t/Tools/AmpliconSearch.t

   1 BEGIN {
   2     use Bio::Root::Test;
   3
   4     test_begin(-tests => 185);
   5
   6     use_ok 'Bio::PrimarySeq';
   7     use_ok 'Bio::SeqFeature::Primer';
   8     use_ok 'Bio::Tools::AmpliconSearch';
   9 }
  10
  11
  12 my ($search, $amplicon, $seq, $seq2, $forward, $forward2, $reverse, $reverse2,
  13     $primer, $primer_seq, $annotated, $num_feats, $template_seq, $rna);
  14
  15
  16 # Basic object
  17
  18 ok $search = Bio::Tools::AmpliconSearch->new(), 'Basic';
  19 isa_ok $search, 'Bio::Tools::AmpliconSearch';
  20
  21
  22 # Forward primer only
  23
  24 $seq = Bio::PrimarySeq->new(
  25    -seq => 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac',
  26 );
  27
  28 $rna = Bio::PrimarySeq->new(
  29    -seq => 'acgAAACUUAAAGGAAUUGACGGacguacguacguGUACACACCGCCCGUacguac',
  30 );
  31
  32
  33 $forward = Bio::PrimarySeq->new(
  34    -seq => 'AAACTTAAAGGAATTGACGG',
  35 );
  36 ok $search = Bio::Tools::AmpliconSearch->new(
  37    -template       => $seq,
  38    -fwd_primer     => $forward,
  39    -attach_primers => 1,
  40 ), 'Forward primer only';
  41 is $search->fwd_primer->seq, 'AAACTTAAAGGAATTGACGG';
  42 is $search->rev_primer, undef;
  43 ok $template_seq = $search->template;
  44 isa_ok $template_seq, 'Bio::Seq';
  45 is $template_seq->seq, 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac';
  46 ok $amplicon = $search->next_amplicon;
  47 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
  48 is $amplicon->start, 4;
  49 is $amplicon->end, 56;
  50 is $amplicon->strand, 1;
  51 is $amplicon->seq->seq, 'AAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac';
  52 ok $primer = $amplicon->fwd_primer;
  53 ok $primer_seq = $primer->seq;
  54 is $primer_seq->seq, 'AAACTTAAAGGAATTGACGG';
  55 is $primer->start, 4;
  56 is $primer->end, 23;
  57 is $primer->strand, 1;
  58 is $amplicon = $search->next_amplicon, undef;
  59
  60
  61 # Reverse primer only
  62
  63 $seq = Bio::PrimarySeq->new(
  64    -seq => 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac',
  65 );
  66 $reverse = Bio::SeqFeature::Primer->new(
  67    -seq => 'ACGGGCGGTGTGTAC',
  68 );
  69 ok $search = Bio::Tools::AmpliconSearch->new(
  70    -template       => $seq,
  71    -rev_primer     => $reverse,
  72    -attach_primers => 0,
  73 ), 'Reverse primer only';
  74 is $search->fwd_primer, undef;
  75 is $search->rev_primer->seq->seq, 'ACGGGCGGTGTGTAC';
  76 is $search->template->seq, 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac';
  77 ok $amplicon = $search->next_amplicon;
  78 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
  79 is $amplicon->start, 1;
  80 is $amplicon->end, 50;
  81 is $amplicon->strand, 1;
  82 is $amplicon->seq->seq, 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGT';
  83 is $amplicon->fwd_primer, undef;
  84 is $amplicon->rev_primer, undef;
  85 is $search->next_amplicon, undef;
  86
  87
  88 # Forward and reverse primers, no amplicon
  89
  90 $seq = Bio::PrimarySeq->new(
  91    -seq => 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac',
  92 );
  93 $forward = Bio::PrimarySeq->new(
  94    -seq => 'AAACTTAAAGGAATTGACGG',
  95 );
  96 $reverse = Bio::PrimarySeq->new(
  97    -seq => 'CCCCCCCCCC',
  98 );
  99 ok $search = Bio::Tools::AmpliconSearch->new(
 100    -template   => $seq,
 101    -fwd_primer => $forward,
 102    -rev_primer => $reverse,
 103 ), 'Two primers, no match';
 104 is $search->fwd_primer->seq, 'AAACTTAAAGGAATTGACGG';
 105 is $search->rev_primer->seq, 'CCCCCCCCCC';
 106 is $search->template->seq, 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac';
 107 is $search->next_amplicon, undef;
 108
 109
 110 # Degenerate forward and reverse primers from file, single amplicon
 111
 112 ok $search = Bio::Tools::AmpliconSearch->new(
 113    -template    => $seq,
 114    -primer_file => test_input_file('forward_reverse_primers.fa'),
 115 ), 'Two degenerate primers from a file';
 116 is $search->fwd_primer->seq, 'AAACTYAAAKGAATTGRCGG';
 117 is $search->rev_primer->seq, 'ACGGGCGGTGTGTRC';
 118 is $search->template->seq, 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac';
 119 ok $amplicon = $search->next_amplicon;
 120 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 121 is $amplicon->start, 4;
 122 is $amplicon->end, 50;
 123 is $amplicon->strand, 1;
 124 is $amplicon->seq->seq, 'AAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGT';
 125 is $search->next_amplicon, undef;
 126
 127
 128 # Same thing but with RNA template
 129
 130 ok $search = Bio::Tools::AmpliconSearch->new(
 131    -template    => $rna,
 132    -primer_file => test_input_file('forward_reverse_primers.fa'),
 133 ), 'RNA template';
 134 is $search->fwd_primer->seq, 'AAACTYAAAKGAATTGRCGG';
 135 is $search->rev_primer->seq, 'ACGGGCGGTGTGTRC';
 136 is $search->template->seq, 'acgAAACUUAAAGGAAUUGACGGacguacguacguGUACACACCGCCCGUacguac';
 137 ok $amplicon = $search->next_amplicon;
 138 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 139 is $amplicon->start, 4;
 140 is $amplicon->end, 50;
 141 is $amplicon->strand, 1;
 142 is $amplicon->seq->seq, 'AAACUUAAAGGAAUUGACGGacguacguacguGUACACACCGCCCGU';
 143 is $search->next_amplicon, undef;
 144
 145
 146 # Forward primer only, in sequence file
 147
 148 ok $search = Bio::Tools::AmpliconSearch->new(
 149    -primer_file => test_input_file('forward_primer.fa'),
 150 ), 'Forward primer from file';
 151 ok $search->template($seq);
 152 is $search->fwd_primer->seq, 'AAACTTAAAGGAATTGACGG';
 153 is $search->rev_primer, undef;
 154 ok $template_seq = $search->template;
 155 isa_ok $template_seq, 'Bio::Seq';
 156 is $template_seq->seq, 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac';
 157 ok $amplicon = $search->next_amplicon;
 158 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 159 is $amplicon->start, 4;
 160 is $amplicon->end, 56;
 161 is $amplicon->strand, 1;
 162 is $amplicon->seq->seq, 'AAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac';
 163 is $amplicon = $search->next_amplicon, undef;
 164
 165
 166 # Multiple amplicons
 167
 168 $seq = Bio::PrimarySeq->new(
 169    -seq => 'acgtaCCCCacgtacgtacTTTTTTacgtaCCCCacgtaTTTTTTacgtacgtac',
 170 );
 171 $forward = Bio::PrimarySeq->new(
 172    -seq => 'CCCC',
 173 );
 174 $reverse = Bio::PrimarySeq->new(
 175    -seq => 'AAAAAA',
 176 );
 177 ok $search = Bio::Tools::AmpliconSearch->new(
 178    -template   => $seq,
 179    -fwd_primer => $forward,
 180    -rev_primer => $reverse,
 181 ), 'Multiple amplicons';
 182 ok $amplicon = $search->next_amplicon;
 183 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 184 is $amplicon->start, 6;
 185 is $amplicon->end, 25;
 186 is $amplicon->strand, 1;
 187 is $amplicon->seq->seq, 'CCCCacgtacgtacTTTTTT';
 188 ok $amplicon = $search->next_amplicon;
 189 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 190 is $amplicon->start, 31;
 191 is $amplicon->end, 45;
 192 is $amplicon->strand, 1;
 193 is $amplicon->seq->seq, 'CCCCacgtaTTTTTT';
 194 is $search->next_amplicon, undef;
 195
 196
 197 # Amplicon on reverse strand
 198
 199 $seq = Bio::PrimarySeq->new(
 200    # Reverse-complement of previous sequence... should have same amplicons
 201    -seq => 'gtacgtacgtAAAAAAtacgtGGGGtacgtAAAAAAgtacgtacgtGGGGtacgt',
 202 );
 203 $forward = Bio::PrimarySeq->new(
 204    -seq => 'CCCC',
 205 );
 206 $reverse = Bio::PrimarySeq->new(
 207    -seq => 'AAAAAA',
 208 );
 209 ok $search = Bio::Tools::AmpliconSearch->new(
 210    -template   => $seq,
 211    -fwd_primer => $forward,
 212    -rev_primer => $reverse,
 213 ), 'Amplicon on reverse strand';
 214 ok $amplicon = $search->next_amplicon;
 215 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 216 is $amplicon->start, 11;
 217 is $amplicon->end, 25;
 218 is $amplicon->strand, -1;
 219 is $amplicon->seq->seq, 'CCCCacgtaTTTTTT';
 220 ok $amplicon = $search->next_amplicon;
 221 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 222 is $amplicon->start, 31;
 223 is $amplicon->end, 50;
 224 is $amplicon->strand, -1;
 225 is $amplicon->seq->seq, 'CCCCacgtacgtacTTTTTT';
 226 is $search->next_amplicon, undef;
 227
 228
 229 # Overlapping amplicons (1)
 230
 231 $seq = Bio::PrimarySeq->new(
 232    -seq => 'acgtaCCCCacgtacgtacgtacgCCCCacgtaTTTTTTacgtacgtac',
 233 );
 234 $forward = Bio::PrimarySeq->new(
 235    -seq => 'CCCC',
 236 );
 237 $reverse = Bio::PrimarySeq->new(
 238    -seq => 'AAAAAA',
 239 );
 240 ok $search = Bio::Tools::AmpliconSearch->new(
 241    -template   => $seq,
 242    -fwd_primer => $forward,
 243    -rev_primer => $reverse,
 244 ), 'Overlapping amplicons (1)';
 245 ok $amplicon = $search->next_amplicon;
 246 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 247 is $amplicon->start, 25;
 248 is $amplicon->end, 39;
 249 is $amplicon->strand, 1;
 250 is $amplicon->seq->seq, 'CCCCacgtaTTTTTT';
 251 is $search->next_amplicon, undef;
 252
 253
 254 # Overlapping amplicons (2)
 255
 256 $seq = Bio::PrimarySeq->new(
 257    -seq => 'acgtaCCCCacgtacgtacgtacgTTTTTTacgtaTTTTTTacgtacgtac',
 258 );
 259 $forward = Bio::PrimarySeq->new(
 260    -seq => 'CCCC',
 261 );
 262 $reverse = Bio::PrimarySeq->new(
 263    -seq => 'AAAAAA',
 264 );
 265 ok $search = Bio::Tools::AmpliconSearch->new(
 266    -template   => $seq,
 267    -fwd_primer => $forward,
 268    -rev_primer => $reverse,
 269 ), 'Overlapping amplicons (2)';
 270 ok $amplicon = $search->next_amplicon;
 271 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 272 is $amplicon->start, 6;
 273 is $amplicon->end, 30;
 274 is $amplicon->strand, 1;
 275 is $amplicon->seq->seq, 'CCCCacgtacgtacgtacgTTTTTT';
 276 is $search->next_amplicon, undef;
 277
 278
 279 # Overlapping amplicons (3)
 280
 281 $seq = Bio::PrimarySeq->new(
 282    -seq => 'acgtaCCCCacgtacgCCCCacgtacgaTTTTTTacgtaTTTTTTaaaaaaaaaa',
 283 );
 284 $forward = Bio::PrimarySeq->new(
 285    -seq => 'CCCC',
 286 );
 287 $reverse = Bio::PrimarySeq->new(
 288    -seq => 'AAAAAA',
 289 );
 290 ok $search = Bio::Tools::AmpliconSearch->new(
 291    -template   => $seq,
 292    -fwd_primer => $forward,
 293    -rev_primer => $reverse,
 294 ), 'Overlapping amplicons (3)';
 295 ok $amplicon = $search->next_amplicon;
 296 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 297 is $amplicon->start, 17;
 298 is $amplicon->end, 34;
 299 is $amplicon->strand, 1;
 300 is $amplicon->seq->seq, 'CCCCacgtacgaTTTTTT';
 301 is $search->next_amplicon, undef;
 302
 303
 304 # Amplicons on both strands
 305
 306 $seq = Bio::PrimarySeq->new(
 307    -seq => 'acgtaCCCCacgtaacgtaTTTTTTacAAAAAAgtGGGGacgtaacgta',
 308 );
 309 $forward = Bio::PrimarySeq->new(
 310    -seq => 'CCCC',
 311 );
 312 $reverse = Bio::PrimarySeq->new(
 313    -seq => 'AAAAAA',
 314 );
 315 ok $search = Bio::Tools::AmpliconSearch->new(
 316    -template   => $seq,
 317    -fwd_primer => $forward,
 318    -rev_primer => $reverse,
 319 ), 'Amplicons on both strands';
 320 ok $amplicon = $search->next_amplicon;
 321 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 322 is $amplicon->start, 6;
 323 is $amplicon->end, 25;
 324 is $amplicon->strand, 1;
 325 is $amplicon->seq->seq, 'CCCCacgtaacgtaTTTTTT';
 326 ok $amplicon = $search->next_amplicon;
 327 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 328 is $amplicon->start, 28;
 329 is $amplicon->end, 39;
 330 is $amplicon->strand, -1;
 331 is $amplicon->seq->seq, 'CCCCacTTTTTT';
 332 is $search->next_amplicon, undef;
 333
 334
 335 # Overlapping amplicons on both strands
 336
 337 $seq = Bio::PrimarySeq->new(
 338    -seq => 'acgtaCCCCacgtacgtacagAAAAAATTTTTTacgtGGGGacgtacgtac',
 339 );
 340 $forward = Bio::PrimarySeq->new(
 341    -seq => 'CCCC',
 342 );
 343 $reverse = Bio::PrimarySeq->new(
 344    -seq => 'AAAAAA',
 345 );
 346 ok $search = Bio::Tools::AmpliconSearch->new(
 347    -template   => $seq,
 348    -fwd_primer => $forward,
 349    -rev_primer => $reverse,
 350 ), 'Overlapping amplicons on both strands';
 351 ok $amplicon = $search->next_amplicon;
 352 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 353 is $amplicon->start, 22;
 354 is $amplicon->end, 41;
 355 is $amplicon->strand, -1;
 356 is $amplicon->seq->seq, 'CCCCacgtAAAAAATTTTTT';
 357 is $search->next_amplicon, undef;
 358
 359
 360 # Annotate template
 361
 362 $seq = Bio::PrimarySeq->new(
 363    -seq => 'acgtaCCCCacgtacgtacTTTTTTacAAAAAAgtGGGGacgtacgtac',
 364 );
 365 $forward = Bio::PrimarySeq->new(
 366    -seq => 'CCCC',
 367 );
 368 $reverse = Bio::PrimarySeq->new(
 369    -seq => 'AAAAAA',
 370 );
 371 ok $search = Bio::Tools::AmpliconSearch->new(
 372    -template   => $seq,
 373    -fwd_primer => $forward,
 374    -rev_primer => $reverse,
 375 ), 'Annotated template';
 376 ok $annotated = $search->annotate_template;
 377 isa_ok $annotated, 'Bio::Seq';
 378 $num_feats = 0;
 379 for my $feat ( $annotated->get_SeqFeatures ) {
 380    $num_feats++;
 381    isa_ok $feat, 'Bio::SeqFeature::Amplicon';
 382 }
 383 is $num_feats, 2;
 384 is $search->next_amplicon, undef;
 385
 386
 387 # Update primers
 388
 389 $seq = Bio::PrimarySeq->new(
 390    -seq => 'acgtaCCCCacgtacgtacTTTTTTacCTCTCTgtTGTGTGacgtacgtac',
 391 );
 392 $forward = Bio::PrimarySeq->new(
 393    -seq => 'CTCTCT',
 394 );
 395 $reverse = Bio::PrimarySeq->new(
 396    -seq => 'CACACA',
 397 );
 398 $forward2 = Bio::PrimarySeq->new(
 399    -seq => 'CCCC',
 400 );
 401 $reverse2 = Bio::PrimarySeq->new(
 402    -seq => 'AAAAAA',
 403 );
 404 ok $search = Bio::Tools::AmpliconSearch->new(
 405    -template   => $seq,
 406 ), 'Update primers';
 407 ok $search->fwd_primer($forward);
 408 is $search->fwd_primer->seq, 'CTCTCT';
 409 ok $search->rev_primer($reverse);
 410 is $search->rev_primer->seq, 'CACACA';
 411 ok $amplicon = $search->next_amplicon;
 412 is $amplicon->seq->seq, 'CTCTCTgtTGTGTG';
 413 is $search->next_amplicon, undef;
 414 ok $search->fwd_primer($forward2);
 415 ok $search->rev_primer($reverse2);
 416 is $search->fwd_primer->seq, 'CCCC';
 417 is $search->rev_primer->seq, 'AAAAAA';
 418 ok $amplicon = $search->next_amplicon;
 419 is $amplicon->seq->seq, 'CCCCacgtacgtacTTTTTT';
 420 is $search->next_amplicon, undef;
 421
 422
 423 # Update template
 424
 425 $seq = Bio::PrimarySeq->new(
 426    -seq => 'acgtaCCCCacgtacgtacTTTTTTa',
 427 );
 428 $seq2 = Bio::PrimarySeq->new(
 429    -seq => 'aCCCCgaTTTTTTgacgtacgtac',
 430 );
 431 $forward = Bio::PrimarySeq->new(
 432    -seq => 'CCCC',
 433 );
 434 $reverse = Bio::PrimarySeq->new(
 435    -seq => 'AAAAAA',
 436 );
 437 ok $search = Bio::Tools::AmpliconSearch->new(
 438    -fwd_primer => $forward,
 439    -rev_primer => $reverse,
 440 ), 'Update template';
 441 ok $search->template($seq);
 442 is $search->template->seq, 'acgtaCCCCacgtacgtacTTTTTTa';
 443 ok $amplicon = $search->next_amplicon;
 444 is $amplicon->seq->seq, 'CCCCacgtacgtacTTTTTT';
 445 is $search->next_amplicon, undef;
 446 ok $search->template($seq2);
 447 is $search->template->seq, 'aCCCCgaTTTTTTgacgtacgtac';
 448 ok $amplicon = $search->next_amplicon;
 449 is $amplicon->seq->seq, 'CCCCgaTTTTTT';
 450 is $search->next_amplicon, undef;
 451