bioperl.org> 
#
# Cared for by Heikki Lehvaslaiho <heikki-at-bioperl-dot-org>
#
# Copyright Joseph Insana
#
# You may distribute this module under the same terms as perl itself
#
# POD documentation - main docs before the code

=head1 NAME

Bio::LiveSeq::Mutator - Package mutating LiveSequences

=head1 SYNOPSIS

  # $gene is a Bio::LiveSeq::Gene object
  my $mutate = Bio::LiveSeq::Mutator->new('-gene' => $gene,
  					  '-numbering' => "coding"
  					   );
  # $mut is a Bio::LiveSeq::Mutation object
  $mutate->add_Mutation($mut);
  # $results is a Bio::Variation::SeqDiff object
  my $results=$mutate->change_gene();
  if ($results) {
      my $out = Bio::Variation::IO->new( '-format' => 'flat');
      $out->write($results);
  }

=head1 DESCRIPTION

This class mutates Bio::LiveSeq::Gene objects and returns a
Bio::Variation::SeqDiff object. Mutations are described as
Bio::LiveSeq::Mutation objects. See L<Bio::LiveSeq::Gene>,
L<Bio::Variation::SeqDiff>, and L<Bio::LiveSeq::Mutation> for details.

=head1 FEEDBACK


User feedback is an integral part of the evolution of this and other
Bioperl modules. Send your comments and suggestions preferably to the
Bioperl mailing lists  Your participation is much appreciated.

  bioperl-l@bioperl.org                  - General discussion
  http://bioperl.org/wiki/Mailing_lists  - About the mailing lists

=head2 Support 

Please direct usage questions or support issues to the mailing list:

I<bioperl-l@bioperl.org>

rather than to the module maintainer directly. Many experienced and 
reponsive experts will be able look at the problem and quickly 
address it. Please include a thorough description of the problem 
with code and data examples if at all possible.

=head2 Reporting Bugs

report bugs to the Bioperl bug tracking system to help us keep track
the bugs and their resolution.  Bug reports can be submitted via the
web:

  http://bugzilla.open-bio.org/

=head1 AUTHOR - Heikki Lehvaslaiho & Joseph A.L. Insana

  Email:  heikki-at-bioperl-dot-org
          insana@ebi.ac.uk, jinsana@gmx.net

=head1 APPENDIX

  The rest of the documentation details each of the object
  methods. Internal methods are usually preceded with a _

=cut

# Let the code begin...

package Bio::LiveSeq::Mutator;
use strict;

use Bio::Variation::SeqDiff;
use Bio::Variation::DNAMutation;
use Bio::Variation::RNAChange;
use Bio::Variation::AAChange;
use Bio::Variation::Allele;
use Bio::LiveSeq::Mutation;

#use integer;
# Object preamble - inheritance


use base qw(Bio::Root::Root);

sub new {
    my($class,@args) = @_;
    my $self;
    $self = {};
    bless $self, $class;

    my ($gene, $numbering) =
	    $self->_rearrange([qw(GENE
				  NUMBERING
				  )],
			      @args);

    $self->{ 'mutations' } = [];

    $gene && $self->gene($gene);
    $numbering && $self->numbering($numbering);

    #class constant;
    $self->{'flanklen'} = 25;
    return $self; # success - we hope!
}

=head2 gene

 Title   : gene
 Usage   : $mutobj = $obj->gene;
         : $mutobj = $obj->gene($objref);
 Function:

           Returns or sets the link-reference to a
           Bio::LiveSeq::Gene object. If no value has ben set, it
           will return undef

 Returns : an object reference  or undef
 Args    : a Bio::LiveSeq::Gene

See L<Bio::LiveSeq::Gene> for more information.

=cut

sub gene {
  my ($self,$value) = @_;
  if (defined $value) {
      if( ! $value->isa('Bio::LiveSeq::Gene') ) {
	  $self->throw("Is not a Bio::LiveSeq::Gene object but a [$value]");
	  return;
      }
      else {
	  $self->{'gene'} = $value;
      }
  }
  unless (exists $self->{'gene'}) {
      return;
  } else {
      return $self->{'gene'};
  }
}


=head2 numbering

 Title   : numbering
 Usage   : $obj->numbering();
 Function:

            Sets and returns coordinate system used in positioning the
            mutations. See L<change_gene> for details.

 Example :
 Returns : string
 Args    : string (coding [transcript number] | gene | entry)

=cut


sub numbering {
    my ($self,$value) = @_;
    if( defined $value) {
	if ($value =~ /(coding)( )?(\d+)?/ or $value eq 'entry' or $value eq 'gene') {
	    $self->{'numbering'} = $value;
	} else { # defaulting to 'coding'
	    $self->{'numbering'} = 'coding';
	}
    }
    unless (exists $self->{'numbering'}) {
	return 'coding';
    } else {
	return $self->{'numbering'};
    }
}

=head2 add_Mutation

 Title   : add_Mutation
 Usage   : $self->add_Mutation($ref)
 Function: adds a Bio::LiveSeq::Mutation object
 Example :
 Returns :
 Args    : a Bio::LiveSeq::Mutation

See L<Bio::LiveSeq::Mutation> for more information.

=cut

sub add_Mutation{
    my ($self,$value) = @_;
    if( $value->isa('Bio::Liveseq::Mutation') ) {
	my $com = ref $value;
	$self->throw("Is not a Mutation object but a [$com]" );
	return;
    }
    if (! $value->pos) {
	$self->warn("No value for mutation position in the sequence!");
	return;
    }
    if (! $value->seq && ! $value->len) {
	$self->warn("Either mutated sequence or length of the deletion must be given!");
	return;
    }
    push(@{$self->{'mutations'}},$value);
}

=head2 each_Mutation

 Title   : each_Mutation
 Usage   : foreach $ref ( $a->each_Mutation )
 Function: gets an array of Bio::LiveSeq::Mutation objects
 Example :
 Returns : array of Mutations
 Args    :

See L<Bio::LiveSeq::Mutation> for more information.

=cut

sub each_Mutation{
   my ($self) = @_;
   return @{$self->{'mutations'}};
}


=head2 mutation

 Title   : mutation
 Usage   : $mutobj = $obj->mutation;
         : $mutobj = $obj->mutation($objref);
 Function:

           Returns or sets the link-reference to the current mutation
           object.  If the value is not set, it will return undef.
           Internal method.

 Returns : an object reference  or undef

=cut


sub mutation {
  my ($self,$value) = @_;
  if (defined $value) {
      if( ! $value->isa('Bio::LiveSeq::Mutation') ) {
	  $self->throw("Is not a Bio::LiveSeq::Mutation object but a [$value]");
	  return;
      }
      else {
	  $self->{'mutation'} = $value;
      }
  }
  unless (exists $self->{'mutation'}) {
      return;
  } else {
      return $self->{'mutation'};
  }
}

=head2 DNA

 Title   : DNA
 Usage   : $mutobj = $obj->DNA;
         : $mutobj = $obj->DNA($objref);
 Function:

           Returns or sets the reference to the LiveSeq object holding
           the reference sequence. If there is no link, it will return
           undef.
           Internal method.

 Returns : an object reference or undef

=cut

sub DNA {
  my ($self,$value) = @_;
  if (defined $value) {
      if( ! $value->isa('Bio::LiveSeq::DNA') and ! $value->isa('Bio::LiveSeq::Transcript') ) {
	  $self->throw("Is not a Bio::LiveSeq::DNA/Transcript object but a [$value]");
	  return;
      }
      else {
	  $self->{'DNA'} = $value;
      }
  }
  unless (exists $self->{'DNA'}) {
      return;
  } else {
      return $self->{'DNA'};
  }
}


=head2 RNA

 Title   : RNA
 Usage   : $mutobj = $obj->RNA;
         : $mutobj = $obj->RNA($objref);
 Function:

           Returns or sets the reference to the LiveSeq object holding
           the reference sequence. If the value is not set, it will return
           undef.
           Internal method.

 Returns : an object reference  or undef

=cut


sub RNA {
  my ($self,$value) = @_;
  if (defined $value) {
      if( ! $value->isa('Bio::LiveSeq::Transcript') ) {
	  $self->throw("Is not a Bio::LiveSeq::RNA/Transcript object but a [$value]");
	  return;
      }
      else {
	  $self->{'RNA'} = $value;
      }
  }
  unless (exists $self->{'RNA'}) {
      return;
  } else {
      return $self->{'RNA'};
  }
}


=head2 dnamut

 Title   : dnamut
 Usage   : $mutobj = $obj->dnamut;
         : $mutobj = $obj->dnamut($objref);
 Function:

           Returns or sets the reference to the current DNAMutation object.
           If the value is not set, it will return undef.
           Internal method.

 Returns : a Bio::Variation::DNAMutation object or undef

See L<Bio::Variation::DNAMutation> for more information.

=cut


sub dnamut {
  my ($self,$value) = @_;
  if (defined $value) {
      if( ! $value->isa('Bio::Variation::DNAMutation') ) {
	  $self->throw("Is not a Bio::Variation::DNAMutation object but a [$value]");
	  return;
      }
      else {
	  $self->{'dnamut'} = $value;
      }
  }
  unless (exists $self->{'dnamut'}) {
      return;
  } else {
      return $self->{'dnamut'};
  }
}


=head2 rnachange

 Title   : rnachange
 Usage   : $mutobj = $obj->rnachange;
         : $mutobj = $obj->rnachange($objref);
 Function:

           Returns or sets the reference to the current RNAChange object.
           If the value is not set, it will return undef.
           Internal method.

 Returns : a Bio::Variation::RNAChange object or undef

See L<Bio::Variation::RNAChange> for more information.

=cut


sub rnachange {
  my ($self,$value) = @_;
  if (defined $value) {
      if( ! $value->isa('Bio::Variation::RNAChange') ) {
	  $self->throw("Is not a Bio::Variation::RNAChange object but a [$value]");
	  return;
      }
      else {
	  $self->{'rnachange'} = $value;
      }
  }
  unless (exists $self->{'rnachange'}) {
      return;
  } else {
      return $self->{'rnachange'};
  }
}


=head2 aachange

 Title   : aachange
 Usage   : $mutobj = $obj->aachange;
         : $mutobj = $obj->aachange($objref);
 Function:

           Returns or sets the reference to the current AAChange object.
           If the value is not set, it will return undef.
           Internal method.

 Returns : a Bio::Variation::AAChange object or undef

See L<Bio::Variation::AAChange> for more information.

=cut


sub aachange {
  my ($self,$value) = @_;
  if (defined $value) {
      if( ! $value->isa('Bio::Variation::AAChange') ) {
	  $self->throw("Is not a Bio::Variation::AAChange object but a [$value]");
	  return;
      }
      else {
	  $self->{'aachange'} = $value;
      }
  }
  unless (exists $self->{'aachange'}) {
      return;
  } else {
      return $self->{'aachange'};
  }
}


=head2 exons

 Title   : exons
 Usage   : $mutobj = $obj->exons;
         : $mutobj = $obj->exons($objref);
 Function:

           Returns or sets the reference to a current array of Exons.
           If the value is not set, it will return undef.
           Internal method.

 Returns : an array of Bio::LiveSeq::Exon objects or undef

See L<Bio::LiveSeq::Exon> for more information.

=cut


sub exons {
  my ($self,$value) = @_;
  if (defined $value) {
      $self->{'exons'} = $value;
  }
  unless (exists $self->{'exons'}) {
      return;
  } else {
      return $self->{'exons'};
  }
}

=head2 change_gene_with_alignment

 Title   : change_gene_with_alignment
 Usage   : $results=$mutate->change_gene_with_alignment($aln);

 Function:

           Returns a Bio::Variation::SeqDiff object containing the
           results of the changes in the alignment. The alignment has
           to be pairwise and have one sequence named 'QUERY', the
           other one is assumed to be a part of the sequence from
           $gene.

           This method offers a shortcut to change_gene and
           automates the creation of Bio::LiveSeq::Mutation objects.
           Use it with almost identical sequnces, e.g. to locate a SNP.

 Args    : Bio::SimpleAlign object representing a short local alignment
 Returns : Bio::Variation::SeqDiff object or 0 on error

See L<Bio::LiveSeq::Mutation>, L<Bio::SimpleAlign>, and
L<Bio::Variation::SeqDiff>  for more information.

=cut

sub change_gene_with_alignment {
    my ($self, $aln) = @_;

    #
    # Sanity checks
    #

    $self->throw("Argument is not a Bio::SimpleAlign object but a [$aln]")
	unless $aln->isa('Bio::SimpleAlign');
    $self->throw("'Pairwise alignments only, please") 
	if $aln->no_sequences != 2;

    # find out the order the two sequences are given
    my $queryseq_pos = 1; #default
    my $refseq_pos = 2;
    unless ($aln->get_seq_by_pos(1)->id eq 'QUERY') {
	carp('Query sequence has to be named QUERY') 
	    if $aln->get_seq_by_pos(2)->id ne 'QUERY';
	$queryseq_pos = 2; # alternative
	$refseq_pos = 1;
    }

    # trim the alignment
    my $start =  $aln->column_from_residue_number('QUERY', 1);
    my $end =  $aln->column_from_residue_number('QUERY', 
						$aln->get_seq_by_pos($queryseq_pos)->end );
    
    my $aln2 = $aln->slice($start, $end);

    #
    # extracting mutations
    #

    my $cs = $aln2->consensus_string(51);
    my $queryseq = $aln2->get_seq_by_pos($queryseq_pos);
    my $refseq = $aln2->get_seq_by_pos($refseq_pos);

    while ( $cs =~ /(\?+)/g) {
	# pos in local coordinates
	my $pos = pos($cs) - length($1) + 1;
	my $mutation = create_mutation($self, 
				       $refseq, 
				       $queryseq, 
				       $pos, 
				       CORE::length($1)
				       );
	# reset pos to refseq coordinates
	$pos +=  $refseq->start - 1;
	$mutation->pos($pos);

        $self->add_Mutation($mutation);
    }
    return $self->change_gene();
}

=head2 create_mutation

 Title   : create_mutation
 Usage   : 
 Function:

           Formats sequence differences from two sequences into
           Bio::LiveSeq::Mutation objects which can be applied to a
           gene.

           To keep it generic, sequence arguments need not to be
           Bio::LocatableSeq. Coordinate change to parent sequence
           numbering needs to be done by the calling code.

           Called from change_gene_with_alignment

 Args    : Bio::PrimarySeqI inheriting object for the reference sequence
           Bio::PrimarySeqI inheriting object for the query sequence
           integer for the start position of the local sequence difference
           integer for the length of the sequence difference
 Returns : Bio::LiveSeq::Mutation object 

=cut

sub create_mutation {
    my ($self, $refseq, $queryseq, $pos, $len) = @_;
    
    $self->throw("Is not a Bio::PrimarySeqI object but a [$refseq]") 
	unless $refseq->isa('Bio::PrimarySeqI');
    $self->throw("Is not a Bio::PrimarySeqI object but a [$queryseq]") 
	unless $queryseq->isa('Bio::PrimarySeqI');
    $self->throw("Position is not a positive integer but [$pos]") 
	unless $pos =~ /^\+?\d+$/;
    $self->throw("Length is not a positive integer but [$len]") 
	unless $len =~ /^\+?\d+$/;

    my $mutation;
    my $refstring = $refseq->subseq($pos, $pos + $len - 1);
    my $varstring = $queryseq->subseq($pos, $pos + $len - 1);
    
    if ($len == 1 and $refstring =~ /[^\.\-\*\?]/ and 
	$varstring  =~ /[^\.\-\*\?]/ ) { #point
	$mutation = Bio::LiveSeq::Mutation->new(-seq => $varstring,
						-pos => $pos,
						);
    }
    elsif ( $refstring =~ /^[^\.\-\*\?]+$/ and 
	    $varstring  !~ /^[^\.\-\*\?]+$/ ) { # deletion
	$mutation = Bio::LiveSeq::Mutation->new(-pos => $pos,
						-len => $len
						);
    }
    elsif ( $refstring !~ /^[^\.\-\*\?]+$/ and 
	    $varstring  =~ /^[^\.\-\*\?]+$/ ) { # insertion
	$mutation = Bio::LiveSeq::Mutation->new(-seq => $varstring,
						-pos => $pos,
						-len => 0
						);
    } else { # complex
	$mutation = Bio::LiveSeq::Mutation->new(-seq => $varstring,
						-pos => $pos,
						-len => $len
						);
    }
    
    return $mutation;
}

=head2 change_gene

 Title   : change_gene
 Usage   : my $mutate = Bio::LiveSeq::Mutator->new(-gene => $gene,
						   numbering => "coding"
						   );
           # $mut is Bio::LiveSeq::Mutation object
           $mutate->add_Mutation($mut);
           my $results=$mutate->change_gene();

 Function:

           Returns a Bio::Variation::SeqDiff object containing the
           results of the changes performed according to the
           instructions present in Mutation(s).  The -numbering
           argument decides what molecule is being changed and what
           numbering scheme being used:

            -numbering => "entry"

               determines the DNA level, using the numbering from the
               beginning of the sequence

            -numbering => "coding"

               determines the RNA level, using the numbering from the
               beginning of the 1st transcript

               Alternative transcripts can be used by specifying
               "coding 2" or "coding 3" ...

            -numbering => "gene"

               determines the DNA level, using the numbering from the
               beginning of the 1st transcript and inluding introns.
               The meaning equals 'coding' if the reference molecule
               is cDNA.

 Args    : Bio::LiveSeq::Gene object
           Bio::LiveSeq::Mutation object(s)
           string specifying a numbering scheme (defaults to 'coding')
 Returns : Bio::Variation::SeqDiff object or 0 on error

=cut

sub change_gene {
    my ($self) = @_;

    #
    # Sanity check
    #
    unless ($self->gene) {
	$self->warn("Input object Bio::LiveSeq::Gene is not given");
	return 0;
    }
    #
    # Setting the reference sequence based on -numbering
    #
    my @transcripts=@{$self->gene->get_Transcripts};
    my $refseq; # will hold Bio::LiveSeq:Transcript object or Bio::LiveSeq::DNA

    # 'gene' eq 'coding' if reference sequence is cDNA
    $self->numbering ('coding') if $self->gene->get_DNA->alphabet eq 'rna' and $self->numbering eq 'gene';

    if ($self->numbering =~ /(coding)( )?(\d+)?/ ) {
	$self->numbering($1);
	my $transnumber = $3;
	$transnumber-- if $3; # 1 -> 0, 2 -> 1
	if ($transnumber && $transnumber >= 0 && $transnumber <= $#transcripts) {
	    $refseq=$transcripts[$transnumber];
	} else {
	    $transnumber && $self->warn("The alternative transcript number ". $transnumber+1 .
	    "- does not exist. Reverting to the 1st transcript\n");
	    $refseq=$transcripts[0];
	}
    } else {
	$refseq=$transcripts[0]->{'seq'};
    }
    #
    # Recording the state: SeqDiff object creation  ?? transcript no.??
    #
    my $seqDiff = Bio::Variation::SeqDiff->new(-verbose => $self->verbose);
    $seqDiff->alphabet($self->gene->get_DNA->alphabet);
    $seqDiff->numbering($self->numbering);
    my ($DNAobj, $RNAobj);
    if ($refseq->isa("Bio::LiveSeq::Transcript")) {
	$self->RNA($refseq);
	$self->DNA($refseq->{'seq'});
	$seqDiff->rna_ori($refseq->seq );
	$seqDiff->aa_ori($refseq->get_Translation->seq);
    } else {
	$self->DNA($refseq);
	$self->RNA($transcripts[0]);
	$seqDiff->rna_ori($self->RNA->seq);
	$seqDiff->aa_ori($self->RNA->get_Translation->seq);
    }
    $seqDiff->dna_ori($self->DNA->seq);
    # put the accession number into the SeqDiff object ID
    $seqDiff->id($self->DNA->accession_number);

    # the atg_offset takes in account that DNA object could be a subset of the
    # whole entry (via the light_weight loader)
    my $atg_label=$self->RNA->start;
    my $atg_offset=$self->DNA->position($atg_label)+($self->DNA->start)-1;
    $seqDiff->offset($atg_offset - 1);
    $self->DNA->coordinate_start($atg_label);

    my @exons = $self->RNA->all_Exons;
    $seqDiff->cds_end($exons[$#exons]->end);

    #
    # Converting mutation positions to labels
    #
    $self->warn("no mutations"), return 0 
	unless $self->_mutationpos2label($refseq, $seqDiff);

    # need to add more than one rna & aa
    #foreach $transcript (@transcripts) {
    #  $seqDiff{"ori_transcript_${i}_seq"}=$transcript->seq;
    #  $seqDiff{"ori_translation_${i}_seq"}=$transcript->get_Translation->seq;
    #}

    # do changes
    my $k;
    foreach my $mutation ($self->each_Mutation) {
	next unless $mutation->label > 0;
	$self->mutation($mutation);

	$mutation->issue(++$k);
	#
	# current position on the transcript
	#
	if ($self->numbering =~ /coding/) {
	    $mutation->transpos($mutation->pos); # transpos given by user
	} else {
	    #transpos of label / It will be 0 if mutating an intron, negative if upstream of ATG
	    $mutation->transpos($self->RNA->position($mutation->label,$atg_label));
	}
	#
	# Calculate adjacent labels based on the position on the current sequence
	#
	$mutation->prelabel($self->DNA->label(-1, $mutation->label)); # 1 before label
	if ($mutation->len == 0) {
	    $mutation->postlabel($mutation->label);
	    $mutation->lastlabel($mutation->label);
	} elsif ($mutation->len == 1) {
	    $mutation->lastlabel($mutation->label); # last nucleotide affected
	    $mutation->postlabel($self->DNA->label(2,$mutation->lastlabel)); # $len after label
	} else {
	    $mutation->lastlabel($self->DNA->label($mutation->len,$mutation->label));
	    $mutation->postlabel($self->DNA->label(2,$mutation->lastlabel));
	}
	my $dnamut = $self->_set_DNAMutation($seqDiff);
	#
	#
	#
	if ($self->_rnaAffected) {
	    $self->_set_effects($seqDiff, $dnamut);
	}
	elsif ($seqDiff->offset != 0 and $dnamut->region ne 'intron') {
	    $self->_untranslated ($seqDiff, $dnamut);
	} else {
	    #$self->warn("Mutation starts outside coding region, RNAChange object not created");
	}

	#########################################################################
	# Mutations are done here!                                              #
	$refseq->labelchange($mutation->seq, $mutation->label, $mutation->len); #
	#########################################################################

	$self->_post_mutation ($seqDiff);

	$self->dnamut(undef);
	$self->rnachange(undef);
	$self->aachange(undef);
	$self->exons(undef);
    }
    # record the final state of all three sequences
    $seqDiff->dna_mut($self->DNA->seq);
    $seqDiff->rna_mut($self->RNA->seq);
    if ($refseq->isa("Bio::LiveSeq::Transcript")) {
	$seqDiff->aa_mut($refseq->get_Translation->seq);
    } else {
	$seqDiff->aa_mut($self->RNA->get_Translation->seq);
    }

    #$seqDiff{mut_dna_seq}=$gene->get_DNA->seq;
    #my $i=1;
    #foreach $transcript (@transcripts) {
    #  $seqDiff{"mut_transcript_${i}_seq"}=$transcript->seq;
    #  $seqDiff{"mut_translation_${i}_seq"}=$transcript->get_Translation->seq;
    #}
    return $seqDiff;
}

=head2 _mutationpos2label

 Title   : _mutationpos2label
 Usage   :
 Function: converts mutation positions into labels
 Example :
 Returns : number of valid mutations
 Args    : LiveSeq sequence object

=cut

sub _mutationpos2label {
    my ($self, $refseq, $SeqDiff) = @_;
    my $count;
    my @bb = @{$self->{'mutations'}};
    my $cc = scalar @bb;
    foreach my $mut (@{$self->{'mutations'}}) {
#	 if ($self->numbering eq 'gene' and $mut->pos < 1) {
#	     my $tmp = $mut->pos;
#	     print STDERR "pos: ", "$tmp\n";
#	     $tmp++ if $tmp < 1;
#	     $tmp += $SeqDiff->offset;
#	     print STDERR "pos2: ", "$tmp\n";
#	     $mut->pos($tmp);
#	 }
#	elsif ($self->numbering eq 'entry') {
	if ($self->numbering eq 'entry') {
	    my $tmp = $mut->pos;
	    $tmp -= $SeqDiff->offset;
	    $tmp-- if $tmp < 1;
	    $mut->pos($tmp);
	}

	my $label = $refseq->label($mut->pos); # get the label for the position
	$mut->label($label), $count++ if $label > 0 ;
	#print STDERR "x", $mut->pos,'|' ,$mut->label, "\n";
    }
    return $count;
}

#
# Calculate labels around mutated nucleotide
#

=head2 _set_DNAMutation

 Title   : _set_DNAMutation
 Usage   :
 Function:

           Stores DNA level mutation attributes before mutation into
           Bio::Variation::DNAMutation object.  Links it to SeqDiff
           object.

 Example :
 Returns : Bio::Variation::DNAMutation object
 Args    : Bio::Variation::SeqDiff object

See L<Bio::Variation::DNAMutation> and L<Bio::Variation::SeqDiff>.

=cut

sub _set_DNAMutation {
    my ($self, $seqDiff) = @_;

    my $dnamut_start = $self->mutation->label - $seqDiff->offset;
    # if negative DNA positions (before ATG)
    $dnamut_start-- if $dnamut_start <= 0;
    my $dnamut_end;
    ($self->mutation->len == 0 or $self->mutation->len == 1) ?
	($dnamut_end = $dnamut_start) :
	($dnamut_end = $dnamut_start+$self->mutation->len);
    #print "start:$dnamut_start, end:$dnamut_end\n";
    my $dnamut = Bio::Variation::DNAMutation->new(-start => $dnamut_start,
						  -end => $dnamut_end,
						  );
    $dnamut->mut_number($self->mutation->issue);
    $dnamut->isMutation(1);
    my $da_m = Bio::Variation::Allele->new;
    $da_m->seq($self->mutation->seq) if $self->mutation->seq;
    $dnamut->allele_mut($da_m);
    $dnamut->add_Allele($da_m);
    # allele_ori
    my $allele_ori = $self->DNA->labelsubseq($self->mutation->prelabel,
					     undef,
					     $self->mutation->postlabel); # get seq
    chop $allele_ori; # chop the postlabel nucleotide
    $allele_ori=substr($allele_ori,1); # away the prelabel nucleotide
    my $da_o = Bio::Variation::Allele->new;
    $da_o->seq($allele_ori) if $allele_ori;
    $dnamut->allele_ori($da_o);
    ($self->mutation->len == 0) ?
	($dnamut->length($self->mutation->len)) : ($dnamut->length(CORE::length $allele_ori));
    #print " --------------- $dnamut_start -$len-  $dnamut_end -\n";
    $seqDiff->add_Variant($dnamut);
    $self->dnamut($dnamut);
    $dnamut->mut_number($self->mutation->issue);
    # setting proof
    if ($seqDiff->numbering eq "entry" or $seqDiff->numbering eq "gene") {
	 $dnamut->proof('experimental');
    } else {
	 $dnamut->proof('computed');
    }
    # how many nucleotides to store upstream and downstream of the change
    my $flanklen = $self->{'flanklen'};
    #print  `date`, " flanking sequences start\n";
    my $uplabel = $self->DNA->label(1-$flanklen,$self->mutation->prelabel); # this could be unavailable!

    my $upstreamseq;
    if ($uplabel > 0) {
	 $upstreamseq =
	     $self->DNA->labelsubseq($uplabel, undef, $self->mutation->prelabel);
    } else { # from start (less than $flanklen nucleotides)
	 $upstreamseq =
	     $self->DNA->labelsubseq($self->DNA->start, undef, $self->mutation->prelabel);
    }
    $dnamut->upStreamSeq($upstreamseq);
    my $dnstreamseq = $self->DNA->labelsubseq($self->mutation->postlabel, $flanklen);
    $dnamut->dnStreamSeq($dnstreamseq); # $flanklen or less nucleotides
    return $dnamut;
}


#
### Check if mutation propagates to RNA (and AA) level
#  
# side effect: sets intron/exon information
# returns a boolean value
#

sub _rnaAffected {
    my ($self) = @_;
    my @exons=$self->RNA->all_Exons;
    my $RNAstart=$self->RNA->start;
    my $RNAend=$self->RNA->end;
    my ($firstexon,$before,$after,$i);
    my ($rnaAffected) = 0;

    # check for inserted labels (that require follows instead of <,>)
    my $DNAend=$self->RNA->{'seq'}->end;
    if ($self->mutation->prelabel > $DNAend or $self->mutation->postlabel > $DNAend) {
	 #this means one of the two labels is an inserted one
	 #(coming from a previous mutation. This would falsify all <,>
	 #checks, so the follow() has to be used
	 $self->warn("Attention, workaround not fully tested yet! Expect unpredictable results.\n");
	 if (($self->mutation->postlabel==$RNAstart) or (follows($self->mutation->postlabel,$RNAstart))) {
	     $self->warn("RNA not affected because change occurs before RNAstart");
	 }
	 elsif (($RNAend==$self->mutation->prelabel) or (follows($RNAend,$self->mutation->prelabel))) {
	     $self->warn("RNA not affected because change occurs after RNAend");
	 }
	 elsif (scalar @exons == 1) {
	     #no introns, just one exon
	     $rnaAffected = 1; # then RNA is affected!
	 } else {
	     # otherwise check for change occurring inside an intron
	     $firstexon=shift(@exons);
	     $before=$firstexon->end;
	
	     foreach $i (0..$#exons) {
		 $after=$exons[$i]->start;
		 if (follows($self->mutation->prelabel,$before) or
			($after==$self->mutation->prelabel) or
			follows($after,$self->mutation->prelabel) or
			follows($after,$self->mutation->postlabel)) {

		     $rnaAffected = 1;
		     # $i is number of exon and can be used for proximity check
		 }
		 $before=$exons[$i]->end;
	     }
	
	 }
    } else {
	my $strand = $exons[0]->strand;
	if (($strand == 1 and $self->mutation->postlabel <= $RNAstart) or
	    ($strand != 1 and $self->mutation->postlabel >= $RNAstart)) {
	    #$self->warn("RNA not affected because change occurs before RNAstart");
	    $rnaAffected = 0;
	}
	elsif (($strand == 1 and $self->mutation->prelabel >= $RNAend) or
		($strand != 1 and $self->mutation->prelabel <= $RNAend)) {
	     #$self->warn("RNA not affected because change occurs after RNAend");
	     $rnaAffected = 0;
	     my $dist;
	     if ($strand == 1){
		 $dist = $self->mutation->prelabel - $RNAend;
	     } else {
		 $dist = $RNAend - $self->mutation->prelabel;
	     }
	     $self->dnamut->region_dist($dist);
	 }
	 elsif (scalar @exons == 1) {
	     #if just one exon -> no introns, 
	     $rnaAffected = 1; # then RNA is affected!
	 } else {	
	     # otherwise check for mutation occurring inside an intron
	     $firstexon=shift(@exons);
	     $before=$firstexon->end;
	     if ( ($strand == 1 and $self->mutation->prelabel < $before) or 
		  ($strand == -1 and $self->mutation->prelabel > $before) 
		  ) {
		 $rnaAffected = 1 ;

		 #print "Exon 1 : ", $firstexon->start, " - ", $firstexon->end, "<br>\n";
		 my $afterdist = $self->mutation->prelabel - $firstexon->start;
		 my $beforedist =  $firstexon->end - $self->mutation->postlabel;
		 my $exonvalue = $i + 1;
		 $self->dnamut->region('exon');
		 $self->dnamut->region_value($exonvalue);
		 if ($afterdist < $beforedist) {
		     $afterdist++; 		  
		     $afterdist++;
		     $self->dnamut->region_dist($afterdist);
		     #print "splice site $afterdist nt upstream!<br>";
		 } else {
		     $self->dnamut->region_dist($beforedist);
		     #print "splice site $beforedist nt downstream!<br>";
		 }
	     } else {
		 #print "first exon  : ", $firstexon->start, " - ", $firstexon->end, "<br>\n";
		 foreach $i (0..$#exons) {
		     $after=$exons[$i]->start;
		     #proximity test for intronic mutations
		     if ( ($strand == 1 and 
			   $self->mutation->prelabel >=  $before and 
			   $self->mutation->postlabel <= $after) 
			  or
			  ($strand == -1 and 
			   $self->mutation->prelabel <=  $before and 
			   $self->mutation->postlabel >= $after)  ) {
			 $self->dnamut->region('intron');
			 #$self->dnamut->region_value($i);
			 my $afterdist = $self->mutation->prelabel - $before;
			 my $beforedist =  $after - $self->mutation->postlabel;
			 my $intronvalue = $i + 1;
			 if ($afterdist < $beforedist) {
			     $afterdist++;
			     $self->dnamut->region_value($intronvalue);
			     $self->dnamut->region_dist($afterdist);
			     #print "splice site $afterdist nt upstream!<br>";
			 } else {
			     $self->dnamut->region_value($intronvalue);
			     $self->dnamut->region_dist($beforedist * -1);
			     #print "splice site $beforedist nt downstream!<br>";
			 }
			 $self->rnachange(undef);
			 last;
		     } 
		     #proximity test for exon mutations
		     #proximity test for exon mutations
		     elsif ( ( $strand == 1 and 
			       $exons[$i]->start < $self->mutation->prelabel and 
			       $exons[$i]->end > $self->mutation->prelabel) or 
			     ( $strand == 1 and 
			       $exons[$i]->start < $self->mutation->postlabel and 
			       $exons[$i]->end > $self->mutation->postlabel) or 
			     ( $strand == -1 and 
			       $exons[$i]->start > $self->mutation->prelabel and 
			       $exons[$i]->end < $self->mutation->prelabel) or
			     ( $strand == -1 and 
			       $exons[$i]->start > $self->mutation->postlabel and 
			       $exons[$i]->end < $self->mutation->postlabel) ) {
			 $rnaAffected = 1;

			 my $afterdist = $self->mutation->prelabel - $exons[$i]->start;
			 my $beforedist =  $exons[$i]->end - $self->mutation->postlabel;
			 my $exonvalue = $i + 1;
			 $self->dnamut->region('exon');
			 if ($afterdist < $beforedist) {
			     $afterdist++;
			     $self->dnamut->region_value($exonvalue+1);
			     $self->dnamut->region_dist($afterdist);
			     #print "splice site $afterdist nt upstream!<br>";
			 } else {
			     #$beforedist;
			     $self->dnamut->region_value($exonvalue+1);
			     $self->dnamut->region_dist($beforedist * -1);
			     #print "splice site $beforedist nt downstream!<br>";
			 }
			 last;
		     }
		     $before=$exons[$i]->end;
		 }
	     }
	 }
     }
    #$self->warn("RNA not affected because change occurs inside an intron");
    #return(0); # if still not returned, then not affected, return 0
    return $rnaAffected;
}

#
# ### Creation of RNA and AA variation objects
#

=head2 _set_effects

 Title   : _set_effects
 Usage   :
 Function:

           Stores RNA and AA level mutation attributes before mutation
           into Bio::Variation::RNAChange and
           Bio::Variation::AAChange objects.  Links them to
           SeqDiff object.

 Example :
 Returns :
 Args    : Bio::Variation::SeqDiff object
           Bio::Variation::DNAMutation object

See L<Bio::Variation::RNAChange>, L<Bio::Variation::RNAChange>,
L<Bio::Variation::SeqDiff>, and L<Bio::Variation::DNAMutation>.

=cut

sub _set_effects {
    my ($self, $seqDiff, $dnamut) = @_;
    my ($rnapos_end, $upstreamseq, $dnstreamseq);
    my $flanklen = $self->{'flanklen'};

    ($self->mutation->len == 0) ?
	($rnapos_end = $self->mutation->transpos) :
	($rnapos_end = $self->mutation->transpos + $self->mutation->len -1);
    my $rnachange = Bio::Variation::RNAChange->new(-start => $self->mutation->transpos,
						    -end =>  $rnapos_end
						    );
    $rnachange->isMutation(1);

    # setting proof
    if ($seqDiff->numbering eq "coding") {
	 $rnachange->proof('experimental');
    } else {
	 $rnachange->proof('computed');
    }

    $seqDiff->add_Variant($rnachange);
    $self->rnachange($rnachange);
    $rnachange->DNAMutation($dnamut);
    $dnamut->RNAChange($rnachange);
    $rnachange->mut_number($self->mutation->issue);

    # setting the codon_position of the "start" nucleotide of the change
    $rnachange->codon_pos(($self->RNA->frame($self->mutation->label))+1); # codon_pos=frame+1

    my @exons=$self->RNA->all_Exons;
    $self->exons(\@exons);
    #print  `date`, " before flank, after exons. RNAObj query\n";
    # if cannot retrieve from Transcript, Transcript::upstream_seq will be used
    # before "fac7 g 65" bug discovered
    # $uplabel=$self->RNA->label(1-$flanklen,$prelabel);
    my $RNAprelabel=$self->RNA->label(-1,$self->mutation->label); # to fix fac7g65 bug
    # for the fix, all prelabel used in the next block have been changed to RNAprelabel
    my $uplabel=$self->RNA->label(1-$flanklen,$RNAprelabel);
    if ($self->RNA->valid($uplabel)) {
	 $upstreamseq = $self->RNA->labelsubseq($uplabel, undef, $RNAprelabel);
    } else {
	$upstreamseq = $self->RNA->labelsubseq($self->RNA->start, undef, $RNAprelabel)
	    if $self->RNA->valid($RNAprelabel);
	my $lacking=$flanklen-length($upstreamseq); # how many missing
	my $upstream_atg=$exons[0]->subseq(-$lacking,-1);
	$upstreamseq=$upstream_atg . $upstreamseq;
    }

    $rnachange->upStreamSeq($upstreamseq);

    # won't work OK if postlabel NOT in Transcript
    # now added RNApostlabel but this has to be /fully tested/
    # for the fix, all postlabel used in the next block have been changed to RNApostlabel
    my $RNApostlabel; # to fix fac7g64 bug
    if ($self->mutation->len == 0) {
      $RNApostlabel=$self->mutation->label;
    } else {
      my $mutlen = 1 + $self->mutation->len;
      $RNApostlabel=$self->RNA->label($mutlen,$self->mutation->label);
    }
    $dnstreamseq=$self->RNA->labelsubseq($RNApostlabel, $flanklen);
    if ($dnstreamseq eq '-1') { # if out of transcript was requested
	 my $lastexon=$exons[-1];
	 my $lastexonlength=$lastexon->length;
	 $dnstreamseq=$self->RNA->labelsubseq($RNApostlabel); # retrieves till RNAend
	 my $lacking=$flanklen-length($dnstreamseq); # how many missing
	 my $downstream_stop=$lastexon->subseq($lastexonlength+1,undef,$lacking);
	 $dnstreamseq .= $downstream_stop;
    } else {
	 $rnachange->dnStreamSeq($dnstreamseq);
    }
    # AAChange creation
    my $AAobj=$self->RNA->get_Translation;
    # storage of prelabel here, to be used in create_mut_objs_after
    my $aachange = Bio::Variation::AAChange->new(-start => $RNAprelabel
						  );
    $aachange->isMutation(1);
    $aachange->proof('computed');

    $seqDiff->add_Variant($aachange);
    $self->aachange($aachange);
    $rnachange->AAChange($aachange);
    $aachange->RNAChange($rnachange);

    $aachange->mut_number($self->mutation->issue);
#    $before_mutation{aachange}=$aachange;

    my $ra_o = Bio::Variation::Allele->new;
    $ra_o->seq($dnamut->allele_ori->seq) if $dnamut->allele_ori->seq;
    $rnachange->allele_ori($ra_o);

    $rnachange->length(CORE::length $rnachange->allele_ori->seq);

    my $ra_m = Bio::Variation::Allele->new;
    $ra_m->seq($self->mutation->seq) if $self->mutation->seq;
    $rnachange->allele_mut($ra_m);
    $rnachange->add_Allele($ra_m);

    #$rnachange->allele_mut($seq);
    $rnachange->end($rnachange->start) if $rnachange->length == 0;

    # this holds the aminoacid sequence that will be affected by the mutation
    my $aa_allele_ori=$AAobj->labelsubseq($self->mutation->label,undef, 
					  $self->mutation->lastlabel);

    my $aa_o = Bio::Variation::Allele->new;
    $aa_o->seq($aa_allele_ori) if $aa_allele_ori;
    $aachange->allele_ori($aa_o);
    #$aachange->allele_ori($aa_allele_ori);

    my $aa_length_ori = length($aa_allele_ori);
    $aachange->length($aa_length_ori); #print "==========$aa_length_ori\n";
    $aachange->end($aachange->start + $aa_length_ori - 1 );
}

=head2 _untranslated

 Title   : _untranslated
 Usage   :
 Function:

           Stores RNA change attributes before mutation
           into Bio::Variation::RNAChange object.  Links it to
           SeqDiff object.

 Example :
 Returns :
 Args    : Bio::Variation::SeqDiff object
           Bio::Variation::DNAMutation object

See L<Bio::Variation::RNAChange>, L<Bio::Variation::SeqDiff> and
L<Bio::Variation::DNAMutation> for details.

=cut

sub  _untranslated {
    my ($self, $seqDiff, $dnamut) = @_;
    my $rnapos_end;
    ($self->mutation->len == 0) ?
	($rnapos_end = $self->mutation->transpos) :
	($rnapos_end = $self->mutation->transpos + $self->mutation->len -1);
    my $rnachange = Bio::Variation::RNAChange->new(-start => $self->mutation->transpos,
						    -end =>  $rnapos_end
						    );
    #my $rnachange = Bio::Variation::RNAChange->new;

    $rnachange->isMutation(1);
    my $ra_o = Bio::Variation::Allele->new;
    $ra_o->seq($dnamut->allele_ori->seq) if $dnamut->allele_ori->seq;
    $rnachange->allele_ori($ra_o);
    my $ra_m = Bio::Variation::Allele->new;
    $ra_m->seq($dnamut->allele_mut->seq) if $dnamut->allele_mut->seq;
    $rnachange->allele_mut($ra_m);
    $rnachange->add_Allele($ra_m);
    $rnachange->upStreamSeq($dnamut->upStreamSeq);
    $rnachange->dnStreamSeq($dnamut->dnStreamSeq);
    $rnachange->length($dnamut->length);
    $rnachange->mut_number($dnamut->mut_number);
    # setting proof
    if ($seqDiff->numbering eq "coding") {
	$rnachange->proof('experimental');
    } else {
	$rnachange->proof('computed');
    }

    my $dist; 
    if ($rnachange->end < 0) {
	$rnachange->region('5\'UTR');
	$dnamut->region('5\'UTR');
	my $dist = $dnamut->end ;
	$dnamut->region_dist($dist);
	$dist = $seqDiff->offset - $self->gene->maxtranscript->start + 1 + $dist;
	$rnachange->region_dist($dist);
	return if $dist < 1; # if mutation is not in mRNA 
    } else {
	$rnachange->region('3\'UTR');
	$dnamut->region('3\'UTR');
	my $dist = $dnamut->start - $seqDiff->cds_end + $seqDiff->offset;
	$dnamut->region_dist($dist);
	$dist = $seqDiff->cds_end - $self->gene->maxtranscript->end -1 + $dist;
	$rnachange->region_dist($dist);
	return if $dist > 0; # if mutation is not in mRNA 
    }
    $seqDiff->add_Variant($rnachange);
    $self->rnachange($rnachange);
    $rnachange->DNAMutation($dnamut);
    $dnamut->RNAChange($rnachange);
}

# args: reference to label changearray, reference to position changearray
# Function: take care of the creation of mutation objects, with
# information AFTER the change takes place
sub _post_mutation {
    my ($self, $seqDiff) = @_;

    if ($self->rnachange and $self->rnachange->region eq 'coding') {

	#$seqDiff->add_Variant($self->rnachange);

	my $aachange=$self->aachange;
	my ($AAobj,$aa_start_prelabel,$aa_start,$mut_translation);
	$AAobj=$self->RNA->get_Translation;
	$aa_start_prelabel=$aachange->start;
	$aa_start=$AAobj->position($self->RNA->label(2,$aa_start_prelabel));
	$aachange->start($aa_start);
	$mut_translation=$AAobj->seq;

	# this now takes in account possible preinsertions
	my $aa_m = Bio::Variation::Allele->new;
	$aa_m->seq(substr($mut_translation,$aa_start-1)) if substr($mut_translation,$aa_start-1);
	$aachange->allele_mut($aa_m);
	$aachange->add_Allele($aa_m);
	#$aachange->allele_mut(substr($mut_translation,$aa_start-1));
	#$aachange->allele_mut($mut_translation);
	my ($rlenori, $rlenmut);
	$rlenori = CORE::length($aachange->RNAChange->allele_ori->seq);
	$rlenmut = CORE::length($aachange->RNAChange->allele_mut->seq);
	#point mutation

	if ($rlenori == 1 and $rlenmut == 1 and $aachange->allele_ori->seq ne '*') {
	     my $alleleseq;
	     if ($aachange->allele_mut->seq) {
		 $alleleseq = substr($aachange->allele_mut->seq, 0, 1);
		 $aachange->allele_mut->seq($alleleseq);
	     }
	     $aachange->end($aachange->start);
	     $aachange->length(1);
	 }
	elsif ( $rlenori == $rlenmut and 
		$aachange->allele_ori->seq ne '*' ) { #complex inframe mutation
	    $aachange->allele_mut->seq(substr $aachange->allele_mut->seq, 
				       0, 
				       length($aachange->allele_ori->seq));
	}
	#inframe mutation
	elsif ((int($rlenori-$rlenmut))%3 == 0) {
	    if ($aachange->RNAChange->allele_mut->seq  and
		$aachange->RNAChange->allele_ori->seq ) {
		# complex
		my $rna_len = length ($aachange->RNAChange->allele_mut->seq);
		my $len = $rna_len/3;
		$len++ unless $rna_len%3 == 0;
		$aachange->allele_mut->seq(substr $aachange->allele_mut->seq, 0, $len );
	    }
	    elsif  ($aachange->RNAChange->codon_pos == 1){
		 # deletion
		if ($aachange->RNAChange->allele_mut->seq eq  '') {
		    $aachange->allele_mut->seq('');
		    $aachange->end($aachange->start + $aachange->length - 1 );
		}
		 # insertion
		 elsif ($aachange->RNAChange->allele_ori->seq eq '' ) {
		     $aachange->allele_mut->seq(substr $aachange->allele_mut->seq, 0,
					   length ($aachange->RNAChange->allele_mut->seq) / 3);
		     $aachange->allele_ori->seq('');
		     $aachange->end($aachange->start + $aachange->length - 1 );
		     $aachange->length(0);
		 }
	    } else {
		#elsif  ($aachange->RNAChange->codon_pos == 2){
		 # deletion
		 if (not $aachange->RNAChange->allele_mut->seq ) {
		     $aachange->allele_mut->seq(substr $aachange->allele_mut->seq, 0, 1);
		 }
		 # insertion
		 elsif (not $aachange->RNAChange->allele_ori->seq) {
		     $aachange->allele_mut->seq(substr $aachange->allele_mut->seq, 0,
						length ($aachange->RNAChange->allele_mut->seq) / 3 +1);
		 }
	     }
	 } else {
	     #frameshift
	     #my $pos = index $aachange->allele_mut
	     #$aachange->allele_mut(substr($aachange->allele_mut, 0, 1));
	     $aachange->length(CORE::length($aachange->allele_ori->seq));
	     my $aaend = $aachange->start + $aachange->length -1;
	     $aachange->end($aachange->start);
	 }

	 # splicing site deletion check
	 my @beforeexons=@{$self->exons};
	 my @afterexons=$self->RNA->all_Exons;
	 my $i;
	 if (scalar(@beforeexons) ne scalar(@afterexons)) {
	     my $mut_number = $self->mutation->issue;
	     $self->warn("Exons have been modified at mutation n.$mut_number!");
	     $self->rnachange->exons_modified(1);
	 } else {
	   EXONCHECK:
	     foreach $i (0..$#beforeexons) {
		 if ($beforeexons[$i] ne $afterexons[$i]) {
	     my $mut_number = $self->mutation->issue;
		     $self->warn("Exons have been modified at mutation n.$mut_number!");
		     $self->rnachange->exons_modified(1);
		     last EXONCHECK;
		 }
	     }
	 }
     } else {
	 #$seqDiff->rnachange(undef);
	 #print "getting here?";
     }
    return 1;
}

1;
