Bio/Tools/SiRNA.pm

   1 #
   2 # BioPerl module for Bio::Tools::SiRNA
   3 #
   4 # Please direct questions and support issues to <bioperl-l@bioperl.org>
   5 #
   6 # Cared for by Donald Jackson, donald.jackson@bms.com
   7 #
   8 # Copyright Bristol-Myers Squibb
   9 #
  10 # You may distribute this module under the same terms as perl itself
  11
  12 # POD documentation - main docs before the code
  13
  14 =head1 NAME
  15
  16 SiRNA - Perl object for designing small inhibitory RNAs.
  17
  18 =head1 SYNOPSIS
  19
  20   use Bio::Tools::SiRNA;
  21
  22   my $sirna_designer = Bio::Tools::SiRNA->new( -target => $bio_seq,
  23                                                -rules  => 'saigo'
  24     );
  25   my @pairs = $sirna_designer->design;
  26
  27   foreach $pair (@pairs) {
  28       my $sense_oligo_sequence = $pair->sense->seq;
  29       my $antisense_oligo_sequence = $pair->antisense->seq;
  30
  31       # print out results
  32       print join ("\t", $pair->start, $pair->end, $pair->rank,
  33                   $sense_oligo_sequence, $antisense_oligo_sequence), "\n";
  34   }
  35
  36 =head1 DESCRIPTION
  37
  38 Package for designing siRNA reagents.
  39
  40 Input is a L<Bio::SeqI>-compliant object (the target).
  41
  42 Output is a list of Bio::SeqFeature::SiRNA::Pair objects, which are
  43 added to the feature table of the target sequence.  Each
  44 Bio::SeqFeature::SiRNA::Pair contains two subfeatures
  45 (Bio::SeqFeature::Oligo objects) which correspond to the individual
  46 oligos.  These objects provide accessors for the information on the
  47 individual reagent pairs.
  48
  49 This version of Bio::Tools::SiRNA represents a major change in architecture.
  50 Specific 'rulesets' for siRNA selection as developed by various groups are
  51 implemented as Bio::Tools::SiRNA::Ruleset objects, which inherit from
  52 Bio::Tools::SiRNA.  This will make it easier to add new rule sets or modify
  53 existing approaches. Currently the Tuschl and Ui-Tei (2004) rules are
  54 implemented. For consistency, the Tuschl rules are implemented by default.
  55
  56 In addition, this module provides three 'extra' rules which can be added
  57 above and beyond any ruleset.
  58
  59 =over 3
  60
  61 =item 1.
  62
  63 SiRNAs that overlap known SNPs (identified as SeqFeatures with
  64 primary tag = variation) can be avoided.
  65
  66 =item 2.
  67
  68 Other regions (with primary tag = 'Excluded') can also be skipped.  I
  69 use this with Bio::Tools::Run::Mdust to avoid low-complexity regions
  70 (must be run separately), but other programs could also be used.
  71
  72 =item 3.
  73
  74 SiRNAs may also be selected in the 3 prime UTR of a gene by setting
  75 $sirna_designer-E<gt>include_3pr() to true.
  76
  77 =back
  78
  79 =head2 EXPORT
  80
  81 None.
  82
  83 =head1 SEE ALSO
  84
  85 L<Bio::Tools::Run::Mdust>, L<Bio::SeqFeature::SiRNA::Pair>,
  86 L<Bio::SeqFeature::SiRNA::Oligo>..
  87
  88 =head1 FEEDBACK
  89
  90 =head2 Mailing Lists
  91
  92 User feedback is an integral part of the evolution of this and other
  93 Bioperl modules. Send your comments and suggestions preferably to
  94 the Bioperl mailing list.  Your participation is much appreciated.
  95
  96   bioperl-l@bioperl.org                  - General discussion
  97   http://bioperl.org/wiki/Mailing_lists  - About the mailing lists
  98
  99 =head2 Support
 100
 101 Please direct usage questions or support issues to the mailing list:
 102
 103 I<bioperl-l@bioperl.org>
 104
 105 rather than to the module maintainer directly. Many experienced and
 106 reponsive experts will be able look at the problem and quickly
 107 address it. Please include a thorough description of the problem
 108 with code and data examples if at all possible.
 109
 110 =head2 Reporting Bugs
 111
 112 Report bugs to the Bioperl bug tracking system to help us keep track
 113 of the bugs and their resolution. Bug reports can be submitted via
 114 the web:
 115
 116   https://github.com/bioperl/bioperl-live/issues
 117
 118 =head1 AUTHOR
 119
 120 Donald Jackson (donald.jackson@bms.com)
 121
 122 =head1 APPENDIX
 123
 124 The rest of the documentation details each of the object methods.
 125 Internal methods are usually preceded with a _
 126
 127 =cut
 128
 129 package Bio::Tools::SiRNA;
 130
 131 use strict;
 132 use warnings;
 133
 134 use vars qw($AUTOLOAD);
 135
 136 use Bio::Seq::RichSeq;
 137 use Bio::SeqFeature::Generic;
 138 use Bio::SeqFeature::SiRNA::Oligo;
 139 use Bio::SeqFeature::SiRNA::Pair;
 140
 141
 142 use base qw(Bio::Root::Root);
 143
 144
 145 our %COMP = ( A => 'T',
 146               T => 'A',
 147               C => 'G',
 148               G => 'C',
 149               N => 'N',
 150               );
 151
 152 our @ARGNAMES = qw(RULES START_PAD END_PAD MIN_GC CUTOFF OLIGOS AVOID_SNPS
 153                    GSTRING TMPDIR TARGET DEBUG);
 154
 155
 156 =head2 new
 157
 158  Title          : new
 159  Usage          : my $sirna_designer = Bio::Tools::SiRNA->new();
 160  Function       : Constructor for designer object
 161  Returns        : Bio::Tools::SiRNA object
 162  Args           : target - the target sequence for the SiRNAs as a Bio::Seq::RichSeq
 163                   start_pad - distance from the CDS start to skip (default 75)
 164                   end_pad - distance from the CDS end to skip (default 50)
 165                   include_3pr - set to true to include SiRNAs in the 3prime UTR (default false)
 166                   rules - rules for selecting siRNAs, currently supporting saigo and tuschl
 167                   min_gc - minimum GC fraction (NOT percent) (default 0.4)
 168                   max_gc - maximum GC fraction (NOT percent) (default 0.6)
 169                   cutoff - worst 'rank' accepted(default 3)
 170                   avoid_snps - boolean - reject oligos that overlap a variation
 171                      SeqFeature in the target (default true)
 172                   gstring - maximum allowed consecutive Gs.
 173                      Too many can cause problems in synthesis (default 4)
 174   Note          : All arguments can also be changed/accessed using autoloaded
 175                  methods such as:
 176
 177     my $start_pad = $sirna_designer->start_pad().
 178
 179 =cut
 180
 181 sub new {
 182     my ($proto, @args) = @_;
 183     my $pkg = ref($proto) || $proto;
 184
 185     my $self = {};
 186     bless ($self, $pkg);
 187
 188     my %args;
 189
 190     @args{@ARGNAMES} = $self->_rearrange(\@ARGNAMES, @args);
 191
 192     if ($args{'RULES'}) {
 193         $self->rules($args{'RULES'});
 194     }
 195
 196     $self->{'start_pad'} = $args{'START_PAD'} || 75; # nt from start to mask
 197     $self->{'end_pad'} = $args{'END_PAD'} || 50; # nt from end to mask
 198     $self->{'include_3pr'} = $args{'INCLUDE_3PR'} || 0; # look for oligos in 3prime UTR
 199     $self->{'min_gc'} = $args{'MIN_GC'} || 0.40;
 200     $self->{'max_gc'} = $args{'MAX_GC'} || 0.60;
 201     $self->{'cutoff'} = $args{'CUTOFF'} || 3; # highest (worst) rank wanted
 202     $self->{'oligos'} = [];
 203     defined($args{'AVOID_SNPS'}) ? $self->{'avoid_snps'} = $args{'AVOID_SNPS'} :
 204         $self->{'avoid_snps'} = 1; # (t/f to avoid or include reagents that cover SNPs)
 205     $self->{'gstring'} = $args{'GSTRING'} || 4; # maximum allowed consecutive Gs - too many can cause problems in oligo synthesis
 206     $self->{'tmpdir'} = $args{'TMPDIR'}  || $ENV{'TMPDIR'} || $ENV{'TMP'} || '';
 207     $self->{'debug'} = $args{'DEBUG'} || 0;
 208
 209     $self->target($args{'TARGET'}) if ($args{'TARGET'});
 210
 211     return $self;
 212 }
 213
 214
 215 =head2 target
 216
 217   Title         : target
 218   Usage         : my $target_seq = $sirna_designer->target(); # get the current target
 219                   OR
 220                   $sirna_designer->target($new_target_seq); # set a new target
 221   Function      : Set/get the target as a Bio::SeqI-compliant object
 222   Returns       : a Bio::SeqI-compliant object
 223   Args          : a Bio::SeqI-compliant object (optional)
 224
 225 =cut
 226
 227 sub target {
 228     my ($self, $target) = @_;
 229
 230     if ($target) {
 231         unless ($target->isa('Bio::SeqI')) {
 232             $self->throw(  -class => 'Bio::Root::BadParameter',
 233                            -text  => "Target must be passed as a Bio::Seq object" );
 234         }
 235         if ($target->can('molecule')) {
 236             ( grep { uc($target->molecule) eq $_ } qw(DNA MRNA CDNA)) or
 237                 $self->throw(  -class => 'Bio::Root::BadParameter',
 238                                -text  =>  "Sequences of type ". $target->molecule. " are not supported"
 239                                );
 240         }
 241         else {
 242             ($target->alphabet eq 'dna') or
 243                 $self->throw(  -class => 'Bio::Root::BadParameter',
 244                                -text  =>  "Sequences of alphabet ". $target->alphabet. " are not supported"
 245                                );
 246         }
 247
 248         $self->{'target'} = $target;
 249         return 1;
 250
 251     }
 252     elsif ($self->{'target'}) {
 253         return $self->{'target'};
 254     }
 255     else {
 256         $self->throw("Target sequence not defined");
 257     }
 258 }
 259
 260 =head2 rules
 261
 262     Title       : rules
 263     Usage       : $sirna->rules('ruleset')
 264     Purpose     : set/get ruleset to use for selecting SiRNA oligo pairs.
 265     Returns     : not sure yet
 266     Args        : a ruleset name (currently supported: Tuschl, Saigo)
 267                   or a Bio::Tools::SiRNA::RulesetI compliant object
 268
 269 =cut
 270
 271 sub rules {
 272     my ($self, $rules) = @_;
 273
 274     if ($rules) {
 275         $self->_load_ruleset($rules);
 276     }
 277     # default: use tuschl rules
 278     unless ($self->{_rules}) {
 279         $self->_load_ruleset('tuschl');
 280     }
 281     return $self->{_rules};
 282 }
 283
 284 sub _load_ruleset {
 285     my ($self, $ruleset) = @_;
 286
 287     my $rule_module = join('::', ref($self), 'Ruleset', lc($ruleset));
 288
 289     eval "require $rule_module";
 290
 291     if ($@) {
 292         #warn join("\n", '@INC contains:', @INC, undef);
 293         $self->throw("Unable to load $rule_module: $@");
 294         return;
 295     }
 296
 297     else {
 298         $self->{_rules} = $rule_module;
 299         bless($self, $rule_module); # recast as subclass
 300     }
 301
 302     return 1;
 303 }
 304
 305 =head2 design
 306
 307   Title         : design
 308   Usage         : my @pairs = $sirna_designer->design();
 309   Purpose       : Design SiRNA oligo pairs.
 310   Returns       : A list of SiRNA pairs as Bio::SeqFeature::SiRNA::Pair objects
 311   Args          : none
 312
 313 =cut
 314
 315 sub design {
 316     my ($self) = @_;
 317
 318     ($self->rules) or $self->throw('Unable to design siRNAs: no rule set specified');
 319
 320 #     unless ( grep { $_->primary_tag eq 'Target' } $self->target->top_SeqFeatures ) {
 321 #       $self->_define_target();
 322 #     }
 323
 324     my @oligos = $self->_get_oligos();
 325
 326     return ( grep { $_->isa('Bio::SeqFeature::SiRNA::Pair') } $self->target->top_SeqFeatures );
 327 }
 328
 329 sub _define_target {
 330     my ($self) = @_;
 331     my ($feat, $cds, $left, $right);
 332
 333     my $target = $self->target or
 334         $self->throw("Unable to design oligos - no target provided");
 335
 336     ($cds) = grep { $_->primary_tag eq 'CDS' } $target->top_SeqFeatures if ($target->can('top_SeqFeatures'));
 337
 338     if ($cds) {
 339         $left = $cds->start + $self->start_pad;
 340         if (!$self->include_3pr) {
 341             $right = $cds->end - $self->end_pad;
 342         }
 343         else {
 344             $right = $target->length - $self->end_pad;
 345         }
 346     }
 347     else {
 348         $left = 0 + $self->start_pad;
 349         $right = $target->length - $self->end_pad;
 350     }
 351
 352
 353     # is there anything left?
 354     if (($right - $left) < 20) {
 355         $self->throw("There isn't enough sequence to design oligos.  Please reduce start_pad and end_pad or supply more sequence");
 356     }
 357     # define target region
 358     my $targregion = Bio::SeqFeature::Generic->new( -start              => $left,
 359                                                     -end                => $right,
 360                                                     -primary            => 'Target' );
 361     $self->target->add_SeqFeature($targregion);
 362
 363     # locate excluded regions
 364     my @excluded = grep { $_->primary_tag eq 'Excluded' } $self->target->top_SeqFeatures;
 365
 366     if ($self->avoid_snps) {
 367         my @snps =  grep { $_->primary_tag eq 'variation' } $self->target->top_SeqFeatures;
 368         push(@excluded, @snps);
 369     }
 370
 371     $self->excluded(\@excluded);
 372
 373     return $targregion;
 374 }
 375
 376 sub _get_targetregion {
 377     my ($self) = @_;
 378
 379     my ($targregion) = grep { $_->primary_tag eq 'Target' } $self->target->top_SeqFeatures;
 380     $targregion ||= $self->_define_target;
 381
 382     $self->throw("Target region for SiRNA design not defined") unless ($targregion);
 383
 384     my $seq = $targregion->seq->seq;
 385     # but this way I loose start info
 386      my $targstart = $targregion->start;
 387
 388     return ($seq, $targstart);
 389 }
 390
 391 # MOVE to SiRNA::Ruleset::tuschl
 392 # sub _regex {
 393 #     my ($self, $rank) = @_;
 394 #     return $PATTERNS{$rank};
 395 # }
 396
 397 # sub _get_oligos {
 398 #     # use regular expressions to pull out oligos
 399
 400 #     my ($self, $rank) = @_;
 401 #     my $regex = $self->_regex($rank);
 402 #     my @exclude;
 403
 404
 405 #     my ($targregion) = grep { $_->primary_tag eq 'Target' } $self->target->top_SeqFeatures;
 406 #     my $seq = $targregion->seq->seq;
 407 #     # but this way I loose start info
 408 #     my $targstart = $targregion->start;
 409
 410 #     # exclude masked region
 411 #     push(@exclude, grep { $_->primary_tag eq 'Excluded' } $self->target->top_SeqFeatures);
 412
 413 #     # add SNP checking
 414 #     if ($self->avoid_snps) {
 415 #       my @snps =  grep { $_->primary_tag eq 'variation' } $self->target->top_SeqFeatures;
 416 #       push(@exclude, @snps);
 417 #     }
 418
 419 #     while ( $seq =~ /$regex/gi ) {
 420 #       my $target = $1;
 421
 422 #       # check for too many Gs (or Cs on the other strand)
 423 #       next if ( $target =~ /G{ $self->gstring,}/io );
 424 #       next if ( $target =~ /C{ $self->gstring,}/io );
 425 #       # skip Ns (for filtering)
 426 #       next if ( $target =~ /N/i);
 427
 428 #       my $start = length($`) + $targstart;
 429 #       my $stop = $start + length($target) -1;
 430
 431 #       my @gc = ( $target =~ /G|C/gi);
 432 #       my $fxGC = sprintf("%2.2f", (scalar(@gc) / length($target)));
 433 #       next if ($fxGC < $self->min_gc);
 434 #       next if ($fxGC > $self->max_gc);
 435
 436 #       my $sense = Bio::SeqFeature::SiRNA::Oligo->new( -start          => $start,
 437 #                                                       -end            => $stop,
 438 #                                                       -strand         => 1,
 439 #                                                       -seq            => _get_sense($target),
 440 #                                                       -source_tag     => ref($self),
 441 #                                                      );
 442
 443 #       my $asense = Bio::SeqFeature::SiRNA::Oligo->new( -start         => $start,
 444 #                                                        -end           => $stop,
 445 #                                                        -strand        => -1,
 446 #                                                        -seq           => _get_anti($target),
 447 #                                                        -source_tag    => ref($self),
 448 #                                                        );
 449
 450 #       my $sirna = Bio::SeqFeature::SiRNA::Pair->new( -rank            => $rank,
 451 #                                                      -fxGC            => $fxGC,
 452 #                                                      -sense           => $sense,
 453 #                                                      -antisense       => $asense,
 454 #                                                      -source_tag      => ref($self),
 455 #                                                      );
 456
 457 #       unless ($self->_has_overlap($sirna, \@exclude)) {
 458 #           $self->target->add_SeqFeature($sirna);
 459 #       }
 460 #     }
 461 # }
 462
 463 =head2 add_oligos
 464
 465   Title         : add_oligos
 466   Usage         : $sirna_designer->add_oligos($sequence, $start, $rank);
 467   Purpose       : Add SiRNA olgos to target Bio::Seq as Bio::SeqFeature::SiRNA::Pair objects
 468   Args          : Oligo sequence and start position (required), rank/score (optional)
 469
 470 =cut
 471
 472 sub add_oligos {
 473     my ($self, $seq, $start, $rank) = @_;
 474
 475     ($seq) or throw ('No sequence supplied for add_oligos');
 476     (defined $start) or throw ('No start position specified for  add_oligos');
 477
 478     my ($end) = $start + length($seq);
 479
 480     my ($sseq) = $self->_get_sense($seq);
 481     my $sense = Bio::SeqFeature::SiRNA::Oligo->new( -start              => $start,
 482                                                     -end                => ($start + length($sseq)),
 483                                                     -strand     => 1,
 484                                                     -seq                => $sseq,
 485                                                     -source_tag => ref($self),
 486                                                     );
 487
 488     my $aseq = $self->_get_anti($seq);
 489     my $asense = Bio::SeqFeature::SiRNA::Oligo->new( -start             => $end,
 490                                                      -end               => ($end - length($aseq)),
 491                                                      -strand            => -1,
 492                                                      -seq               => $aseq,
 493                                                      -source_tag        => ref($self),
 494                                                      );
 495
 496     my $sirna = Bio::SeqFeature::SiRNA::Pair->new( -rank                => $rank,
 497                                                   # -fxGC               => $fxGC,
 498                                                    -sense               => $sense,
 499                                                    -antisense   => $asense,
 500                                                    -source_tag  => ref($self),
 501                                                    );
 502
 503     unless ($self->_has_overlap($sirna, $self->excluded)) {
 504         $self->target->add_SeqFeature($sirna);
 505     }
 506 }
 507
 508 sub _has_overlap {
 509     # flag any pairs that overlap an UNDESIRED feature (eg SNP)
 510     # return true if there is overlap, false if not
 511
 512     my ($self, $test, $flist) = @_;
 513     print STDERR "Checking oligo at ", $test->start, " to ",$test->end, "\n"
 514         if ($self->debug);
 515
 516     foreach my $feat (@$flist) {
 517         if (($test->start <= $feat->end) and ($test->end >= $feat->start)) {
 518             print STDERR "Overlaps ", $feat->primary_tag, " at ",
 519             $feat->start, " to ", $feat->end, "\n" if ($self->debug);
 520             return 1;
 521         }
 522     }
 523     return 0; # default - no overlap
 524 }
 525
 526 # MOVE to SiRNA::Ruleset::tuschl
 527
 528 # sub _get_sense {
 529 #     my ($target) = @_;
 530 #     # trim off 1st 2 nt to get overhang
 531 #     $target =~ s/^..//;
 532 #     # convert T's to U's (transcribe)
 533 #     $target =~ s/T/U/gi;
 534 #     # force last 2 nt to be T's
 535 #     $target =~ s/..$/TT/;
 536
 537 #     return $target;
 538 # }
 539
 540 # sub _get_anti {
 541 #     my ($target) = @_;
 542 #     my @target = split(//, $target);
 543 #     my ($nt,@antitarget);
 544
 545 #     while ($nt = pop @target) {
 546 #       push(@antitarget, $COMP{$nt});
 547 #     }
 548 #     my $anti = join('', @antitarget);
 549 #     # trim off 1st 2 nt to get overhang
 550 #     $anti =~ s/^..//;
 551 #     # convert T's to U's
 552 #     $anti =~ s/T/U/gi;
 553 #     # convert last 2 NT's to T
 554 #     $anti =~ s/..$/TT/;
 555
 556 #     return $anti;
 557 # }
 558
 559
 560 sub AUTOLOAD {
 561     my ($self, $value) = @_;
 562     my $name = $AUTOLOAD;
 563     $name =~ s/.+:://;
 564
 565     return if ($name eq 'DESTROY');
 566
 567
 568     if (defined $value) {
 569         $self->{$name} = $value;
 570     }
 571
 572     unless (exists $self->{$name}) {
 573         $self->throw("Attribute $name not defined for ". ref($self));
 574     }
 575
 576     return $self->{$name};
 577 }
 578
 579 sub _comp {
 580     my ($self, $char) = @_;
 581
 582     return unless ($char);
 583     $char = uc($char);
 584     return $COMP{ $char };
 585 }
 586 1;