examples/sirna/rnai_finder.cgi

   1 #!/usr/bin/perl
   2
   3 =pod
   4
   5 =head1 NAME
   6
   7 rnai_finder.cgi
   8
   9 =head1 DESCRIPTION
  10
  11 A CGI script using the Bio::Tools::SiRNA package to design RNAi reagents.
  12 Retrieves sequences from NCBI and generates output in graphic and tabular form.
  13
  14 =head1 INSTALLATION
  15
  16 To use this script, place it in an appropriate cgi-bin directory on a web server.
  17 The script needs to write its graphic maps to a temporary directory.  Please update
  18 $TMPDIR and $TMPURL to suit your local configuation.
  19
  20 =head1 AUTHOR
  21
  22 Donald Jackson (donald.jackson@bms.com)
  23
  24 =head1 SEE ALSO
  25
  26 L<Bio::Tools::SIRNA>, L<Bio::Graphics::Panel>, L<Bio::DB::NCBIHelper>, L<CGI>
  27
  28 =cut
  29
  30 use Bio::Tools::SiRNA;
  31
  32 use Bio::Graphics::Panel;
  33 use Bio::DB::NCBIHelper;
  34 use Bio::Seq::RichSeq; # for hand-entry
  35 use Bio::SeqFeature::Generic;
  36
  37 use GD::Text::Align;
  38 use Clone qw(clone);
  39
  40 use CGI;
  41 use CGI::Carp qw (fatalsToBrowser carpout);
  42
  43 my $q = CGI->new;
  44
  45
  46
  47 # define a bunch of constants
  48 my %COLORRANKS = ( 1 => 'red',
  49                    2 => 'orchid',
  50                    3 => 'blue' );
  51 my $TMPDIR = '/var/www/htdocs/tmp/';
  52 my $TMPURL = '/tmp/';
  53 my $ATGPAD = 75; # how far from start do we wait?
  54 my $NOLIGOS = 3;
  55
  56 my $log = $TMPDIR . 'RNAiFinder.log';
  57 open (LOG, ">>$log") or die $!;
  58 carpout(LOG);
  59
  60 print $q->header,
  61     $q->start_html;
  62
  63 print $q->h1('RNAi Finder');
  64
  65
  66 if ($q->param('Design')) {
  67     if ($q->param('accession') and !$q->param('seq')) {
  68         $target = get_target();
  69     }
  70     else {
  71         $target = make_target();
  72     }
  73     get_rnai($target);
  74 }
  75 else {
  76     get_settings();
  77 }
  78
  79
  80 sub get_settings {
  81     print <<EOM1;
  82 <P>Oligos are designed as described on the <A HREF="http://www.mpibpc.gwdg.de/abteilungen/100/105/sirna.html" TARGET="Tuschl">Tuschl lab web page</A> and are ranked as follows:
  83 <UL>
  84 <LI><B>New:</B> Selecting 'Pol3-compatible targets' looks for oligos with the
  85 pattern NAR(N17)YNN which can be synthesized or expressed from a Pol3 promoter.
  86 <br>This selection <b>overrides</b> the 'Cutoff' rank.
  87 <LI>Oligos with Rank = 1 (best) match the AAN(19)TT rule.
  88 <LI>Oligos with Rank = 2 match the AAN(21) rule
  89 <LI>Oligos with Rank = 3 match the NAN(21) rule.
  90 </UL>
  91 <P>If percent GC and specificity are similar, Rank 1 oligos are better. All 3 prime overhangs are converted to TT; the rest of the sequence is transcribed into RNA</P>
  92
  93 <h3>Modifications to published rules:</h3>
  94 <ul>
  95 <li>
  96 Runs of 3 or more consecutive Gs on either strand are skipped - these can cause problems in synthesis.
  97 <li>Users may choose to exclude oligos that overlap single nucleotide polymorphisms (ON by default).  SNP data comes from the NCBI dbSNP database.
  98 <li>'Low-complexity' regions (such as runs of a single nucleotide) are also excluded.
  99 </ul>
 100
 101 EOM1
 102
 103     print $q->start_form;
 104     print $q->h2('Enter your sequence and other parameters:'), "\n";
 105     print $q->p('The values already here are DEFAULTS - you should change them to suit YOUR sequence');
 106     print $q->start_table();
 107     print $q->TR( $q->td({-align=> 'left'},
 108                        [
 109                         $q->textfield(-name     => 'mingc', -default => '0.40'),
 110                         $q->textfield(-name     => 'maxgc', -default => '0.60'),
 111                         ]
 112                        ),
 113                       $q->td({-align=> 'left'},
 114                              $q->popup_menu(-name        => 'worstrank',
 115                                       -values   => [1,2,3],
 116                                       -default  => 2,
 117                                       ),
 118                              $q->b('OR'),
 119                              $q->checkbox(-name                 => 'pol3',
 120                                     -label              => 'Pol3 compatible',
 121                                     -default    => 0,
 122                                     ),
 123                              ),
 124                       );
 125     print    $q->TR( $q->th({-align=> 'left'}, 'Exclude oligos with SNPs?'),
 126                      $q->td($q->radio_group(-name => 'avoid_snps',
 127                                             -values => [1,0],
 128                                             -default => 1,
 129                                             -labels => {1 => 'Yes', 0 => 'No'}
 130                                             )),
 131                      );
 132
 133     print $q->TR( $q->th({-align=> 'left'}, 'Sequence Name:'),
 134                   $q->td({-align=> 'left'},$q->textfield('accession')),
 135                   $q->td({-align=> 'left'},
 136                          $q->em( q(Enter an accession and you won't have to enter the <br>sequence or start/stop. Use accessions beginning with NM_ if possible.))),
 137                   );
 138
 139     print $q->TR( $q->th({-align=> 'left'}, ['Position of initiator ATG:',
 140                                              'NT after start to exclude:',
 141                                              'Position of Stop codon:' ]));
 142     print $q->TR( $q->td({-align=> 'left'},
 143                                            [$q->textfield(-name => 'cdstart', -default => 1),
 144                                             $q->textfield(-name => 'atgpad', -default => $ATGPAD),
 145                                             $q->textfield('cdend'), ]));
 146     print $q->TR( $q->th({-align=> 'left'}, ['Minimum Fraction GC:',
 147                                              'Maximum Fraction GC:',
 148                                              'Rank cutoff',
 149                                              ]));
 150     print $q->TR($q->th({-align=> 'left', -colspan=>2},'cDNA Sequence in plain text or FASTA format'),
 151                  $q->td( $q->a({-href =>'Fasta_format.html', -target => 'Fasta_desc'}, 'What is FASTA format?')),
 152                  );
 153     print $q->TR($q->td({-align => 'left', -colspan=>3},
 154                   $q->textarea( -name =>'seq',
 155                                 -rows => 4,
 156                                 -columns => 80,
 157                                 -wrap => 'virtual',
 158                                 )));
 159     print $q->TR( $q->th({-align => 'left', -colspan=>3},
 160                          'Output options: '));
 161     print $q->TR( $q->td({-align=> 'left'},
 162                    [ $q->checkbox(-name => 'Graphic', -checked => 'checked'),
 163                      $q->checkbox(-name =>  'Table',  -checked => 'checked'),
 164                                                  ]));
 165     print $q->TR($q->td({-align=> 'left', -colspan=>3}, $q->submit('Design')));
 166     print $q->end_table();
 167     print $q->end_form;
 168
 169 }
 170
 171 sub get_rnai {
 172     # design and output RNAi reagents
 173     my ($gene) = @_;
 174
 175     my $factory = Bio::Tools::SiRNA->new( -target       => $gene,
 176                                           -tmpdir       => $TMPDIR,
 177                                           -cutoff       => $q->param('worstrank') || 2,
 178                                           -avoid_snps   => $q->param('avoid_snps') || 1,
 179                                           -min_gc       => $q->param('min_gc') || 0.40,
 180                                           -max_gc       => $q->param('max_gc') || 0.60,
 181                                           -pol3         => $q->param('pol3') || 0,
 182                                           );
 183
 184     print $q->p('Designing Pol3-compatible oligos') if ($q->param('pol3'));
 185
 186     my @pairs = $factory->design;
 187
 188     draw_gene($gene) if ($q->param('Graphic'));
 189     print_table($gene->accession, \@pairs) if ($q->param('Table'));
 190     print_text($gene->accession, \@pairs) if ($q->param('Text'));
 191 }
 192
 193 sub get_target {
 194     my ($acc) = $q->param('accession');
 195     my $gb = Bio::DB::NCBIHelper->new();
 196     my $seq = $gb->get_Seq_by_acc($acc);
 197
 198     if ($seq) {
 199         return $seq;
 200     }
 201     else {
 202         print_error("Unable to retrieve sequence from GenBank using accession $acc");
 203         return;
 204     }
 205
 206 }
 207
 208 sub make_target {
 209       # sanity chex - do we have the necessary info?
 210       $q->param('seq') or print_error("Please supply a sequence", 1);
 211       my $seq = $q->param('seq');
 212       my $name;
 213
 214       # is sequence in fasta format?
 215       if ($seq =~ /^>/) {
 216           my ($head, $realseq) = split (/\n/, $seq, 2);
 217           $head =~ /^>(.+?) /;
 218           $name = $1;
 219           $realseq =~ s/[\n|\r|\s]//g;
 220           $seq = $realseq;
 221       }
 222       elsif ($q->param('accession')) {
 223         $name = $q->param('accession');
 224         $seq =~ s/[\n|\r|\s]//g;
 225       }
 226       else {
 227         print_error('Please supply a sequence name!');
 228         return;
 229       }
 230
 231       $cds_start = $q->param('cds_start') || 1;
 232       $cds_end = $q->param('cds_end') || length($seq);
 233
 234       # create a new Bio::Seq::RichSeq object from parameters
 235       my $seqobj = Bio::Seq::RichSeq->new( -seq                 => $seq,
 236                                            -accession_number    => $name,
 237                                            -molecule            => 'DNA',
 238
 239                                      );
 240       my $cds = Bio::SeqFeature::Generic->new( -start   => $cds_start,
 241                                                -end     => $cds_end,
 242                                              );
 243       $cds->primary_tag('CDS');
 244       $seqobj->add_SeqFeature($cds);
 245       return $seqobj;
 246
 247 }
 248 sub draw_gene {
 249 # now draw a pretty picture
 250     my ($gene) = @_;
 251
 252     my $panel = Bio::Graphics::Panel->new( -segment     => $gene,
 253                                            -width       => 600,
 254                                            -pad_top     => 100,
 255                                            -pad_bottom  => 20,
 256                                            -pad_left    => 50,
 257                                            -pad_right   => 50,
 258                                            -fontcolor   => 'black',
 259                                            -fontcolor2  => 'black',
 260                                            -key_color   => 'white',
 261                                            -grid        => 1,
 262                                            -key_style   => 'between',
 263                                            #-gridcolor  => 'lightgray',
 264                                            );
 265
 266     my $genefeat = Bio::SeqFeature::Generic->new( -start        => 1,
 267                                                   -end          => $gene->length);
 268
 269     $panel->add_track( arrow    => $genefeat,
 270                        -bump    => 0,
 271                        -tick    => 2,
 272                        -label   => 1,
 273                        );
 274
 275     my %feature_classes;
 276
 277     foreach $feat($gene->top_SeqFeatures) {
 278         $feature_classes{ $feat->primary_tag } ||= [];
 279
 280         push(@{ $feature_classes{ $feat->primary_tag } }, $feat);
 281     }
 282
 283 # for some reason, Bio::Graphics insists on drawing subfeatures for SiRNA::Pair objects...
 284     $cleanpairs = cleanup_feature($feature_classes{'SiRNA::Pair'});
 285
 286 # draw
 287     $panel->add_track( transcript       => $feature_classes{'gene'},
 288                        -bgcolor => 'green',
 289                        -fgcolor => 'black',
 290                        -fontcolor2  => 'black',
 291                        -key             => 'Gene',
 292                        -bump    => +1,
 293                        -height  => 8,
 294                        -label   => \&feature_label,
 295                        -description     => 1,
 296                        );
 297
 298     $panel->add_track( transcript2      => $feature_classes{'CDS'},
 299                        -bgcolor         => 'blue',
 300                        -fontcolor2  => 'black',
 301                        -fgcolor         => 'black',
 302                        -key             => 'CDS',
 303                        -bump            => +1,
 304                        -height          => 8,
 305                        -label           => \&feature_label,
 306                        -description             => \&feature_desc,
 307                        );
 308
 309     $panel->add_track( $feature_classes{'variation'},
 310                        -bgcolor => 'black',
 311                        -fgcolor => 'black',
 312                        -fontcolor2  => 'black',
 313                        -key     => 'SNPs',
 314                        -bump    => +1,
 315                        -height  => 8,
 316                        -label   => \&snp_label,
 317                        #-glyph  => 'triangle',
 318                        -glyph   => 'diamond',
 319                        -description             => \&feature_desc,
 320                        );
 321
 322     $panel->add_track( generic  => $feature_classes{'Excluded'},
 323                        -bgcolor => 'silver',
 324                        -fgcolor => 'black',
 325                        -fontcolor  => 'black',
 326                        -fontcolor2  => 'black',
 327                        -key     => 'Excluded Regions',
 328                        -bump    => +1,
 329                        -height  => 6,
 330                        -label   => \&feature_label,
 331                        -description             => \&feature_desc,
 332                        );
 333
 334     $panel->add_track(
 335                        generic => $cleanpairs,
 336                        -bgcolor => \&feature_color,
 337                        -fgcolor => \&feature_color,
 338                        -fontcolor  => 'black',
 339                        -fontcolor2  => 'black',
 340                        -key     => 'SiRNA Reagents',
 341                        -bump    => +1,
 342                        -height  => 8,
 343                        -label   => \&feature_label,
 344                        -glyph   => 'generic',
 345                        -description             => \&feature_desc,
 346                        );
 347
 348     my $gd = $panel->gd;
 349     my $black = $gd->colorAllocate(0,0,0);
 350     my $txt = GD::Text::Align->new($gd);
 351     $txt->set( valign => 'center', align => 'center', color => $black);
 352     #$txt->set_font(['/usr/share/fonts/truetype/VERDANA.TTF',gdGiantFont ], 10);
 353     $txt->set_font(gdGiantFont);
 354     $txt->set_text("RNAi Reagents for ".$gene->accession );
 355     $txt->draw(200, 50, 0);
 356
 357     my $pngfile = $TMPDIR . $gene->accession . '.png';
 358     my $pngurl = $TMPURL . $gene->accession . '.png';
 359     open (IMG, ">$pngfile") or die $!;
 360     binmode IMG;
 361     print IMG $gd->png;
 362     close IMG;
 363
 364     # also get the imagemap boxes
 365     my @pairboxes = extract_pairs($panel->boxes);
 366
 367     print $q->img({-src => $pngurl, -usemap=>"#MAP"});
 368     print $q->p('Oligos are color coded: rank 1 in ',
 369                 $q->font({-color => $COLORRANKS{1}}, $COLORRANKS{1}),
 370                 ', rank 2 in ',
 371                 $q->font({-color => $COLORRANKS{2}}, $COLORRANKS{2}),
 372                 ' and rank 3 in ',
 373                 $q->font({-color => $COLORRANKS{3}}, $COLORRANKS{3}),
 374                 '. Click on an oligo to bring it up in the table below');
 375
 376     print_imagemap(@pairboxes);
 377
 378 }
 379
 380 sub feature_label {
 381     my ($feature) = @_;
 382     my (@notes, @label);
 383     #$label = ucfirst($feature->primary_tag);
 384     foreach (qw(note name product gene)) {
 385         if ($feature->has_tag($_)) {
 386             @notes = $feature->each_tag_value($_);
 387             #$label .= ': ' . $notes[0];
 388             push(@label, $notes[0]);
 389             last;
 390         }
 391     }
 392     return join(': ', @label);
 393     #return $label;
 394 }
 395
 396 sub feature_color {
 397     my ($feature) = @_;
 398     my ($rank) = $feature->each_tag_value('rank');
 399     #print STDERR "Feature rank: $rank COLOR $COLORRANKS{$rank}\n";
 400     return $COLORRANKS{$rank};
 401     #return 'red';
 402 }
 403
 404
 405 sub print_table {
 406     my ($accession, $pairs) = @_;
 407
 408     print $q->h2("RNAi Reagents for $accession");
 409     print $q->start_table({-border => 1, -cellpadding => 2});
 410     print $q->TR( $q->th(['Reagent #', 'Start', 'Stop', 'Rank', 'Fxn GC', 'Sense Oligo', 'Antisense Oligo', 'Target' ]) ), "\n";
 411
 412
 413     my $i = 1;
 414
 415     foreach $pair ( sort { $a->start <=> $b->start } @$pairs ) {
 416         my $sense = $pair->sense;
 417         my $anti = $pair->antisense;
 418         my $color = feature_color($pair);
 419
 420 #       my $blasturl = "http://nunu.hpw.pri.bms.com/biocgi/versablast.pl?p=blastn&sequence=";
 421 #       $blasturl .= $pair->seq->seq;
 422 #       $blasturl .= "&action=Nucleotide Databases";
 423
 424         print
 425             $q->TR( $q->td( [ $q->a({-name => 'RNAi' . $pair->start}) . $i,
 426                               $pair->start,
 427                               $pair->end,
 428                               $q->font({-color => $color},$pair->rank),
 429                               $pair->fxGC,
 430                               $q->tt($sense->seq),
 431                               $q->tt($anti->seq),
 432                               $q->tt($pair->seq->seq),
 433 #                             $q->a({-href=>$blasturl,
 434 #                                    -target=>"blastn"},
 435 #                                   "BLAST this target"),
 436                               ] ) ),
 437         "\n";
 438         $i++;
 439     }
 440     print $q->end_table;
 441 }
 442
 443
 444
 445
 446
 447 sub print_text {
 448     my ($accession,  $pairs ) = @_;
 449     my ($pair);
 450
 451     print "RNAi reagents for $accession \n";
 452
 453     print join("\t", qw(Start Stop Rank Sense Antisense)), "\n";
 454     foreach $pair (@$pairs ) {
 455         my $sense = $pair->sense;
 456         my $anti = $pair->antisense;
 457
 458         print join("\t", $pair->start, $pair->end, $pair->rank, $sense->seq, $anti->seq), "\n";
 459
 460
 461     }
 462
 463
 464 }
 465
 466 sub cleanup_feature {
 467     my ($flist) = @_;
 468
 469     my ($feat, @clean, $cfeat);
 470
 471     foreach $feat(@$flist) {
 472         $cfeat = clone($feat);
 473 #       $cfeat = $feat->clone;
 474         $cfeat->flush_sub_SeqFeature;
 475         push (@clean, $cfeat); # will they
 476     }
 477     return \@clean;
 478 }
 479
 480
 481 sub extract_pairs {
 482     # get SiRNA::Pair features ONLY for imagemap
 483     return ( grep {ref($_->[0]) eq "Bio::SeqFeature::SiRNA::Pair"} @_ );
 484 }
 485
 486 sub print_imagemap {
 487     my @items = @_;
 488
 489     print q(<MAP NAME="MAP">), "\n";
 490
 491     my $i = 1;
 492
 493     foreach $item (@items) {
 494         my ($feature, $x1, $y1, $x2, $y2) = @$item;
 495         my $fstart = $feature->start; # should be unique
 496         my $text = 'RNAi #' . $i. ' Start=' . $feature->start . ' Rank='.$feature->rank;
 497         print qq(<AREA SHAPE="RECT" COORDS="$x1,$y1,$x2,$y2" TITLE="$text" HREF="#RNAi$fstart">), "\n";
 498         warn "Mouseover text: $text\n";
 499
 500         $i++;
 501     }
 502     print "</MAP>\n";
 503 }
 504
 505
 506 sub print_error {
 507     # print error messages in big red type. Provide more graceful die/warn to end user
 508     my ($msg, $fatal) = @_;
 509     print $q->h3($q->font({-color=>'RED'}, $msg));
 510
 511     if ($fatal) {
 512         print $q->end_html;
 513         die "$msg \n";
 514     }
 515     else {
 516         warn $msg;
 517     }
 518 }
 519
 520 sub dump {
 521     print $q->start_ul;
 522
 523     foreach ($q->param) {
 524         print $q->li($_),
 525         $q->ul($q->li([ $q->param($_) ]));
 526     }
 527 }
 528
 529 sub snp_label {
 530     # special format for SNPs
 531     my ($feature) = @_;
 532     my $label;
 533
 534     if ( $feature->has_tag('db_xref') ) {
 535         my @notes = $feature->each_tag_value('db_xref');
 536         $label .= $notes[0];
 537         $label .= ' ';
 538     }
 539     if ( $feature->has_tag('allele') ) {
 540         my ($nt1, $nt2) = $feature->each_tag_value('allele');
 541         $label .=  $nt1 . '->' . $nt2;
 542     }
 543     return $label;
 544 }
 545
 546 sub feature_desc {
 547     my ($feature) = @_;
 548     my $desc = $feature->start;
 549     $desc .= '-' . $feature->end unless ($feature->start == $feature->end);
 550     return $desc;
 551 }