cgi-bin/tools/align_viewer/show_align.pl

   1
   2 =head1 NAME
   3
   4 show_align.pl - a cgi-based graphical alignment viewer
   5
   6 =head1 DESCRIPTION
   7
   8 Generates an alignment view from data submitted through the input page (index.pl in this directory). It is a web script that supports the following cgi parameters:
   9
  10 =over 5
  11
  12 =item format
  13
  14 format may be either 'clustalw', 'fasta', or 'fasta_unaligned', and describes the format of the uploaded sequences. If format is 'fasta_unaligned', the sequences will be aligned using t_coffee.
  15
  16 =item seq_data
  17
  18 seq_data is the actual data uploaded, in the format described by the format parameter.
  19
  20 =item temp_file
  21
  22 if no seq_data is given, a temp_file name is supplied that specifies a previously parsed input. The temp_file specifies a file name, without the directory information, which will be in the default directory location, as defined in the conf object.
  23
  24 =item start_value
  25
  26 denotes the first position of the section of the alignment that should be displayed.
  27
  28 =item end_value
  29
  30 denotes the end position of the section of the alignment that should be displayed.
  31
  32 =item title
  33
  34 the title of the alignment, shown on the top of the page.
  35
  36 =item type
  37
  38 =over 5
  39
  40 =item 'nt' for nucleotide
  41
  42 =item 'pep' for peptide
  43
  44 =back 5
  45
  46 Not sure about this one. Defaults to peptide as default.
  47
  48 =item hide_seqs
  49
  50 a parameter that specifies a list of sequence ids, separated by whitespace, that should not be shown in the alignment.
  51
  52 =item run
  53
  54 'local' means run on local host. 'cluster' means run job on cluster (if an alignment needs to be run).
  55
  56 =back
  57
  58 =head1 DEPENDENCIES
  59
  60 Depends on the CXGN code base, in particular heavily on CXGN::Alignment and others, and on GD for the graphics routines and File::Temp for the temp file handling.
  61
  62 =head1 AUTHOR(S)
  63
  64 Code by Chenwei Lin (cl295@cornell.edu). Documentation and minor modifications (ability to run T-coffee from the page) by Lukas Mueller (lam87@cornell.edu).
  65
  66 =cut
  67
  68 use strict;
  69 use Storable qw /store/;
  70 use File::Copy;
  71 use CXGN::Page;
  72 use CXGN::Page::FormattingHelpers qw/ page_title_html blue_section_html  /;
  73 use CXGN::Page::Widgets qw/ collapser /;
  74 use File::Temp qw/tempfile/;
  75 use File::Basename qw /basename/;
  76 use CXGN::Phylo::Alignment;
  77 use Bio::AlignIO;
  78 use CXGN::DB::Connection;
  79 use CXGN::Tools::Run;
  80 use CXGN::Tools::Gene;
  81 use CXGN::Tools::Identifiers qw/ identifier_namespace identifier_url /;
  82 use CXGN::Tools::Entrez;
  83 use HTML::Entities;
  84 use CXGN::Tools::Param qw/ hash2param /;
  85 use CatalystX::GlobalContext '$c';
  86
  87 our $ALIGN_SEQ_COUNT_LIMIT        = 500;
  88 our $ALIGN_SEQ_TOTAL_LENGTH_LIMIT = 500_000;
  89
  90 my $page = CXGN::Page->new( "SGN Alignment Analyzer", "Chenwei-Carpita" );
  91 $page->jsan_use("CXGN.Effects");
  92
  93 our (
  94     $format,    $seq_data,       $id_data,   $start_value,
  95     $end_value, $title,          $type,      $hide_seqs,
  96     $run,       $sv_similarity,  $sv_shared, $sv_indel,
  97     $show_sv,   $show_breakdown, $maxiters,  $force_gap_cds,
  98     $show_domains
  99   )
 100   = $page->get_arguments(
 101     "format",    "seq_data",       "id_data",   "start_value",
 102     "end_value", "title",          "type",      "hide_seqs",
 103     "run",       "sv_similarity",  "sv_shared", "sv_indel",
 104     "show_sv",   "show_breakdown", "maxiters",  "force_gap_cds",
 105     "show_domains"
 106   );
 107
 108 my ( $image_content, $sum_content, $seq_sum_content, $sv_content, $al_content,
 109     $combined_content );
 110
 111 our ( $temp_file, $cds_temp_file ) =
 112   $page->get_arguments( "temp_file", "cds_temp_file" );
 113
 114 our ( $family_nr, $i_value, $family_id ) =
 115   $page->get_arguments( "family_nr", "i_value", "family_id" );
 116
 117 if ( $family_id && !( $family_nr || $i_value ) ) {
 118     my $dbh = $c->dbc->dbh;
 119     my $sth =
 120       $dbh->prepare(
 121 "SELECT family_nr, i_value FROM sgn.family LEFT JOIN sgn.family_build USING (family_build_id) WHERE family_id=?"
 122       );
 123     $sth->execute($family_id);
 124     ( $family_nr, $i_value ) = $sth->fetchrow_array();
 125 }
 126
 127 $show_breakdown = 1    if $show_breakdown;
 128 $show_sv        = 1    if $show_sv;
 129 $maxiters       = 1000 if ( $maxiters > 1000 );
 130 $maxiters ||= 2;
 131
 132 $show_domains = 1 unless ( $show_domains eq '0' );
 133
 134 #Run locally by default, cluster will run if the computation
 135 #is found to be intensive (later in script)
 136 unless ( $run =~ /^(local)|(cluster)$/ ) { $run = "local"; }
 137
 138 #############################################################
 139 #Check if the input sequence is empty
 140 unless ( $seq_data
 141     || $id_data
 142     || $temp_file
 143     || $family_id
 144     || ( $family_nr && $i_value ) )
 145 {
 146     &input_err_page( $page, "No sequence data provided!\n" );
 147 }
 148
 149 #############################################################
 150 #If a $seq_data is passed to the page, write the content of $seq_data into a temp file
 151
 152 our $HTML_ROOT_PATH = $c->config->{'basepath'};
 153 our $PATH     = $page->path_to( $page->tempfiles_subdir('align_viewer') );
 154 unless( -d $PATH ) {
 155     mkdir $PATH
 156         or die "temp dir '$PATH' does not exist, and could not create";
 157 }
 158 our $tmp_fh;
 159 our $CLUSTER_SHARED_TEMPDIR = $c->config->{'cluster_shared_tempdir'};
 160 our $NOCLUSTER              = $c->config->{'nocluster'};
 161
 162 #You can send the temp_file as a filename and not a full path, good for security
 163 unless ( $temp_file =~ /\// ) {
 164     $temp_file = $PATH . "/" . $temp_file;
 165 }
 166 unless ( $cds_temp_file =~ /\// ) {
 167     $cds_temp_file = $PATH . "/" . $cds_temp_file;
 168
 169     #If a temp file is set, we are definitely CDS
 170     #Fixes imagemap problem:
 171     $type = "cds" if ( -f $cds_temp_file );
 172 }
 173
 174 if ( -f $cds_temp_file && $force_gap_cds ) {
 175
 176     #The CDS temp file needs to be gapped
 177     $cds_temp_file = build_gapped_cds_file( $temp_file, $cds_temp_file );
 178 }
 179
 180 # upload file, if requested... (Lukas, 2006/09/05)
 181 #
 182 my $upload = $page->get_upload("upload");
 183 my $upload_fh;
 184
 185 # print STDERR "Uploading file $args{upload_file}...\n";
 186 if ( defined $upload ) {
 187     $upload_fh = $upload->fh();
 188     while (<$upload_fh>) {
 189         $seq_data .= $_;
 190     }
 191 }
 192
 193 my @good_ids = ();
 194 if ( $seq_data =~ /\S+/ ) {
 195     $tmp_fh = File::Temp->new(
 196         DIR    => $PATH,
 197         UNLINK => 0,
 198     );
 199
 200     $temp_file = $tmp_fh->filename;
 201     print $tmp_fh "$seq_data";
 202
 203     close $tmp_fh;
 204
 205     # Convert the input file into fasta if it is clustal
 206     if ( $format eq 'clustalw' ) {
 207         my $temp_file_fasta = $temp_file . ".fasta";
 208         convert_clustal_fasta( $temp_file, $temp_file_fasta );
 209
 210         # continue with the converted file...
 211         $temp_file = $temp_file_fasta;
 212     }
 213 }
 214 elsif ( $id_data =~ /\S+/ ) {
 215     unless ( -f $temp_file ) {
 216         my $tmp_fh = File::Temp->new(
 217             DIR    => $PATH,
 218             UNLINK => 0,
 219         );
 220         $temp_file = $tmp_fh->filename;
 221         $tmp_fh->close();
 222     }
 223     $format = "fasta_unaligned";
 224
 225     open( my $temp_fh, '>', $temp_file ) or die $!;
 226
 227     my @ids = split /\s+/, $id_data;
 228     foreach my $id (@ids) {
 229         if ( $id =~ /^gi:\d+$/i
 230             || CXGN::Tools::Identifiers::is_genbank_accession($id) )
 231         {
 232             ($id) = $id =~ /^gi:(\d+)$/i if $id =~ /^gi:\d+$/;
 233             my $database = "protein";
 234             $database = "nucleotide" if $type ne "pep";
 235             my $entrez = CXGN::Tools::Entrez->new( { db => $database } );
 236             my $seq = $entrez->get_sequence($id);
 237             $id = "gi:$id" if $id =~ /^\d+$/;
 238             print $temp_fh ">$id\n$seq" if $seq;
 239             push( @good_ids, $id ) if $seq;
 240         }
 241         else {
 242             eval {
 243                 my $gene    = CXGN::Tools::Gene->new($id, $c->dbc->dbh );
 244                 my $seqtype = "";
 245                 $seqtype = "protein" if $type eq "pep";
 246                 $seqtype = "cds"     if $type eq "cds";
 247                 $seqtype = "genomic" if $type eq "nt";
 248                 my $seq = $gene->getSequence($seqtype);
 249                 print $temp_fh ">$id\n$seq\n" if $seq;
 250             };
 251             if( my $err = $@ ) {
 252                 $err =~ s/\n\s*Catalyst::.+//;
 253                 warn $err;
 254             } else {
 255                 push @good_ids, $id;
 256
 257             }
 258         }
 259     }
 260     $id_data = join " ", @good_ids;
 261     close($temp_fh);
 262 }
 263 elsif ( $family_nr && $i_value ) {
 264     my $family_basedir = "/data/prod/public/family/$i_value";
 265     unless ( -d $family_basedir ) {
 266         &input_err_page( $page,
 267             "No current family build corresponds to an i_value of $i_value" );
 268     }
 269     unless ( -f "$family_basedir/pep_align/$family_nr.pep.aligned.fasta" ) {
 270         &input_err_page( $page,
 271             "Alignment was not calculated for this family" );
 272     }
 273
 274     unless ( -f $temp_file ) {
 275         my $tmp_fh = File::Temp->new(
 276             DIR    => $PATH,
 277             UNLINK => 0,
 278         );
 279         $temp_file = $tmp_fh->filename;
 280         $tmp_fh->close();
 281     }
 282     $format = "fasta_aligned";
 283     $type   = "cds";
 284
 285     #disable cds until kinks worked out
 286     if ( 0 && -f "$family_basedir/cds_align/$family_nr.cds.align.fasta" ) {
 287         $type = "cds";
 288         system
 289           "cp $family_basedir/cds_align/$family_nr.cds.align.fasta $temp_file";
 290     }
 291     else {
 292         $type = "pep";
 293         system
 294 "cp $family_basedir/pep_align/$family_nr.pep.aligned.fasta $temp_file";
 295     }
 296 }
 297
 298 #Check that the input file is a good FASTA, throw error page otherwise
 299 our ( $SEQ_COUNT, $MAX_LENGTH, $TOTAL_LENGTH ) = fasta_check( $temp_file, $page );
 300
 301 #Also, use seq_count, maxiters, and max_length parameters for Muscle to see if we
 302 #should run it on the cluster, otherwise allow running on the web server
 303 if (   !$NOCLUSTER && ( $maxiters * $SEQ_COUNT * $MAX_LENGTH > 30000 )
 304     || !$maxiters )
 305 {
 306     $run = "cluster";
 307 }
 308 ##Convert cds to protein, but if cds_temp_file exists,
 309 #the conversion was already done on the previous page (or before)
 310 if ( $type eq "cds" && $temp_file && !( -f $cds_temp_file ) ) {
 311     my $new_temp_fh = File::Temp->new(
 312         DIR    => $PATH,
 313         UNLINK => 0,
 314     );
 315     my $new_temp = $new_temp_fh->filename;
 316     my $instream = Bio::SeqIO->new( -file => $temp_file, -format => 'fasta' );
 317     while ( my $in = $instream->next_seq() ) {
 318         my $seq = $in->seq();
 319         my $id  = $in->id();
 320         my $len = length $seq;
 321         my $member;
 322         eval {
 323             $member = CXGN::Phylo::Alignment::Member->new(
 324                 id          => $id,
 325                 seq         => $seq,
 326                 type        => "cds",
 327                 start_value => 1,
 328                 end_value   => $len
 329             );
 330             $member->translate_cds();
 331         };
 332         if ($@) {
 333             input_err_page( $page, $@ );
 334         }
 335
 336         my $prot_seq = $member->{translated_protein_seq};
 337         print $new_temp_fh ">$id\n$prot_seq\n";
 338     }
 339     close($new_temp_fh);
 340
 341     #Important transition happens here.  The temp_file
 342     #is now the translated protein sequence.  Maybe it's
 343     #gapped, maybe not, depends on the format parameter
 344     #and if the CDS was gapped
 345     $cds_temp_file = $temp_file;
 346     $temp_file     = $new_temp;
 347 }
 348
 349 #############################################################
 350 #Read the alignment sequence and store them in an align object
 351
 352 #First parse $hide_seqs
 353 my %hidden_seqs = ();
 354 my @ids = split /\s+/, $hide_seqs;
 355 foreach (@ids) {
 356     $hidden_seqs{$_} = 1;
 357 }
 358
 359 # align sequences if they are in the unaligned fasta format
 360 # (added by Lukas, 9/5/2006)
 361 if ( $format eq "fasta_unaligned" ) {
 362
 363     if ( $SEQ_COUNT > $ALIGN_SEQ_COUNT_LIMIT ) {
 364         my $message =
 365 "<center><span style='font-size:1.2em;'>You submitted <b>$SEQ_COUNT</b> sequences. Due to limited computational resources, we only <br>allow <b>$ALIGN_SEQ_COUNT_LIMIT or fewer</b> sequences to be aligned through our web interface.</span>";
 366         $message .=
 367 "<br /><br /><span style='font-size:1.1em'>You can download and run your own copy of the <br />alignment software at <a href='http://www.drive5.com/muscle/'>www.drive5.com/muscle/</a></span></center>";
 368         input_err_page( $page, $message );
 369     }
 370     elsif( $TOTAL_LENGTH > $ALIGN_SEQ_TOTAL_LENGTH_LIMIT ) {
 371         input_err_page( $page, <<"" );
 372 <center><span style='font-size:1.2em;'>You submitted <b>$TOTAL_LENGTH</b> bases of sequence. Due to limited computational resources, we only <br>allow <b>$ALIGN_SEQ_TOTAL_LENGTH_LIMIT or fewer</b> sequences to be aligned through our web interface.</span>
 373 <br /><br /><span style='font-size:1.1em'>You can download and run your own copy of the <br />alignment software at <a href='http://www.drive5.com/muscle/'>www.drive5.com/muscle/</a></span></center>
 374
 375     }
 376
 377     run_muscle( $page, $run );
 378 }
 379
 380 #die "\n$temp_file\n$cds_temp_file";
 381
 382 #Let's see the toolbar while we're just building the image
 383
 384 #######################################
 385 ## Construct Alignment ################
 386 #######################################
 387
 388 my $disp_type = $type;
 389 $disp_type = "pep" if ( $disp_type eq "cds" );
 390
 391 my $alignment = CXGN::Phylo::Alignment->new(
 392     name   => $title,
 393     width  => 800,
 394     height => 2000,
 395     type   => $disp_type
 396 );
 397 $alignment->{page} = $page;
 398 my $instream;
 399 my $len;
 400
 401 $sv_similarity = 95 unless ($sv_similarity);
 402 $sv_indel      = 4  unless ($sv_indel);
 403 $sv_shared     = 20 unless ($sv_shared);
 404
 405 $alignment->set_sv_criteria( $sv_shared, $sv_similarity, $sv_indel );
 406
 407 #die $temp_file;
 408 $instream = Bio::SeqIO->new( -file => $temp_file, -format => 'fasta' );
 409 while ( my $in = $instream->next_seq() ) {
 410     my $seq = $in->seq();
 411     my ( $id, $species ) = $in->id() =~ m/(.+)\/(.+)/;
 412     $id = $in->id();
 413     ( !$species ) and $species = ();
 414
 415     my $hidden = 0;
 416     $hidden = 1
 417       if ( exists $hidden_seqs{$id} )
 418       ;    #skip the sequence if it is in the hide_seq
 419
 420     chomp $seq;
 421     $len = length $seq;
 422     my $member;
 423     eval {
 424
 425         $member = CXGN::Phylo::Alignment::Member->new(
 426             start_value => 1,
 427             end_value   => $len,
 428             id          => $id,
 429             seq         => $seq,
 430             hidden      => $hidden,
 431             species     => $species,
 432             type        => $disp_type
 433         );
 434         $alignment->add_member($member);
 435     };
 436     $@ and input_err_page( $page, $@ );
 437
 438     my ( $gene, $annot, $sigpos, $cleavage );
 439     eval {
 440         $gene  = CXGN::Tools::Gene->new($id);
 441         $annot = $gene->getAnnotation;
 442         $annot = HTML::Entities::encode($annot);
 443     };
 444     unless ($@) {
 445         $member->set_tooltip($annot);
 446     }
 447
 448     $member->show_remove();    #draw X for removal
 449     $member->show_link();
 450 }
 451
 452 #Set the start value and end value#######
 453 ( !$start_value ) and $start_value = 1;
 454 $alignment->set_start_value($start_value);
 455 ( !$end_value )       and $end_value = $len;
 456 ( $len < $end_value ) and $end_value = $len;
 457 $alignment->set_end_value($end_value);
 458
 459 ## Domain Highlighting ########################
 460 $alignment->highlight_domains() if $show_domains;
 461
 462 ### If cds_temp exists, create a $cds_alignment object, for producing CDS files
 463
 464 our $cds_alignment;
 465 if ( -f $cds_temp_file && $type eq "cds" ) {
 466     $cds_alignment = CXGN::Phylo::Alignment->new(
 467         name => $title,
 468         type => "cds"
 469     );
 470     my $instream =
 471       Bio::SeqIO->new( -file => $cds_temp_file, -format => 'fasta' );
 472     while ( my $in = $instream->next_seq() ) {
 473         my $seq = $in->seq();
 474         my ( $id, $species ) = $in->id() =~ m/(.+)\/(.+)/;
 475         $id = $in->id();
 476         ( !$species ) and $species = ();
 477
 478         my $hidden = 0;
 479         $hidden = 1
 480           if ( exists $hidden_seqs{$id} )
 481           ;    #skip the sequence if it is in the hide_seq
 482
 483         chomp $seq;
 484         my $len = length $seq;
 485         my $member;
 486         eval {
 487             $member = CXGN::Phylo::Alignment::Member->new(
 488                 start_value => 1,
 489                 end_value   => $len,
 490                 id          => $id,
 491                 seq         => $seq,
 492                 hidden      => $hidden,
 493                 species     => $species,
 494                 type        => $disp_type
 495             );
 496             $cds_alignment->add_member($member);
 497         };
 498         $@ and input_err_page( $page, $@ );
 499         $member->show_remove();    #draw X for removal
 500         $member->show_link();
 501     }
 502     $cds_alignment->set_start_value( $start_value * 3 - 2 );
 503     $cds_alignment->set_end_value( $end_value * 3 );
 504 }
 505
 506 ### Do page header AFTER all eval{} statements
 507 $page->header;
 508
 509 ############################################################
 510 #Draw alignment image, unless we are at the original parameters and we
 511 #have already drawn the most computationally intensive image
 512
 513 my $tmp_image = File::Temp->new(
 514     DIR    => $PATH,
 515     SUFFIX => '.png',
 516     UNLINK => 0,
 517 );
 518
 519 #Render image
 520 $alignment->render_png_file( $tmp_image, 'c' );
 521 close $tmp_image;
 522 $tmp_image =~ s/$HTML_ROOT_PATH//;
 523
 524 #Important for Ruler Image-Map Generation:
 525 our ($temp_file_name)     = $temp_file     =~ /([^\/]+)$/;
 526 our ($cds_temp_file_name) = $cds_temp_file =~ /([^\/]+)$/;
 527
 528 #Parameter hash for imagemap and other links on the page
 529 our %PARAM = (
 530     format         => $format,
 531     start_value    => $start_value,
 532     end_value      => $end_value,
 533     title          => $title,
 534     type           => $type,
 535     hide_seqs      => $hide_seqs,
 536     sv_similarity  => $sv_similarity,
 537     sv_shared      => $sv_shared,
 538     sv_indel       => $sv_indel,
 539     show_sv        => $show_sv,
 540     show_breakdown => $show_breakdown,
 541     maxiters       => $maxiters,
 542     force_gap_cds  => $force_gap_cds,
 543     temp_file      => $temp_file_name,
 544     cds_temp_file  => $cds_temp_file_name,
 545     show_domains   => $show_domains
 546 );
 547
 548 $alignment->set_fasta_temp_file($temp_file_name);
 549 $alignment->set_cds_temp_file($cds_temp_file_name);
 550 $alignment->set_param( \%PARAM );
 551
 552 $image_content = "<tr><td align=\"center\">";
 553
 554 $image_content .=
 555 "<img border=0 src='$tmp_image' alt='Alignment Image' usemap='#align_image_map'/>";
 556 $image_content .= $alignment->get_image_map( $show_sv, $show_breakdown );
 557 $image_content .=
 558   "<div style=\"width:100%;text-align:left;padding-left:40px;\">";
 559 if ($show_domains) {
 560     $image_content .=
 561         "<a href=\"show_align.pl?"
 562       . hash2param( \%PARAM, { show_domains => 0 } )
 563       . "\">Hide Domain Information</a>";
 564 }
 565 else {
 566     $image_content .=
 567         "<a href=\"show_align.pl?"
 568       . hash2param( \%PARAM, { show_domains => 1 } )
 569       . "\">Show Domain Information</a>";
 570 }
 571 $image_content .= "</div>";
 572
 573 if ( $start_value == 1 && $end_value == $len ) {
 574     $image_content .=
 575 "<b>Click on a region of the image to zoom in.</b><br />You must submit the form below to change alignment parameters or remove members from analysis.";
 576 }
 577 else {
 578     $image_content .= "<a href='show_align.pl?";
 579     $image_content .=
 580       hash2param( \%PARAM, { start_value => 1, end_value => "" } );
 581     $image_content .= "'>&lt;&lt; See Whole Alignment &gt;&gt;</a>";
 582 }
 583
 584 $image_content .= "</td></tr>";
 585
 586 my $term_help_content = <<HTML;
 587 <table width="100%" summary="" cellpadding="3" cellspacing="3" border="0"><tr><td><p style='font-size:1.1em'>SGN Alignment Viewer analyzes and provides useful information that can be used to optimize the overlapping region of the alignment.</p></td></tr></table>
 588 <table width="100%" summary="" cellpadding="3" cellspacing="0" border="0">
 589 <tr style='background-color:#ddf'><th valign='top'>Coverage&nbsp;Range</th>
 590 <td>The distance between the first and last element of the un-gapped sequence in the current alignment.</td></tr>
 591 <tr><th valign='top'>Bases</th><td>Number of "real sequence characters" (non gap) in the sequence.</td></tr>
 592 <tr style='background-color:#ddf'><th valign='top'>Gaps</th><td>Number of gaps in the aligment</td></tr>
 593 <tr><th valign='top'>Medium</th><td>The middle position of all non-gap characters of the alignment sequence</td></tr>
 594 <tr style='background-color:#ddf'><th valign='top'>Putative Splice Variant Pairs</th><td>Sequence pairs that are from the same species, that, by default <ol>
 595 <li>Share at least 60 bases if they are nucleotides or 20 bases if they are peptides.
 596 <li>Share at least 95% sequence similarity in the overlapping region.
 597 <li>Have insertion/deletion of at least 4 amino acids or 12 nucleotides in their common region.</ol> </td></tr>
 598 <tr><th valign='top'>Putative Alleles</th><td>Sequence pairs that are from the same species, that, by default
 599 <ol>
 600 <li>Share at least 60 bases if they are nucleotides or 20 bases if they are peptides.
 601 <li>Share at least 95% sequence similarity in the overlapping region.
 602 <li>Have insertion/deletion of <b>not more than </b> 4 amino acids or 12 nucleotides in their common region.
 603 </ol>
 604 </td></tr>
 605 <tr style='background-color:#ddf'><th valign='top'>Overlap Score</th><td>An indication of how well the sequence overlaps with other members in the alignment.  If a non-gap character of a sequence overlaps with a character in another alignment sequence, it gets a point.<br><br>Sometimes a few sequences share very little overlap with the rest, significantly reducing the overall overlapping sequence of the alignment.  Usually these sequences are short and won't help with the understanding of overall alignment.  We suggest the user leaves out these sequences before further analysis of the aligment sequence. </td></tr></table>
 606 HTML
 607 $term_help_content =
 608   blue_section_html( "<em>Help With Terms</em>", $term_help_content );
 609
 610 my ( $help_link, $hidden_help_content ) = collapser(
 611     {
 612         id                  => "alignment_help_content",
 613         alt_href            => "/about/align_term.pl",
 614         alt_target          => "align_about",
 615         linktext            => "(-) Help me understand terms on this page",
 616         hide_state_linktext => "(?) Help me understand terms on this page",
 617         linkstyle           => "font-size:1.1em",
 618         collapsed           => 1,
 619         content             => $term_help_content,
 620     }
 621 );
 622
 623 ##########################################################
 624 #Write sequence output to temp files and create links to them
 625
 626 ###########################################################
 627 #Get summary information
 628
 629 $combined_content = get_combined_content($alignment);
 630
 631 ############################################################
 632 #Page printout: Nav Section
 633
 634 print
 635 "<a href=\"index.pl?id_data=$id_data\" style=\"margin-left:10px;font-size:1.1em\">&lt;&lt; Start over</a>";
 636 my $disp_title = "";
 637 $disp_title = "View and Analyze Alignment of <em>$title</em>" if ($title);
 638 $disp_title ||= "View and Analyze Alignment";
 639 print page_title_html("$disp_title");
 640
 641 print "&nbsp;&nbsp;" . $help_link . "<br><br>";
 642 print $hidden_help_content; #this is actually a blue_section_html, but the whole thing is hidden
 643
 644 my $image_title = "Alignment Image";
 645 $image_title .= ":&nbsp;&nbsp; CDS Translated" if ( $type eq "cds" );
 646 print blue_section_html( $image_title,
 647         '<table width="100%" cellpadding="0" cellspacing="0" border="0">'
 648       . $image_content
 649       . '</table>' );
 650
 651 print blue_section_html(
 652     'Summary, Files, Change Parameters',
 653     '<table width="100%" cellpadding="5" cellspacing="0" border="0">'
 654       . $combined_content
 655       . '</table>'
 656 );
 657
 658 if ($show_breakdown) {
 659     $seq_sum_content = get_seq_sum_content($alignment);
 660 }
 661 else {
 662     $seq_sum_content .= "Not Calculated &nbsp;&nbsp;( <a href=\"show_align.pl?";
 663     $seq_sum_content .= hash2param( \%PARAM, { show_breakdown => 1 } );
 664     $seq_sum_content .=
 665       "\">showing this table</a> will increase page load time )";
 666 }
 667
 668 ###########################################################
 669 #Find splice variants
 670
 671 if ($show_sv) {
 672     ( $sv_content, $al_content ) = get_sv_al_content($alignment);
 673 }
 674 else {
 675     $al_content = "Not Calculated &nbsp;&nbsp;( <a href=\"show_align.pl?";
 676     $al_content .= hash2param( \%PARAM, { show_sv => 1 } );
 677     $al_content .= "\">showing this table</a> will increase page load time )";
 678     $sv_content = "Not Calculated &nbsp;&nbsp;( <a href=\"show_align.pl?";
 679     $sv_content .= hash2param( \%PARAM, { show_sv => 1 } );
 680     $sv_content .= "\">showing this table</a> will increase page load time )";
 681 }
 682
 683 ###########################################################
 684 ##Page Printout: Breakdown and Alleles
 685 print blue_section_html( 'Alignment Breakdown', $seq_sum_content );
 686
 687 print blue_section_html( 'Putative Splice Variants', $sv_content );
 688
 689 print blue_section_html( 'Putative Alleles', $al_content );
 690
 691 #print blue_section_html('Output Sequences','<table width="100%" cellpadding="5" cellspacing="0" border="0">'.$seq_output_content.'</table>');
 692
 693 $page->footer();
 694
 695 sub get_combined_content {
 696     my $alignment       = shift;
 697     my $align_member_nr = $alignment->get_member_nr();
 698     my $ol_nr           = $alignment->get_overlap_num();
 699
 700     my $id_list_ref   = $alignment->get_nonhidden_member_ids;
 701     my $seq_ref       = $alignment->get_nopad_seqs();
 702     my $align_seq_ref = $alignment->get_seqs();
 703     my $ol_seq_ref    = $alignment->get_overlap_seqs();
 704     my $sp_ref        = $alignment->get_member_species();
 705
 706     my ( $cds_ids_ref, $cds_seq_ref, $cds_align_seq_ref, $cds_ol_seq_ref ) =
 707       ( undef, undef, undef, undef );
 708     if ($cds_alignment) {
 709         $cds_seq_ref       = $cds_alignment->get_nopad_seqs();
 710         $cds_align_seq_ref = $cds_alignment->get_seqs();
 711         $cds_ol_seq_ref    = $cds_alignment->get_overlap_seqs();
 712     }
 713
 714     my %sp = %$sp_ref;
 715
 716     my $file_links     = "";
 717     my $cds_file_links = "";
 718     my $tmp_seq_file;
 719     my ( $seq_file, $cds_file, $seq_align_file );
 720     ## Protein/Genomic Files:
 721     if ($seq_ref) {
 722         my $link;
 723         ( $seq_file, $link ) =
 724           build_seq_file( $id_list_ref, $seq_ref, "All Sequences (No Gaps)" );
 725         $file_links .= $link . "<br />";
 726     }
 727     else { $file_links .= "All Sequences (No Gaps)<br>" }
 728
 729     if ($align_seq_ref) {
 730         my $link;
 731         ( $seq_align_file, $link ) =
 732           build_seq_file( $id_list_ref, $align_seq_ref,
 733             "All Alignment Sequences (Gapped)" );
 734         $file_links .= $link . "<br />";
 735     }
 736     else { $file_links .= "All Alignment Sequences (Gapped)<br/>"; }
 737
 738     if ( $ol_nr > 0 ) {
 739         ($file_links) .=
 740           build_seq_file( $id_list_ref, $ol_seq_ref, "Overlap of Sequences" );
 741     }
 742     else { $file_links .= "Overlap of Sequences" }
 743
 744     ## CDS Files, if the CDS alignment was made from cds_file:
 745     if ($cds_seq_ref) {
 746         my $link;
 747         ( $cds_file, $link ) =
 748           build_seq_file( $id_list_ref, $cds_seq_ref,
 749             "Coding Sequences (No Gaps)" );
 750         $cds_file_links .= $link . "<br />";
 751     }
 752     if ($cds_align_seq_ref) {
 753         $cds_file_links .=
 754           build_seq_file( $id_list_ref, $cds_align_seq_ref,
 755             "Coding Aligned Sequences (Gapped)" )
 756           . "<br />";
 757     }
 758     if ( $ol_nr > 0 && $cds_ol_seq_ref ) {
 759         $cds_file_links .=
 760           build_seq_file( $id_list_ref, $cds_ol_seq_ref,
 761             "Overlap of Coding Sequences" );
 762     }
 763     elsif ($cds_ol_seq_ref) { $cds_file_links .= "Overlap of Coding Sequences" }
 764
 765     ############################################################
 766     # Summary, Files, And Alignment Parameter Changes
 767
 768     my $combined_content = "<tr><td>";
 769
 770     $combined_content .= "<table style='width:100\%;border-spacing:0px'><tr>";
 771
 772     #Summary Content
 773     $combined_content .=
 774       "<td style='width:50%;vertical-align:middle;'><table width='100%'>";
 775     $combined_content .= "<tr><th>Type</th><td>";
 776     if ( $type eq 'nt' ) {
 777         $combined_content .= "Nucleotide</td></tr>";
 778     }
 779     else {
 780         $combined_content .= "Peptide</td></tr>";
 781     }
 782     $combined_content .=
 783       "<tr><th>No. Alignment Members</th><td>$align_member_nr</td></tr>";
 784     $combined_content .=
 785       "<tr><th>Selected Range</th><td>$start_value - $end_value</td></tr>";
 786     $combined_content .= "<tr><th>Overlap</th><td>$ol_nr";
 787     if ( $type eq 'nt' ) {
 788         $combined_content .= " bp</td></tr>";
 789     }
 790     else {
 791         $combined_content .= " aa</td></tr>";
 792     }
 793
 794     if ($cds_file_links) {
 795         $combined_content .=
 796           "<tr><th>Protein Files:</th><td>$file_links</td></tr>";
 797         $combined_content .=
 798           "<tr><th>CDS Files:</th><td>$cds_file_links</td></tr>";
 799     }
 800     else {
 801         $combined_content .=
 802           "<tr><th>Sequence Files:</th><td>$file_links</td></tr>";
 803     }
 804
 805     my $rerun_file;
 806     ($cds_file) ? ( $rerun_file = $cds_file ) : ( $rerun_file = $seq_file );
 807     my ($tmp_seq_name)       = $rerun_file     =~ /([^\/]+)$/;
 808     my ($tmp_seq_align_name) = $seq_align_file =~ /([^\/]+)$/;
 809     my $target = "_SGN_TREE_" . int( rand(10000000) );
 810
 811     my $available_seq_count = 0;
 812     while ( my ( $id, $seq ) = each %$seq_ref ) {
 813         $available_seq_count++ if $seq =~ /\S/;
 814     }
 815     if ( $available_seq_count > 1 ) {
 816         $combined_content .= <<HTML;
 817
 818         <tr><td colspan=2><br />
 819         <a href='index.pl?temp_file=$tmp_seq_name&maxiters=$maxiters&type=$type&format=fasta_unaligned&title=$title'>&gt;&gt; Re-run Alignment On Selected Range ($available_seq_count)</a>
 820         <br />This will allow you to specify greater accuracy by increasing the number of iterations performed by Muscle<br /><br />
 821         <a target="$target" href="/tools/tree_browser/index.pl?align_temp_file=$tmp_seq_align_name&align_type=pep&tree_from_align=1">
 822         &gt;&gt; Calculate Tree from Selected Range ($available_seq_count)</a><br />
 823         Use a quick neighbor-joining algorithm (Kimura) to calculate a tree based on the protein alignment, and analyze both in the combined alignment/tree browser.
 824         </td></tr>
 825 HTML
 826     }
 827     else {
 828         $combined_content .= <<HTML;
 829         <tr><td colspan=2><br />
 830         Re-alignment and Tree Calculation cannot be performed, since there are less than two sequences for which data is available at this range
 831         </td></tr>
 832 HTML
 833     }
 834     $combined_content .= "</table></td>";
 835
 836     ### Modify and submit alignment parameters
 837     ##########################################
 838
 839     $combined_content .=
 840       "<td><form method='post' action='show_align.pl' name='show_align'>";
 841
 842     #hidden parameters
 843     $combined_content .= "<input type='hidden' name='title' value='$title' />";
 844     $combined_content .=
 845       "<input type='hidden' name='format' value='$format' />";
 846     $combined_content .=
 847       "<input type='hidden' name='temp_file' value='$temp_file' />";
 848     $combined_content .=
 849 "<input type='hidden' name='cds_temp_file' value='$cds_temp_file_name' />";
 850     $combined_content .= "<input type='hidden' name='type' value='$type' />";
 851
 852     #start and end value
 853     $combined_content .=
 854 "<table style='border:0px;border-spacing:0px'><tr><th>Start Value</th><th>End Value</th></tr>";
 855     $combined_content .=
 856 "<tr><td><input type='text' name='start_value' value='$start_value' /></td>";
 857     $combined_content .=
 858 "<td><input type='text' name='end_value' value='$end_value' /></td></tr></table>";
 859
 860     #Sequences to leave out
 861     $combined_content .=
 862       "<b>IDs of Sequences to Leave Out</b> (separated by spaces)<br>";
 863     $combined_content .=
 864 "<textarea name='hide_seqs' id='hide_seqs' rows='2' cols='50'>$hide_seqs</textarea><br>";
 865
 866     my $show_breakdown_checked = "";
 867     $show_breakdown_checked = "CHECKED" if ($show_breakdown);
 868     $combined_content .=
 869 "<b><input type='checkbox' name='show_breakdown' $show_breakdown_checked> Calculate Coverage and Overlap Details</b> (Time Intensive)<br />";
 870
 871     my $show_sv_checked = "";
 872     $show_sv_checked = "CHECKED" if ($show_sv);
 873     $combined_content .=
 874 "<b><input type='checkbox' name='show_sv' $show_sv_checked> Calculate Splice Variants and Alleles</b> (Time Intensive)";
 875     $combined_content .= "<table style='border:0px;text-align:center'>";
 876     $combined_content .=
 877 "<tr><th>% Similarity</th><th></th><th>Shared</th><th></th><th>InDel Limit</th></tr>";
 878     $combined_content .=
 879 "<tr><td><input type='text' size=10 name='sv_similarity' value='$sv_similarity'></td><td width=3>&nbsp;</td>";
 880     $combined_content .=
 881 "<td><input type='text' size=10 name='sv_shared' value='$sv_shared'></td><td width=3>&nbsp;</td>";
 882     $combined_content .=
 883 "<td><input type='text' size=10 name='sv_indel' value='$sv_indel'></td></tr></table>";
 884
 885     #submit
 886     $combined_content .=
 887       "<input type='submit' value='Change Alignment Details' /></form></td>";
 888
 889     $combined_content .= "</tr></table>";
 890     $combined_content .= "</td></tr>";
 891
 892     return $combined_content;
 893 }
 894
 895 sub get_sv_al_content {
 896     my $alignment = shift;
 897     my ( $ob_ref, $pi_ref, $sv_sp_ref ) = $alignment->get_sv_candidates();
 898     my %ob    = %$ob_ref;
 899     my %pi    = %$pi_ref;
 900     my %sv_sp = %$sv_sp_ref;
 901     my ( $sv_content, $al_content ) = ( "", "" );
 902     if ( keys %ob ) {
 903         $sv_content =
 904           "<table width='100%' cellpadding='5' cellspacing='0' border='1'>";
 905         $sv_content .= "<tr>";
 906         $sv_content .= "<th>Species</th>";    # if $show_species;
 907         $sv_content .=
 908 "<th>Sequence id</th><th>Sequence id</th><th>Overlap Bases</th><th>\% Similarity</th></tr>";
 909         foreach my $first_key ( keys %ob ) {
 910             foreach my $second_key ( keys %{ $ob{$first_key} } ) {
 911                 $sv_content .= "<tr>";
 912                 $sv_content .=
 913                   "<td>$sv_sp{$first_key}</td>";    # if $show_species;
 914                 $sv_content .=
 915 "<td>$first_key</td><td>$second_key</td><td>$ob{$first_key}{$second_key}</td><td>$pi{$first_key}{$second_key}</td></tr>";
 916             }
 917         }
 918         $sv_content .= "</table>";
 919         $sv_content .=
 920           "&nbsp;Reduce page load time by <a href=\"show_align.pl?";
 921         $sv_content .= hash2param( \%PARAM, { show_sv => 0 } );
 922         $sv_content .= "\">hiding this table</a>";
 923
 924     }
 925     else {
 926         $sv_content =
 927           "None were found.  Reduce page load time by <a href=\"show_align.pl?";
 928         $sv_content .= hash2param( \%PARAM, { show_sv => 0 } );
 929         $sv_content .= "\">hiding this table</a>";
 930
 931     }
 932
 933     ###########################################################
 934     #Find alleles
 935
 936     my ( $al_ob_ref, $al_pi_ref, $al_sp_ref ) =
 937       $alignment->get_allele_candidates();
 938     my %al_ob = %$al_ob_ref;
 939     my %al_pi = %$al_pi_ref;
 940     my %al_sp = %$al_sp_ref;
 941
 942     if ( keys %al_ob ) {
 943         $al_content =
 944           "<table width='100%' cellpadding='5' cellspacing='0' border='1'>";
 945         $al_content .= "<tr>";
 946         $al_content .= "<th>Species</th>";    # if $show_species;
 947         $al_content .=
 948 "<th>Sequence id</th><th>Sequence id</th><th>Overlap Bases</th><th>\% Similarity</th></tr>";
 949         foreach my $first_key ( keys %al_ob ) {
 950             foreach my $second_key ( keys %{ $al_ob{$first_key} } ) {
 951                 $al_content .= "<tr>";
 952                 $al_content .=
 953                   "<td>$al_sp{$first_key}</td>";    # if $show_species;
 954                 $al_content .=
 955 "<td>$first_key</td><td>$second_key</td><td>$al_ob{$first_key}{$second_key}</td><td>$al_pi{$first_key}{$second_key}</td></tr>";
 956             }
 957         }
 958         $al_content .= "</table>";
 959         $al_content .=
 960           "&nbsp;Reduce page load time by <a href=\"show_align.pl?";
 961         $al_content .= hash2param( \%PARAM, { show_sv => 0 } );
 962         $al_content .= "\">hiding this table</a>";
 963     }
 964     else {
 965         $al_content =
 966           "None were found.  Reduce page load time by <a href=\"show_align.pl?";
 967         $al_content .= hash2param( \%PARAM, { show_sv => 0 } );
 968         $al_content .= "\">hiding this table</a>";
 969     }
 970     return ( $sv_content, $al_content );
 971 }
 972
 973 sub get_seq_sum_content {
 974
 975     my $alignment = shift;
 976     ###########################################################
 977     #Analyze sequences and create analysis content
 978     my $member_ids_ref = $alignment->get_member_ids();
 979     my $ov_score_ref   = $alignment->get_all_overlap_score();
 980     my $medium_ref     = $alignment->get_all_medium();
 981     my ( $head_ref, $tail_ref ) = $alignment->get_all_range();
 982     my $ng_ref = $alignment->get_all_nogap_length();
 983     my $sp_ref = $alignment->get_member_species();
 984
 985     my @member_ids = @$member_ids_ref;
 986     my %ov_score   = %$ov_score_ref;
 987     my %medium     = %$medium_ref;
 988     my %head       = %$head_ref;
 989     my %tail       = %$tail_ref;
 990     my %ng         = %$ng_ref;
 991     my %gap        = ();
 992     my %species    = %$sp_ref;
 993
 994     foreach ( keys %ng ) {
 995         $gap{$_} = $tail{$_} - $head{$_} + 1 - $ng{$_};
 996     }
 997     my $show_species = 0;
 998     foreach ( keys %species ) {
 999         $show_species = 1 if $species{$_};
1000     }
1001
1002     my $seq_sum_content = "";
1003     $seq_sum_content .=
1004       "<table width='100%' cellpadding='5' cellspacing='0' border='1'>";
1005     $seq_sum_content .= "<tr><th>Sequence ID</th>";
1006     $seq_sum_content .= "<th>Species</th>" if $show_species;
1007     $seq_sum_content .=
1008 "<th>Coverage Range</th><th>Bases</th><th>Gaps</th><th>Medium</th><th>Overlap Score</th></tr>";
1009
1010     foreach (@member_ids)
1011     { ##Access the align_seqs members this way, instead of by the keys of the hashes,  so that the sequences are grouped together
1012         $seq_sum_content .= "<tr><td>$_</td>";
1013         $seq_sum_content .= "<td>$species{$_}</td>" if $show_species;
1014         $seq_sum_content .=
1015 "<td>$head{$_} - $tail{$_}</td><td>$ng{$_}</td><td>$gap{$_}</td><td>$medium{$_}</td><td>$ov_score{$_}</td></tr>";
1016     }
1017     $seq_sum_content .= "</table>";
1018     $seq_sum_content .=
1019       "&nbsp;Reduce page load time by <a href=\"show_align.pl?";
1020     $seq_sum_content .= hash2param( \%PARAM, { show_breakdown => 0 } );
1021     $seq_sum_content .= "\">hiding this table</a>";
1022     return $seq_sum_content;
1023 }
1024
1025 sub build_gapped_cds_file {
1026     my $prot_file = shift;
1027     my $cds_file  = shift;
1028     my $instream  = Bio::SeqIO->new( -file => $prot_file, -format => 'fasta' );
1029     my $cds_instream =
1030       Bio::SeqIO->new( -file => $cds_file, -format => 'fasta' );
1031
1032     my %cds_seq;
1033     my %prot_seq;
1034
1035     while ( my $in = $instream->next_seq ) {
1036         my $id = $in->id();
1037         $prot_seq{$id} = $in->seq();
1038     }
1039     while ( my $in = $cds_instream->next_seq ) {
1040         my $id = $in->id();
1041         $cds_seq{$id} = $in->seq();
1042     }
1043     my $temp_fh = File::Temp->new(
1044         DIR    => $PATH,
1045         SUFFIX => '.txt',
1046         UNLINK => 0,
1047     );
1048     my $new_cds_temp_file = $temp_fh->filename;
1049     my $i                 = 0;
1050     my $st                = time();
1051     while ( my ( $id, $prot_seq ) = each %prot_seq ) {
1052         $i++;
1053         my $cds = $cds_seq{$id};
1054
1055         my $member = CXGN::Phylo::Alignment::Member->new(
1056             id          => $id,
1057             seq         => $cds,
1058             type        => "cds",
1059             cds_nocheck => 1
1060         );
1061         $member->cds_insert_gaps($prot_seq);
1062         my $newseq = $member->get_seq();
1063         print $temp_fh ">$id\n$newseq\n";
1064     }
1065     my $et   = time();
1066     my $rate = ( $et - $st ) / $i;
1067
1068     #die $rate ." seconds/member\n";
1069
1070     $temp_fh->close();
1071     return $new_cds_temp_file;
1072 }
1073
1074 sub build_seq_file {
1075     my $id_list_ref = shift;
1076     my $seq_ref     = shift;
1077     my $linkname    = shift;
1078     my $temp_fh     = File::Temp->new(
1079         DIR    => $PATH,
1080         SUFFIX => '.txt',
1081         UNLINK => 0,
1082     );
1083     my $outstream = Bio::SeqIO->new( -fh => $temp_fh, -format => 'fasta' );
1084     foreach (@$id_list_ref) {
1085         my $seq = Bio::Seq->new( -seq => $seq_ref->{$_}, -id => $_ );
1086         $outstream->write_seq($seq) if $seq_ref->{$_} =~ /\w/;
1087     }
1088     my $tempname = $temp_fh->filename;
1089     close $temp_fh;
1090     $tempname =~ s/$HTML_ROOT_PATH//;
1091     return ( $tempname,
1092         "<a target=\"sgn_align_file\" href=\"$tempname\">$linkname</a>" );
1093 }
1094
1095 sub input_err_page {
1096     my $input_err_page = shift;
1097     my $err_message    = shift;
1098     $input_err_page->header();
1099     my ($disp_err_message) = $err_message =~ /(.*) at \/usr\/local\//;
1100     $disp_err_message = $err_message unless $disp_err_message;
1101     print page_title_html("Combined Tree/Alignment Error");
1102     print "$disp_err_message<br><br><a href=\"index.pl\">Start over</a><br>";
1103     $input_err_page->footer();
1104     exit;
1105 }
1106
1107 sub fasta_check {
1108     my ( $file, $page, $n ) = @_;
1109     my ($filename) = $file =~ /([^\/]+)$/;
1110     my $count        = 0;
1111     my $maxlen       = 0;
1112     my $total_length = 0;
1113     my $instream = Bio::SeqIO->new( -file => $file, -format => 'fasta' );
1114     my $entry = $instream->next_seq();
1115     unless ( $entry && $entry->id && $entry->seq ) {
1116         input_err_page( $page, "FASTA needs IDs and Sequences [$filename]" );
1117     }
1118     $count++;
1119     $maxlen = $entry->length;
1120     $total_length += $entry->length;
1121
1122     $entry  = $instream->next_seq;
1123     unless ( $entry && $entry->id && $entry->seq ) {
1124         input_err_page( $page, "FASTA must have at least two valid sequences" );
1125     }
1126     $count++;
1127     $maxlen = $entry->length if $entry->length > $maxlen;
1128     $total_length += $entry->length;
1129
1130     while ( $entry = $instream->next_seq() ) {
1131         unless ( $entry->id && $entry->seq ) {
1132             input_err_page( $page, "Every entry must have ID AND sequence" );
1133         }
1134         $maxlen = $entry->length if $entry->length > $maxlen;
1135         $total_length += $entry->length;
1136         $count++;
1137     }
1138     return ( $count, $maxlen, $total_length );
1139 }
1140
1141 sub run_muscle {
1142
1143     my ( $page, $run ) = @_;
1144     my @local_run_output = "";
1145     my $command_line     = "";
1146     my $old_temp_file    = $temp_file;
1147     if ( $run eq "local" ) {
1148         my $wd = `pwd`;
1149         chomp $wd;
1150         my $result_file = $temp_file;
1151         $result_file .= ".aligned.fasta" unless $result_file =~ /\.aligned\.fasta$/;
1152         chdir $PATH;
1153
1154 #Within a limit of less than 50 sequences, muscle should run within 3 seconds. Wawa-woo-a!
1155         $command_line =
1156           "muscle -in $temp_file -out $result_file -maxiters $maxiters";
1157         print STDERR "Running: $command_line\n";
1158         @local_run_output = `$command_line `;
1159         $temp_file        = $result_file;
1160         if ( -f $cds_temp_file ) {
1161
1162             #The CDS temp file needs to be gapped
1163             $cds_temp_file =
1164               build_gapped_cds_file( $temp_file, $cds_temp_file );
1165         }
1166         chdir $wd;
1167     }
1168
1169     ## RUN ON CLUSTER NODES ###################################################
1170
1171     if ( $run eq "cluster" ) {
1172
1173         my ( undef, $filename ) = tempfile(
1174             TEMPLATE => "jobXXXXXX",
1175             DIR      => $CLUSTER_SHARED_TEMPDIR
1176         );
1177
1178         copy( $temp_file, $filename )
1179           || die "Can't copy $temp_file to $filename";
1180
1181         chdir $CLUSTER_SHARED_TEMPDIR;
1182
1183         print STDERR "Running on cluster: $command_line\n";
1184
1185         CXGN::Tools::Run->temp_base($CLUSTER_SHARED_TEMPDIR);
1186
1187         # generate a .req file that will indicate to the wait.pl script
1188         # that such a request has been made
1189         #
1190         system("touch $filename.req");
1191
1192         my $old_wd = `pwd`;
1193
1194         my $job = CXGN::Tools::Run->run_cluster(
1195             "muscle",
1196             -in       => "$filename",
1197             -out      => "$filename.aln",
1198             -maxiters => $maxiters,
1199             { #if the next line is uncommented, we get the weird STDERR problem I was talking about --ccarpita
1200                     #out_file => "$filename.aln",
1201                 err_file    => $filename . ".STDERR",
1202                 working_dir => $CLUSTER_SHARED_TEMPDIR,
1203                 temp_base   => $CLUSTER_SHARED_TEMPDIR,
1204                 # don't block and wait if the cluster looks full
1205                 max_cluster_jobs => 1_000_000_000,
1206             }
1207         );
1208
1209         my ( undef, $job_file ) =
1210           tempfile( DIR => $PATH, TEMPLATE => "object_XXXXXX" );
1211
1212         store( $job, $job_file )
1213           or $page->message_page(
1214             "An error occurred in the serializaton of the job object");
1215
1216         my $job_file_base = File::Basename::basename($job_file);
1217
1218         print STDERR "SUBMITTED JOB WITH JOBID: " . $job->job_id() . "\n";
1219         my $cds_temp_filename = File::Basename::basename($cds_temp_file);
1220
1221         # url encode the destination pass_back page.
1222         my $pass_back =
1223 "./align_viewer/show_align.pl?&amp;title=$title&amp;type=$type&amp;force_gap_cds=1&amp;cds_temp_file=$cds_temp_filename&amp;temp_file=";
1224         $pass_back =~ s/([^A-Za-z0-9])/sprintf("%%%02X", ord($1))/seg;
1225         my $message =
1226           "Running Muscle v3.6 ($SEQ_COUNT sequences), please wait ...";
1227
1228         chdir $old_wd;
1229
1230         $page->client_redirect(
1231 "../wait.pl?tmp_app_dir=/align_viewer&amp;job_file=$job_file_base&amp;out_file_override=$filename.aln&amp;message=$message&amp;redirect=$pass_back"
1232         );
1233
1234     }
1235 }
1236
1237 sub convert_clustal_fasta {
1238     my $clustal_file = shift;
1239     my $fasta_file   = shift;
1240
1241     my $in = Bio::AlignIO->new(
1242         -file   => $clustal_file,
1243         -format => 'clustalw'
1244     );
1245     my $cl_out = Bio::AlignIO->new(
1246         -file   => ">$fasta_file",
1247         -format => 'fasta',
1248     );
1249
1250     while ( my $aln = $in->next_aln() ) {
1251         $aln->set_displayname_flat();
1252         $cl_out->write_aln($aln);
1253     }
1254     $cl_out->close();
1255     $in->close();
1256 }
1257