Bio/Tools/Sigcleave.pm

   1 #-----------------------------------------------------------------------------
   2 # PACKAGE : Bio::Tools::Sigcleave
   3 # AUTHOR  : Chris Dagdigian, dag@sonsorol.org
   4 # CREATED : Jan 28 1999
   5 # REVISION: $Id$
   6 #
   7 # Copyright (c) 1997-9 bioperl, Chris Dagdigian and others. All Rights Reserved.
   8 #           This module is free software; you can redistribute it and/or
   9 #           modify it under the same terms as Perl itself.
  10 #
  11 # _History_
  12 #
  13 # Object framework ripped from Steve Chervits's SeqPattern.pm
  14 #
  15 # Core EGCG Sigcleave emulation from perl code developed by
  16 # Danh Nguyen & Kamalakar Gulukota which itself was based
  17 # loosely on Colgrove's signal.c program.
  18 #
  19 # The overall idea is to replicate the output of the sigcleave
  20 # program which was distributed with the EGCG extension to the GCG sequence
  21 # analysis package. There is also an accessor method for just getting at
  22 # the raw results.
  23 #
  24 #-----------------------------------------------------------------------------
  25
  26 =head1 NAME
  27
  28 Bio::Tools::Sigcleave - Bioperl object for sigcleave analysis
  29
  30 =head1 SYNOPSIS
  31
  32 =head2 Object Creation
  33
  34   use Bio::Tools::Sigcleave ();
  35
  36   # to keep the module backwar compatible, you can pass it a sequence string, but
  37   # there recommended say is to pass it a Seq object
  38
  39   # this works
  40   $seq = "MVLLLILSVLLLKEDVRGSAQSSERRVVAHMPGDIIIGALFSVHHQPTVDKVHERKCGAVREQYGI";
  41   $sig = Bio::Tools::Sigcleave->new(-seq  => $seq,
  42                                                 -type => 'protein',
  43                                                 -threshold=>'3.5',
  44                                                 );
  45   # but you do:
  46   $seqobj = Bio::PrimarySeq->new(-seq => $seq);
  47
  48   $sig = Bio::Tools::Sigcleave->new(-seq  => $seqobj,
  49                                                 -threshold=>'3.5',
  50                                                 );
  51
  52   # now you can detect procaryotic signal sequences as well as eucaryotic
  53   $sig->matrix('eucaryotic'); # or 'procaryotic'
  54
  55
  56 =head2 Object Methods & Accessors
  57
  58   # you can use this method to fine tune the threshod before printing out the results
  59   $sig->result_count:
  60
  61   %raw_results      = $sig->signals;
  62   $formatted_output = $sig->pretty_print;
  63
  64 =head1 DESCRIPTION
  65
  66 "Sigcleave" was a program distributed as part of the free EGCG add-on
  67 to earlier versions of the GCG Sequence Analysis package. A new
  68 implementation of the algorithm is now part of EMBOSS package.
  69
  70 From the EGCG documentation:
  71
  72   SigCleave uses the von Heijne method to locate signal sequences, and
  73   to identify the cleavage site. The method is 95% accurate in
  74   resolving signal sequences from non-signal sequences with a cutoff
  75   score of 3.5, and 75-80% accurate in identifying the cleavage
  76   site. The program reports all hits above a minimum value.
  77
  78 The EGCG Sigcleave program was written by Peter Rice (E-mail:
  79 pmr@sanger.ac.uk Post: Informatics Division, The Sanger Centre,
  80 Wellcome Trust Genome Campus, Hinxton, Cambs, CB10 1SA, UK).
  81
  82 Since EGCG is no longer distributed for the latest versions of GCG,
  83 this code was developed to emulate the output of the original program
  84 as much as possible for those who lost access to sigcleave when
  85 upgrading to newer versions of GCG.
  86
  87 There are 2 accessor methods for this object. "signals" will return a
  88 perl associative array containing the sigcleave scores keyed by amino
  89 acid position.  "pretty_print" returns a formatted string similar to
  90 the output of the original sigcleave utility.
  91
  92 In both cases, the "threshold" setting controls the score reporting
  93 level. If no value for threshold is passed in by the user, the code
  94 defaults to a reporting value of 3.5.
  95
  96 In this implemntation the accessor will never return any
  97 score/position pair which does not meet the threshold limit. This is
  98 the slightly different from the behaviour of the 8.1 EGCG sigcleave
  99 program which will report the highest of the under-threshold results
 100 if nothing else is found.
 101
 102
 103 Example of pretty_print output:
 104
 105         SIGCLEAVE of sigtest from: 1 to 146
 106
 107         Report scores over 3.5
 108         Maximum score 4.9 at residue 131
 109
 110          Sequence:  FVILAAMSIQGSA-NLQTQWKSTASLALET
 111                     | (signal)    | (mature peptide)
 112                 118            131
 113
 114          Other entries above 3.5
 115
 116         Maximum score 3.7 at residue 112
 117
 118          Sequence:  CSRQLFGWLFCKV-HPGAIVFVILAAMSIQGSANLQTQWKSTASLALET
 119                    | (signal)    | (mature peptide)
 120                 99            112
 121
 122
 123 =head1 FEEDBACK
 124
 125 When updating and maintaining a module, it helps to know that people
 126 are actually using it. Let us know if you find a bug, think this code
 127 is useful or have any improvements/features to suggest.
 128
 129 =head2 Reporting Bugs
 130
 131 Report bugs to the Bioperl bug tracking system to help us keep track
 132 the bugs and their resolution. Bug reports can be submitted via the
 133 web:
 134
 135   http://bugzilla.open-bio.org/
 136
 137 =head1 AUTHOR
 138
 139 Chris Dagdigian, dag-at-sonsorol.org  & others
 140
 141 =head1 CONTRIBUTORS
 142
 143 Heikki Lehvaslaiho, heikki-at-bioperl-dot-org
 144
 145 =head1 VERSION
 146
 147 Bio::Tools::Sigcleave, $Id$
 148
 149 =head1 COPYRIGHT
 150
 151 Copyright (c) 1999 Chris Dagdigian & others. All Rights Reserved.
 152 This module is free software; you can redistribute it and/or modify it
 153 under the same terms as Perl itself.
 154
 155 =head1 REFERENCES / SEE ALSO
 156
 157 von Heijne G. (1986) "A new method for predicting signal sequences
 158 cleavage sites."  Nucleic Acids Res. 14, 4683-4690.
 159
 160 von Heijne G. (1987) in "Sequence Analysis in Molecular Biology:
 161 Treasure Trove or Trivial Pursuit" (Acad. Press, (1987), 113-117).
 162
 163
 164 =head1 APPENDIX
 165
 166 The following documentation describes the various functions
 167 contained in this module. Some functions are for internal
 168 use and are not meant to be called by the user; they are
 169 preceded by an underscore ("_").
 170
 171 =cut
 172
 173 #
 174 ##
 175 ###
 176 #### END of main POD documentation.
 177 ###
 178 ##
 179 #
 180
 181 package Bio::Tools::Sigcleave;
 182
 183 use Bio::PrimarySeq;
 184
 185 use base qw(Bio::Root::Root);
 186 use strict;
 187 use vars qw ($ID %WeightTable_euc  %WeightTable_pro );
 188 $ID  = 'Bio::Tools::Sigcleave';
 189
 190   %WeightTable_euc = (
 191 #Sample: 161 aligned sequences
 192 # R     -13 -12 -11 -10  -9  -8  -7  -6  -5  -4  -3  -2  -1  +1  +2 Expect
 193  'A' => [16, 13, 14, 15, 20, 18, 18, 17, 25, 15, 47,  6, 80, 18,  6, 14.5],
 194  'C' => [ 3,  6,  9,  7,  9, 14,  6,  8,  5,  6, 19,  3,  9,  8,  3,  4.5],
 195  'D' => [ 0,  0,  0,  0,  0,  0,  0,  0,  5,  3,  0,  5,  0, 10, 11,  8.9],
 196  'E' => [ 0,  0,  0,  1,  0,  0,  0,  0,  3,  7,  0,  7,  0, 13, 14, 10.0],
 197  'F' => [13,  9, 11, 11,  6,  7, 18, 13,  4,  5,  0, 13,  0,  6,  4,  5.6],
 198  'G' => [ 4,  4,  3,  6,  3, 13,  3,  2, 19, 34,  5,  7, 39, 10,  7, 12.1],
 199  'H' => [ 0,  0,  0,  0,  0,  1,  1,  0,  5,  0,  0,  6,  0,  4,  2,  3.4],
 200  'I' => [15, 15,  8,  6, 11,  5,  4,  8,  5,  1, 10,  5,  0,  8,  7,  7.4],
 201  'K' => [ 0,  0,  0,  1,  0,  0,  1,  0,  0,  4,  0,  2,  0, 11,  9, 11.3],
 202  'L' => [71, 68, 72, 79, 78, 45, 64, 49, 10, 23,  8, 20,  1,  8,  4, 12.1],
 203  'M' => [ 0,  3,  7,  4,  1,  6,  2,  2,  0,  0,  0,  1,  0,  1,  2,  2.7],
 204  'N' => [ 0,  1,  0,  1,  1,  0,  0,  0,  3,  3,  0, 10,  0,  4,  7,  7.1],
 205  'P' => [ 2,  0,  2,  0,  0,  4,  1,  8, 20, 14,  0,  1,  3,  0, 22,  7.4],
 206  'Q' => [ 0,  0,  0,  1,  0,  6,  1,  0, 10,  8,  0, 18,  3, 19, 10,  6.3],
 207  'R' => [ 2,  0,  0,  0,  0,  1,  0,  0,  7,  4,  0, 15,  0, 12,  9,  7.6],
 208  'S' => [ 9,  3,  8,  6, 13, 10, 15, 16, 26, 11, 23, 17, 20, 15, 10, 11.4],
 209  'T' => [ 2, 10,  5,  4,  5, 13,  7,  7, 12,  6, 17,  8,  6,  3, 10,  9.7],
 210  'V' => [20, 25, 15, 18, 13, 15, 11, 27,  0, 12, 32,  3,  0,  8, 17, 11.1],
 211  'W' => [ 4,  3,  3,  1,  1,  2,  6,  3,  1,  3,  0,  9,  0,  2,  0,  1.8],
 212  'Y' => [ 0,  1,  4,  0,  0,  1,  3,  1,  1,  2,  0,  5,  0,  1,  7,  5.6]
 213 );
 214
 215   %WeightTable_pro = (
 216 #Sample: 36 aligned sequences
 217 #  R    -13 -12 -11 -10  -9  -8  -7  -6  -5  -4  -3  -2  -1  +1  +2 Expect
 218   'A' => [0,  8,  8,  9,  6,  7,  5,  6,  7,  7, 24,  2, 31, 18,  4,  3.2],
 219   'C' => [1,  0,  0,  1,  1,  0,  0,  1,  1,  0,  0,  0,  0,  0,  0,  1.0],
 220   'D' => [0,  0,  0,  0,  0,  0,  0,  0,  0,  0,  0,  0,  0,  2,  8,  2.0],
 221   'E' => [0,  0,  0,  0,  0,  0,  0,  0,  0,  0,  0,  1,  0,  4,  8,  2.2],
 222   'F' => [2,  4,  3,  4,  1,  1,  8,  0,  4,  1,  0,  7,  0,  1,  0,  1.3],
 223   'G' => [4,  2,  2,  2,  3,  5,  2,  4,  2,  2,  0,  2,  2,  1,  0,  2.7],
 224   'H' => [0,  0,  1,  0,  0,  0,  0,  1,  1,  0,  0,  7,  0,  1,  0,  0.8],
 225   'I' => [3,  1,  5,  1,  5,  0,  1,  3,  0,  0,  0,  0,  0,  0,  2,  1.7],
 226   'K' => [0,  0,  0,  0,  0,  0,  0,  0,  0,  1,  0,  2,  0,  3,  0,  2.5],
 227   'L' => [8, 11,  9,  8,  9, 13,  1,  0,  2,  2,  1,  2,  0,  0,  1,  2.7],
 228   'M' => [0,  2,  1,  1,  3,  2,  3,  0,  1,  2,  0,  4,  0,  0,  1,  0.6],
 229   'N' => [0,  0,  0,  0,  0,  0,  0,  1,  1,  1,  0,  3,  0,  1,  4,  1.6],
 230   'P' => [0,  1,  1,  1,  1,  1,  2,  3,  5,  2,  0,  0,  0,  0,  5,  1.7],
 231   'Q' => [0,  0,  0,  0,  0,  0,  0,  0,  2,  2,  0,  3,  0,  0,  1,  1.4],
 232   'R' => [0,  0,  0,  0,  0,  0,  0,  0,  0,  0,  0,  0,  0,  1,  0,  1.7],
 233   'S' => [1,  0,  1,  4,  4,  1,  5, 15,  5,  8,  5,  2,  2,  0,  0,  2.6],
 234   'T' => [2,  0,  4,  2,  2,  2,  2,  2,  5,  1,  3,  0,  1,  1,  2,  2.2],
 235   'V' => [5,  7,  1,  3,  1,  4,  7,  0,  0,  4,  3,  0,  0,  2,  0,  2.5],
 236   'W' => [0,  0,  0,  0,  0,  0,  0,  0,  0,  0,  0,  0,  0,  1,  0,  0.4],
 237   'Y' => [0,  0,  0,  0,  0,  0,  0,  0,  0,  3,  0,  1,  0,  0,  0,  1.3]
 238 );
 239
 240
 241 ##
 242 ## Now we calculate the _real_ values for the weight tables
 243 ##
 244 ##
 245 ## yeah yeah yeah there is lots of math here that gets repeated
 246 ## every single time a sigcleave object gets created. This is
 247 ## a quick hack to make sure that we get the scores as accurate as
 248 ## possible. Need all those significant digits....
 249 ##
 250 ## suggestions for speedup aproaches welcome
 251 ##
 252
 253
 254 foreach my $i (keys %WeightTable_euc) {
 255         my $expected = $WeightTable_euc{$i}[15];
 256         if ($expected > 0) {
 257                 for (my $j=0; $j<16; $j++) {
 258                         if ($WeightTable_euc{$i}[$j] == 0) {
 259                                 $WeightTable_euc{$i}[$j] = 1;
 260                                 if ($j == 10 || $j == 12) {
 261                                         $WeightTable_euc{$i}[$j] = 1.e-10;
 262                                 }
 263                         }
 264                         $WeightTable_euc{$i}[$j] = log($WeightTable_euc{$i}[$j]/$expected);
 265                 }
 266         }
 267 }
 268
 269
 270 foreach my $i (keys %WeightTable_pro) {
 271         my $expected = $WeightTable_pro{$i}[15];
 272         if ($expected > 0) {
 273                 for (my $j=0; $j<16; $j++) {
 274                         if ($WeightTable_pro{$i}[$j] == 0) {
 275                                 $WeightTable_pro{$i}[$j] = 1;
 276                                 if ($j == 10 || $j == 12) {
 277                                         $WeightTable_pro{$i}[$j] = 1.e-10;
 278                                 }
 279                         }
 280                         $WeightTable_pro{$i}[$j] = log($WeightTable_pro{$i}[$j]/$expected);
 281                 }
 282         }
 283 }
 284
 285 #####################################################################################
 286 ##                                 CONSTRUCTOR                                     ##
 287 #####################################################################################
 288
 289
 290 sub new {
 291     my ($class, @args) = @_;
 292
 293     my $self = $class->SUPER::new(@args);
 294     #my $self = Bio::Seq->new(@args);
 295
 296     my ($seq, $threshold, $matrix) = $self->_rearrange([qw(SEQ THRESHOLD MATRIX)],@args);
 297
 298     defined $threshold && $self->threshold($threshold);
 299     $matrix && $self->matrix($matrix);
 300     $seq && $self->seq($seq);
 301
 302     return $self;
 303 }
 304
 305
 306
 307 =head1 threshold
 308
 309  Title     : threshold
 310  Usage     : $value = $self->threshold
 311  Purpose   : Read/write method sigcleave score reporting threshold.
 312  Returns   : float.
 313  Argument  : new value, float
 314  Throws    : on non-number argument
 315  Comments  : defaults to 3.5
 316  See Also   : n/a
 317
 318 =cut
 319
 320 #----------------
 321 sub threshold {
 322 #----------------
 323         my ($self, $value) = @_;
 324         if( defined $value) {
 325                 $self->throw("I need a number, not [$value]")
 326                   if  $value !~ /^[+-]?[\d\.]+$/;
 327                 $self->{'_threshold'} = $value;
 328         }
 329         return $self->{'_threshold'} || 3.5 ;
 330 }
 331
 332 =head1 matrix
 333
 334  Title     : matrix
 335  Usage     : $value = $self->matrix('procaryotic')
 336  Purpose   : Read/write method sigcleave matrix.
 337  Returns   : float.
 338  Argument  : new value: 'eucaryotic' or 'procaryotic'
 339  Throws    : on non-number argument
 340  Comments  : defaults to 3.5
 341  See Also   : n/a
 342
 343 =cut
 344
 345 #----------------
 346 sub matrix {
 347 #----------------
 348         my ($self, $value) = @_;
 349         if( defined $value) {
 350                 $self->throw("I need 'eucaryotic' or 'procaryotic', not [$value]")
 351                   unless  $value eq 'eucaryotic' or $value eq 'procaryotic';
 352                 $self->{'_matrix'} = $value;
 353         }
 354         return $self->{'_matrix'} || 'eucaryotic' ;
 355 }
 356
 357 =head1 seq
 358
 359  Title     : seq
 360  Usage     : $value = $self->seq($seq_object)
 361  Purpose   : set the Seq object to be used
 362  Returns   : Seq object
 363  Argument  : protein sequence or Seq object
 364  See Also   : n/a
 365
 366 =cut
 367
 368 #----------------
 369 sub seq {
 370 #----------------
 371         my ($self, $value) = @_;
 372         if( defined $value) {
 373                 if ($value->isa('Bio::PrimarySeqI')) {
 374                         $self->{'_seq'} = $value;
 375                 } else {
 376                         $self->{'_seq'} = Bio::PrimarySeq->new(-seq => $value,
 377                                                                                                                                 -alphabet => 'protein');
 378                 }
 379         }
 380         return $self->{'_seq'};
 381 }
 382
 383 =head1 _Analyze
 384
 385  Title     : _Analyze
 386  Usage     : N/A This is an internal method. Not meant to be called from outside
 387            : the package
 388            :
 389  Purpose   : calculates sigcleave score and amino acid position for the
 390            : given protein sequence. The score reporting threshold can
 391            : be adjusted by passing in the "threshold" parameter during
 392            : object construction. If no threshold is passed in, the code
 393            : defaults to reporting any scores equal to or above 3.5
 394            :
 395  Returns   : nothing. results are added to the object
 396  Argument  : none.
 397  Throws    : nothing.
 398  Comments  : nothing.
 399 See Also   : n/a
 400
 401 =cut
 402
 403 #----------------
 404 sub _Analyze {
 405 #----------------
 406     my($self) = @_;
 407
 408     my %signals;
 409     my @hitWeight = ();
 410     my @hitsort   = ();
 411     my @hitpos    = ();
 412     my $maxSite   = "";
 413     my $seqPos    = "";
 414     my $istart    = "";
 415     my $iend      = "";
 416     my $icol      = "";
 417     my $i         = "";
 418     my $weight    = "";
 419     my $k         = 0;
 420     my $c         = 0;
 421     my $seqBegin  = 0;
 422     my $pVal      = -13;
 423     my $nVal      = 2;
 424     my $nHits     = 0;
 425     my $seqEnd    = $self->seq->length;
 426     my $pep       = $self->seq->seq;
 427     my $minWeight = $self->threshold;
 428     my $matrix    = $self->matrix;
 429
 430     ## The weight table is keyed by UPPERCASE letters so we uppercase
 431     ## the pep string because we don't want to alter the actual object
 432     ## sequence.
 433
 434     $pep =~ tr/a-z/A-Z/;
 435
 436     for ($seqPos = $seqBegin; $seqPos < $seqEnd; $seqPos++) {
 437                  $istart = (0 > $seqPos + $pVal)? 0 : $seqPos + $pVal;
 438                  $iend = ($seqPos + $nVal - 1 < $seqEnd)? $seqPos + $nVal - 1 : $seqEnd;
 439                  $icol= $iend - $istart + 1;
 440                  $weight = 0.00;
 441                  for ($k=0; $k<$icol; $k++) {
 442                          $c = substr($pep, $istart + $k, 1);
 443
 444                          ## CD: The if(defined) stuff was put in here because Sigcleave.pm
 445                          ## CD: kept getting warnings about undefined vals during 'make test' ...
 446                          if ($matrix eq 'eucaryotic') {
 447                                  $weight += $WeightTable_euc{$c}[$k] if defined $WeightTable_euc{$c}[$k];
 448                          } else {
 449                                  $weight += $WeightTable_pro{$c}[$k] if defined $WeightTable_pro{$c}[$k];
 450                          }
 451                  }
 452                  $signals{$seqPos+1} = sprintf ("%.1f", $weight)        if $weight >= $minWeight;
 453     }
 454     $self->{"_signal_scores"} = { %signals };
 455 }
 456
 457
 458 =head1 signals
 459
 460  Title     : signals
 461  Usage     : %sigcleave_results = $sig->signals;
 462            :
 463  Purpose   : Accessor method for sigcleave results
 464            :
 465  Returns   : Associative array. The key value represents the amino acid position
 466            : and the value represents the score. Only scores that
 467            : are greater than or equal to the THRESHOLD value are reported.
 468            :
 469  Argument  : none.
 470  Throws    : none.
 471  Comments  : none.
 472 See Also   : THRESHOLD
 473
 474 =cut
 475
 476 #----------------
 477 sub signals {
 478 #----------------
 479         my $self = shift;
 480         my %results;
 481         my $position;
 482
 483         # do the calculations
 484         $self->_Analyze;
 485
 486         foreach $position ( sort keys %{ $self->{'_signal_scores'} } ) {
 487                 $results{$position} = $self->{'_signal_scores'}{$position};
 488         }
 489         return %results;
 490 }
 491
 492
 493 =head1 result_count
 494
 495  Title     : result_count
 496  Usage     : $count = $sig->result_count;
 497            :
 498  Purpose   : Accessor method for sigcleave results
 499            :
 500  Returns   : Integer, number of results above the threshold
 501            :
 502  Argument  : none.
 503  Throws    : none.
 504  Comments  : none.
 505
 506 See Also   : THRESHOLD
 507
 508 =cut
 509
 510 #----------------
 511 sub result_count {
 512 #----------------
 513         my $self = shift;
 514         $self->_Analyze;
 515         return keys %{ $self->{'_signal_scores'} };
 516 }
 517
 518
 519 =head1 pretty_print
 520
 521  Title     : pretty_print
 522  Usage     : $output = $sig->pretty_print;
 523            : print $sig->pretty_print;
 524            :
 525  Purpose   : Emulates the output of the EGCG Sigcleave
 526            : utility.
 527            :
 528  Returns   : A formatted string.
 529  Argument  : none.
 530  Throws    : none.
 531  Comments  : none.
 532 See Also   : n/a
 533
 534 =cut
 535
 536 #----------------
 537 sub pretty_print {
 538 #----------------
 539     my $self = shift;
 540     my $pos;
 541     my $output;
 542     my $cnt = 1;
 543     my %results  = $self->signals;
 544     my @hits     = keys %results;
 545     my $hitcount = $#hits; $hitcount++;
 546     my $thresh   = $self->threshold;
 547     my $seqlen   = $self->seq->length || 0;
 548     my $name     = $self->seq->id || 'NONAME';
 549     my $pep      = $self->seq->seq;
 550     $pep      =~ tr/a-z/A-Z/;
 551
 552     $output = "SIGCLEAVE of $name from: 1 to $seqlen\n\n";
 553
 554     if ($hitcount > 0) {
 555                  $output .= "Report scores over $thresh\n";
 556                  foreach $pos ((sort { $results{$b} cmp $results{$a} } keys %results)) {
 557                          my $start = $pos - 15;
 558                          $start = 1 if $start < 1;
 559                          my $sig = substr($pep,$start -1,$pos-$start );
 560
 561                          $output .= sprintf ("Maximum score %1.1f at residue %3d\n",$results{$pos},$pos);
 562                          $output .= "\n";
 563                          $output .= " Sequence:  ";
 564                          $output .= $sig;
 565                          $output .= "-" x (15- length($sig));
 566                          $output .= "-";
 567                          $output .= substr($pep,$pos-1,50);
 568                          $output .= "\n";
 569                          $output .= " " x 12;
 570                          $output .= "| \(signal\)      | \(mature peptide\)\n";
 571                          $output .= sprintf("          %3d             %3d\n\n",$start,$pos);
 572
 573                          if (($hitcount > 1) && ($cnt == 1)) {
 574                                  $output .= " Other entries above $thresh\n\n";
 575                          }
 576                          $cnt++;
 577                  }
 578     }
 579     $output;
 580 }
 581
 582
 583 1;
 584 __END__
 585
 586
 587 #########################################################################
 588 #  End of class
 589 #########################################################################