#---------------------------------------------------------
=head1 NAME
Bio::Matrix::PSM::ProtMatrix - SiteMatrixI implementation, holds a
position scoring matrix (or position weight matrix) with log-odds scoring
information.
=head1 SYNOPSIS
use Bio::Matrix::PSM::ProtMatrix;
# Create from memory by supplying probability matrix hash both as strings or
# arrays where the frequencies Hash entries of the form lN refer to an array
# of position-specific log-odds scores for amino acid N. Hash entries of the
# form pN represent the position-specific probability of finding amino acid N.
my %param = (
'id' => 'A. thaliana protein atp1',
'-e_val' => $score,
'lS' => [ '-2', '3', '-3', '2', '-3', '1', '1', '3' ],
'lF' => [ '-1', '-4', '0', '-5', '0', '-5', '-4', '-4' ],
'lT' => [ '-1', '1', '0', '1', '-2', '-1', '0', '1' ],
'lN' => [ '-3', '-1', '-2', '3', '-5', '5', '-2', '0' ],
'lK' => [ '-2', '0', '-3', '2', '-3', '2', '-3', '-1' ],
'lY' => [ '-2', '-3', '-3', '-4', '-3', '-4', '-4', '-4' ],
'lE' => [ '-3', '4', '-3', '2', '-4', '-2', '-3', '2' ],
'lV' => [ '0', '-2', '1', '-4', '1', '-4', '-1', '-3' ],
'lQ' => [ '-1', '0', '-2', '3', '-4', '1', '-3', '0' ],
'lM' => [ '8', '-3', '8', '-3', '1', '-3', '-3', '-3' ],
'lC' => [ '-2', '-3', '-3', '-4', '-3', '-4', '-3', '-3' ],
'lL' => [ '1', '-3', '1', '-4', '3', '-4', '-2', '-4' ],
'lA' => [ '-2', '1', '-2', '0', '-2', '-2', '2', '2' ],
'lW' => [ '-2', '-4', '-3', '-5', '-4', '-5', '-5', '-5' ],
'lP' => [ '-3', '-2', '-4', '-3', '-1', '-3', '6', '-3' ],
'lH' => [ '-2', '-2', '-3', '-2', '-5', '-2', '-2', '-3' ],
'lD' => [ '-4', '-1', '-3', '1', '-3', '-1', '-3', '4' ],
'lR' => [ '-2', '-1', '-3', '0', '-4', '4', '-4', '-3' ],
'lI' => [ '0', '-3', '0', '-4', '6', '-4', '-2', '-2' ],
'lG' => [ '-4', '-2', '-4', '-2', '-5', '-3', '-1', '-2' ],
'pS' => [ '0', '33', '0', '16', '1', '12', '11', '25' ],
'pF' => [ '0', '0', '2', '0', '3', '0', '0', '0' ],
'pT' => [ '0', '8', '7', '10', '1', '2', '7', '8' ],
'pN' => [ '0', '0', '2', '13', '0', '36', '1', '4' ],
'pK' => [ '0', '5', '0', '13', '1', '15', '0', '2' ],
'pY' => [ '0', '0', '0', '0', '0', '0', '0', '0' ],
'pE' => [ '0', '41', '1', '12', '0', '0', '0', '15' ],
'pV' => [ '0', '3', '9', '0', '2', '0', '3', '1' ],
'pQ' => [ '0', '0', '0', '15', '0', '4', '0', '3' ],
'pM' => [ '100', '0', '66', '0', '2', '0', '0', '0' ],
'pC' => [ '0', '0', '0', '0', '0', '0', '0', '0' ],
'pL' => [ '0', '0', '8', '0', '25', '0', '4', '0' ],
'pA' => [ '0', '10', '1', '9', '2', '0', '22', '16' ],
'pW' => [ '0', '0', '0', '0', '0', '0', '0', '0' ],
'pP' => [ '0', '0', '0', '0', '3', '1', '45', '0' ],
'pH' => [ '0', '0', '0', '0', '0', '0', '1', '0' ],
'pD' => [ '0', '0', '1', '7', '2', '2', '0', '22' ],
'pR' => [ '0', '0', '0', '3', '0', '27', '0', '0' ],
'pI' => [ '0', '0', '3', '0', '59', '1', '2', '3' ],
'pG' => [ '0', '0', '0', '1', '0', '0', '4', '1' ],
);
my $matrix = Bio::Matrix::PSM::ProtMatrix( %param );
my $site = Bio::Matrix::PSM::ProtMatrix->new(%param);
# Or get it from a file:
use Bio::Matrix::PSM::IO;
my $psmIO = Bio::Matrix::PSM::IO->new(-file => $file, -format => 'psi-blast');
while (my $psm = $psmIO->next_psm) {
#Now we have a Bio::Matrix::PSM::Psm object,
# see Bio::Matrix::PSM::PsmI for details
#This is a Bio::Matrix::PSM::ProtMatrix object now
my $matrix = $psm->matrix;
}
# Get a simple consensus, where alphabet is:
# {A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V,}
# choosing the highest probability or N if prob is too low
my $consensus = $site->consensus;
# Retrieving and using regular expressions:
my $regexp = $site->regexp;
my $count = grep($regexp,$seq);
my $count = ($seq=~ s/$regexp/$1/eg);
print "Motif $mid is present $count times in this sequence\n";
=head1 DESCRIPTION
ProtMatrix is designed to provide some basic methods when working with
position scoring (weight) matrices related to protein sequences. A
protein PSM consists of 20 vectors with 20 frequencies (one per amino
acid per position). This is the minimum information you should
provide to construct a PSM object. The vectors can be provided as
strings with frequencies where the frequency is {0..a} and a=1. This
is the way MEME compressed representation of a matrix and it is quite
useful when working with relational DB. If arrays are provided as an
input (references to arrays actually) they can be any number, real or
integer (frequency or count).
When creating the object the constructor will check for positions that
equal 0. If such is found it will increase the count for all
positions by one and recalculate the frequency. Potential bug - if
you are using frequencies and one of the positions is 0 it will change
significantly. However, you should never have frequency that equals
0.
Throws an exception if: You mix as an input array and string (for
example A matrix is given as array, C - as string). The position
vector is (0,0,0,0). One of the probability vectors is shorter than
the rest.
Summary of the methods I use most frequently (details below):
iupac - return IUPAC compliant consensus as a string
score - Returns the score as a real number
IC - information content. Returns a real number
id - identifier. Returns a string
accession - accession number. Returns a string
next_pos - return the sequence probably for each letter, IUPAC
symbol, IUPAC probability and simple sequence
consenus letter for this position. Rewind at the end. Returns a hash.
pos - current position get/set. Returns an integer.
regexp - construct a regular expression based on IUPAC consensus.
For example AGWV will be [Aa][Gg][AaTt][AaCcGg]
width - site width
get_string - gets the probability vector for a single base as a string.
get_array - gets the probability vector for a single base as an array.
get_logs_array - gets the log-odds vector for a single base as an array.
New methods, which might be of interest to anyone who wants to store
PSM in a relational database without creating an entry for each
position is the ability to compress the PSM vector into a string with
losing usually less than 1% of the data. this can be done with:
my $str=$matrix->get_compressed_freq('A');
or
my $str=$matrix->get_compressed_logs('A');
Loading from a database should be done with new, but is not yet implemented.
However you can still uncompress such string with:
my @arr=Bio::Matrix::PSM::_uncompress_string ($str,1,1); for PSM
or
my @arr=Bio::Matrix::PSM::_uncompress_string ($str,1000,2); for log odds
=head1 FEEDBACK
=head2 Mailing Lists
User feedback is an integral part of the evolution of this and other
Bioperl modules. Send your comments and suggestions preferably to one
of 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:
https://github.com/bioperl/bioperl-live/issues
=head1 AUTHOR - James Thompson
Email tex@biosysadmin.com
=head1 APPENDIX
=cut
# Let the code begin...
package Bio::Matrix::PSM::ProtMatrix;
use strict;
use base qw(Bio::Root::Root Bio::Matrix::PSM::SiteMatrixI);
=head2 new
Title : new
Usage : my $site = Bio::Matrix::PSM::ProtMatrix->new(
%probs,
%logs,
-IC => $ic,
-e_val => $score,
-id => $mid
-model => \%model
);
Function : Creates a new Bio::Matrix::PSM::ProtMatrix object from memory
Throws : If inconsistent data for all vectors (all 20 amino acids) is
provided, if you mix input types (string vs array) or if a
position freq is 0.
Example :
Returns : Bio::Matrix::PSM::ProtMatrix object
Args : Hash references to log-odds scores and probabilities for
position-specific scoring info, e-value (optional), information
content (optional), id (optional), model for background distribution
of proteins (optional).
=cut
sub new {
my ($class, @args) = @_;
my $self = $class->SUPER::new(@args);
my $consensus;
#Too many things to rearrange, and I am creating simultanuously >500
# such objects routinely, so this becomes performance issue
my %input;
while( @args ) {
(my $key = shift @args) =~ s/-//gi; #deletes all dashes (only dashes)!
$input{$key} = shift @args;
}
# get a protein alphabet for processing log-odds scores and probabilities
# maybe change this later on to allow for non-standard aa lists?
my @alphabet = qw/A R N D C Q E G H I L K M F P S T W Y V/;
foreach my $aa (@alphabet) {
$self->{"log$aa"} = defined($input{"l$aa"}) ? $input{"l$aa"}
: $self->throw("Error: No log-odds information for $aa!");
$self->{"prob$aa"} = defined($input{"p$aa"}) ? $input{"p$aa"}
: $self->throw("Error: No probability information for $aa!");
}
$self->{_position} = 0;
$self->{IC} = $input{IC};
$self->{e_val} = $input{e_val};
$self->{sites} = $input{sites};
$self->{width} = $input{width};
$self->{accession_number} = $input{accession_number};
$self->{_correction} = defined($input{correction}) ?
$input{correction} : 1 ; # Correction might be unwanted- supply your own
# No id provided, null for the sake of rel db
$self->{id} = defined($input{id}) ? $input{id} : 'null';
$self->{_alphabet} = \@alphabet;
#Make consensus, throw if any one of the vectors is shorter
$self = _calculate_consensus($self,$input{model});
return $self;
}
=head2 alphabet
Title : Returns an array (or array reference if desired) to the alphabet
Usage :
Function : Returns an array (or array reference) containing all of the
allowable characters for this matrix.
Throws :
Example :
Returns : Array or arrary reference.
Args :
=cut
sub alphabet {
my $self = shift;
if ( wantarray ) {
return $self->{_alphabet};
} else {
return @{$self->{_alphabet}};
}
}
=head2 _calculate_consensus
Title : _calculate_consensus
Usage :
Function : Calculates the consensus sequence for this matrix.
Throws :
Example :
Returns :
Args :
=cut
sub _calculate_consensus {
my $self = shift;
my $thresh = shift;
# verify that all of the array lengths in %probs are the same
my @lengths = map { scalar(@$_) } map {$self->{"prob$_"}} @{ $self->{_alphabet} };
my $len = shift @lengths;
for ( @lengths ) {
if ( $_ ne $len ) { $self->throw( "Probability matrix is damaged!\n" ) };
}
# iterate over probs, generate the most likely sequence and put it into
# $self->{seq}. Put the probability of this sequence into $self->{seqp}.
for ( my $i = 0; $i < $len; $i++ ) {
# get a list of all the probabilities at position $i, ordered by $self->{_alphabet}
my @probs = map { ${$self->{"prob$_"}}[$i] } @{ $self->{_alphabet} };
# calculate the consensus of @probs, put sequence into seqp and probabilities into seqp
(${$self->{seq}}[$i],${$self->{seqp}}[$i]) = $self->_to_cons( @probs, $thresh );
}
return $self;
}
=head2 next_pos
Title : next_pos
Usage :
Function : Retrieves the next position features: frequencies for all 20 amino
acids, log-odds scores for all 20 amino acids at this position,
the main (consensus) letter at this position, the probability
for the consensus letter to occur at this position and the relative
current position as an integer.
Throws :
Example :
Returns : hash (or hash reference) (pA,pR,pN,pD,...,logA,logR,logN,logD,aa,prob,rel)
- pN entries represent the probability for amino acid N
to be at this position
- logN entries represent the log-odds score for having amino acid
N at this position
- aa is the consensus amino acid
- prob is the probability for the consensus amino acid to be at this
position
- rel is the relative index of the current position (integer)
Args : none
=cut
sub next_pos {
my $self = shift;
$self->throw("instance method called on class") unless ref $self;
my $len = @{$self->{seq}};
my $pos = $self->{_position};
# return a PSM if we're still within range
if ($pos<$len) {
my %probs = map { ("p$_", ${$self->{"prob$_"}}[$pos]) } @{$self->{_alphabet}};
my %logs = map { ("l$_", ${$self->{"log$_"}}[$pos]) } @{$self->{_alphabet}};
my $base = ${$self->{seq}}[$pos];
my $prob = ${$self->{seqp}}[$pos];
$self->{_position}++;
my %hash = ( %probs, %logs, base => $base, rel => $pos, prob => $prob );
# decide whether to return the hash or a reference to it
if ( wantarray ) {
return %hash;
} else {
return \%hash;
}
} else { # otherwise, reset $self->{_position} and return nothing
$self->{_position} = 0;
return;
}
}
=head2 curpos
Title : curpos
Usage :
Function : Gets/sets the current position.
Throws :
Example :
Returns : Current position (integer).
Args : New position (integer).
=cut
sub curpos {
my $self = shift;
if (@_) { $self->{_position} = shift; }
return $self->{_position};
}
=head2 e_val
Title : e_val
Usage :
Function : Gets/sets the e-value
Throws :
Example :
Returns :
Args : real number
=cut
sub e_val {
my $self = shift;
if (@_) { $self->{e_val} = shift; }
return $self->{e_val};
}
=head2 IC
Title : IC
Usage :
Function : Position-specific information content.
Throws :
Example :
Returns : Information content for current position.
Args : Information content for current position.
=cut
sub IC {
my $self = shift;
if (@_) { $self->{IC} = shift; }
return $self->{IC};
}
=head2 accession_number
Title : accession_number
Usage :
Function: accession number, this will be unique id for the ProtMatrix object as
well for any other object, inheriting from ProtMatrix.
Throws :
Example :
Returns : New accession number (string)
Args : Accession number (string)
=cut
sub accession_number {
my $self = shift;
if (@_) { $self->{accession_number} = shift; }
return $self->{accession_number};
}
=head2 consensus
Title : consensus
Usage :
Function : Returns the consensus sequence for this PSM.
Throws : if supplied with thresold outisde 5..10 range
Example :
Returns : string
Args : (optional) threshold value 5 to 10 (corresponds to 50-100% at each position
=cut
sub consensus {
my $self = shift;
my $thresh=shift;
$self->_calculate_consensus($thresh) if ($thresh); #Change of threshold
my $consensus='';
foreach my $letter (@{$self->{seq}}) {
$consensus .= $letter;
}
return $consensus;
}
sub IUPAC {
my $self = shift;
return $self->consensus;
}
=head2 get_string
Title : get_string
Usage :
Function: Returns given probability vector as a string. Useful if you want to
store things in a rel database, where arrays are not first choice
Throws : If the argument is outside {A,C,G,T}
Example :
Returns : string
Args : character {A,C,G,T}
=cut
sub get_string {
my $self = shift;
my $base = shift;
my $string = '';
my @prob = @{$self->{"prob$base"}};
if ( ! @prob ) {
$self->throw( "No such base: $base\n");
}
foreach my $prob (@prob) {
my $corrected = $prob*10;
my $next = sprintf("%.0f",$corrected);
$next = 'a' if ($next eq '10');
$string .= $next;
}
return $string;
}
=head2 width
Title : width
Usage :
Function : Returns the length of the site
Throws :
Example :
Returns : number
Args :
=cut
sub width {
my $self = shift;
my $width = @{$self->{probA}};
return $width;
}
=head2 get_array
Title : get_array
Usage :
Function : Returns an array with frequencies for a specified amino acid.
Throws :
Example :
Returns : Array representing frequencies for specified amino acid.
Args : Single amino acid (character).
=cut
sub get_array {
my $self = shift;
my $letter = uc(shift);
$self->throw ("No such base: $letter!\n") unless grep { /$letter/ } @{$self->{_alphabet}};
return @{$self->{"prob$letter"}};
}
=head2 get_logs_array
Title : get_logs_array
Usage :
Function : Returns an array with log_odds for a specified base
Throws :
Example :
Returns : Array representing log-odds scores for specified amino acid.
Args : Single amino acid (character).
=cut
sub get_logs_array {
my $self = shift;
my $letter = uc(shift);
$self->throw ("No such base: $letter!\n") unless grep { /$letter/ } @{$self->{_alphabet}};
return @{$self->{"log$letter"}};
}
=head2 id
Title : id
Usage :
Function : Gets/sets the site id
Throws :
Example :
Returns : string
Args : string
=cut
sub id {
my $self = shift;
if (@_) { $self->{id} = shift; }
return $self->{id};
}
=head2 regexp
Title : regexp
Usage :
Function : Returns a case-insensitive regular expression which matches the
IUPAC convention. X's in consensus sequence will match anything.
Throws :
Example :
Returns : string
Args : Threshold for calculating consensus sequence (number in range 0-100
representing a percentage). Threshold defaults to 20.
=cut
sub regexp {
my $self = shift;
my $threshold = 20;
if ( @_ ) { my $threshold = shift };
my @alphabet = @{$self->{_alphabet}};
my $width = $self->width;
my (@regexp, $i);
for ( $i = 0; $i < $width; $i++ ) {
# get an array of the residues at this position with p > $threshold
my @letters = map { uc($_).lc($_) } grep { $self->{"prob$_"}->[$i] >= $threshold } @alphabet;
my $reg;
if ( scalar(@letters) == 0 ) {
$reg = '\.';
} else {
$reg = '['.join('',@letters).']';
}
push @regexp, $reg;
}
if ( wantarray ) {
return @regexp;
} else {
return join '', @regexp;
}
}
=head2 regexp_array
Title : regexp_array
Usage :
Function : Returns an array of position-specific regular expressions.
X's in consensus sequence will match anything.
Throws :
Example :
Returns : Array of position-specific regular expressions.
Args : Threshold for calculating consensus sequence (number in range 0-100
representing a percentage). Threshold defaults to 20.
Notes : Simply calls regexp method in list context.
=cut
sub regexp_array {
my $self = shift;
return @{ $self->regexp };
}
=head2 _compress_array
Title : _compress_array
Usage :
Function : Will compress an array of real signed numbers to a string (ie vector of bytes)
-127 to +127 for bi-directional(signed) and 0..255 for unsigned ;
Throws :
Example : Internal stuff
Returns : String
Args : array reference, followed by max value and direction (optional, defaults to 1),
direction of 1 is unsigned, anything else is signed.
=cut
sub _compress_array {
my ($array,$lm,$direct)=@_;
my $str;
return unless(($array) && ($lm));
$direct=1 unless ($direct);
my $k1= ($direct==1) ? (255/$lm) : (127/$lm);
foreach my $c (@{$array}) {
$c=$lm if ($c>$lm);
$c=-$lm if (($c<-$lm) && ($direct !=1));
$c=0 if (($c<0) && ($direct ==1));
my $byte=int($k1*$c);
$byte=127+$byte if ($direct !=1);#Clumsy, should be really shift the bits
my $char=chr($byte);
$str.=$char;
}
return $str;
}
=head2 _uncompress_string
Title : _uncompress_string
Usage :
Function : Will uncompress a string (vector of bytes) to create an array of real
signed numbers (opposite to_compress_array)
Throws :
Example : Internal stuff
Returns : string, followed by max value and direction (optional, defaults to 1),
direction of 1 is unsigned, anything else is signed.
Args : array
=cut
sub _uncompress_string {
my ($str,$lm,$direct)=@_;
my @array;
return unless(($str) && ($lm));
$direct=1 unless ($direct);
my $k1= ($direct==1) ? (255/$lm) : (127/$lm);
while (my $c=chop($str)) {
my $byte=ord($c);
$byte=$byte-127 if ($direct !=1);#Clumsy, should be really shift the bits
my $num=$byte/$k1;
unshift @array,$num;
}
return @array;
}
=head2 get_compressed_freq
Title : get_compressed_freq
Usage :
Function: A method to provide a compressed frequency vector. It uses one byte to
code the frequence for one of the probability vectors for one position.
Useful for relational database. Improvement of the previous 0..a coding.
Throws :
Example : my $strA=$self->get_compressed_freq('A');
Returns : String
Args : char
=cut
sub get_compressed_freq {
my $self=shift;
my $base=shift;
my $string='';
my @prob;
BASE: {
if ($base eq 'A') {
@prob = @{$self->{probA}} unless (!defined($self->{probA}));
last BASE;
}
if ($base eq 'G') {
@prob = @{$self->{probG}} unless (!defined($self->{probG}));
last BASE;
}
if ($base eq 'C') {
@prob = @{$self->{probC}} unless (!defined($self->{probC}));
last BASE;
}
if ($base eq 'T') {
@prob = @{$self->{probT}} unless (!defined($self->{probT}));
last BASE;
}
$self->throw ("No such base: $base!\n");
}
my $str= _compress_array(\@prob,1,1);
return $str;
}
=head2 sequence_match_weight
Title : sequence_match_weight
Usage :
Function : This method will calculate the score of a match, based on the PSM
if such is associated with the matrix object. Returns undef if no
PSM data is available.
Throws : if the length of the sequence is different from the matrix width
Example : my $score=$matrix->sequence_match_weight('ACGGATAG');
Returns : Floating point
Args : string
=cut
sub sequence_match_weight {
my ($self,$seq)=@_;
return unless ($self->{logA});
my $seqlen = length($seq);
my $width = $self->width;
$self->throw("Error: Input sequence size ($seqlen) not equal to PSM size ($width)!\n")
unless (length($seq) == $self->width);
my ($score,$i) = (0,0);
foreach my $letter ( split //, $seq ) {
# add up the score for this position
$score += $self->{"log$letter"}->[$i];
$i++;
}
return $score;
}
=head2 _to_IUPAC
Title : _to_IUPAC
Usage :
Function: Converts a single position to IUPAC compliant symbol and returns its probability.
Currently returns the most likely amino acid/probability combination.
Throws :
Example :
Returns : char, real number representing an amino acid and a probability.
Args : real numbers for all 20 amino acids (ordered by alphabet contained
in $self->{_alphabet}, minimum probability threshold.
=cut
sub _to_IUPAC {
my ($self,@probs,$thresh) = @_;
# provide a default threshold of 5, corresponds to 5% threshold for
# inferring that the aa at any position is the true aa
$thresh = 5 unless ( defined $thresh );
my ($IUPAC_aa,$max_prob) = ('X',$thresh);
for my $aa ( @{$self->{_alphabet}} ) {
my $prob = shift @probs;
if ( $prob > $max_prob ) {
$IUPAC_aa = $aa;
$max_prob = $prob;
}
}
return $IUPAC_aa, $max_prob;
}
=head2 _to_cons
Title : _to_cons
Usage :
Function: Converts a single position to simple consensus character and returns
its probability. Currently just calls the _to_IUPAC subroutine.
Throws :
Example :
Returns : char, real number
Args : real numbers for A,C,G,T (positional)
=cut
sub _to_cons {
return _to_IUPAC( @_ );
}
=head2 get_all_vectors
Title : get_all_vectors
Usage :
Function : returns all possible sequence vectors to satisfy the PFM under
a given threshold
Throws : If threshold outside of 0..1 (no sense to do that)
Example : my @vectors = $self->get_all_vectors(4);
Returns : Array of strings
Args : (optional) floating
=cut
#sub get_all_vectors {
# my $self = shift;
# my $thresh = shift;
#
# $self->throw("Out of range. Threshold should be >0 and 1<.\n") if (($thresh<0) || ($thresh>1));
#
# my @seq = split(//,$self->consensus($thresh*10));
# my @perm;
# for my $i (0..@{$self->{probA}}) {
# push @{$perm[$i]},'A' if ($self->{probA}->[$i]>$thresh);
# push @{$perm[$i]},'C' if ($self->{probC}->[$i]>$thresh);
# push @{$perm[$i]},'G' if ($self->{probG}->[$i]>$thresh);
# push @{$perm[$i]},'T' if ($self->{probT}->[$i]>$thresh);
# push @{$perm[$i]},'N' if ($seq[$i] eq 'N');
# }
# my $fpos=shift @perm;
# my @strings=@$fpos;
# foreach my $pos (@perm) {
# my @newstr;
# foreach my $let (@$pos) {
# foreach my $string (@strings) {
# my $newstring = $string . $let;
# push @newstr,$newstring;
# }
# }
# @strings=@newstr;
# }
# return @strings;
#}
1;