t/Tools/AmpliconSearch.t

   1 BEGIN {
   2     use lib '.';
   3     use Bio::Root::Test;
   4
   5     test_begin(-tests => 185);
   6
   7     use_ok 'Bio::PrimarySeq';
   8     use_ok 'Bio::SeqFeature::Primer';
   9     use_ok 'Bio::Tools::AmpliconSearch';
  10 }
  11
  12
  13 my ($search, $amplicon, $seq, $seq2, $forward, $forward2, $reverse, $reverse2,
  14     $primer, $primer_seq, $annotated, $num_feats, $template_seq, $rna);
  15
  16
  17 # Basic object
  18
  19 ok $search = Bio::Tools::AmpliconSearch->new(), 'Basic';
  20 isa_ok $search, 'Bio::Tools::AmpliconSearch';
  21
  22
  23 # Forward primer only
  24
  25 $seq = Bio::PrimarySeq->new(
  26    -seq => 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac',
  27 );
  28
  29 $rna = Bio::PrimarySeq->new(
  30    -seq => 'acgAAACUUAAAGGAAUUGACGGacguacguacguGUACACACCGCCCGUacguac',
  31 );
  32
  33
  34 $forward = Bio::PrimarySeq->new(
  35    -seq => 'AAACTTAAAGGAATTGACGG',
  36 );
  37 ok $search = Bio::Tools::AmpliconSearch->new(
  38    -template       => $seq,
  39    -fwd_primer     => $forward,
  40    -attach_primers => 1,
  41 ), 'Forward primer only';
  42 is $search->fwd_primer->seq, 'AAACTTAAAGGAATTGACGG';
  43 is $search->rev_primer, undef;
  44 ok $template_seq = $search->template;
  45 isa_ok $template_seq, 'Bio::Seq';
  46 is $template_seq->seq, 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac';
  47 ok $amplicon = $search->next_amplicon;
  48 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
  49 is $amplicon->start, 4;
  50 is $amplicon->end, 56;
  51 is $amplicon->strand, 1;
  52 is $amplicon->seq->seq, 'AAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac';
  53 ok $primer = $amplicon->fwd_primer;
  54 ok $primer_seq = $primer->seq;
  55 is $primer_seq->seq, 'AAACTTAAAGGAATTGACGG';
  56 is $primer->start, 4;
  57 is $primer->end, 23;
  58 is $primer->strand, 1;
  59 is $amplicon = $search->next_amplicon, undef;
  60
  61
  62 # Reverse primer only
  63
  64 $seq = Bio::PrimarySeq->new(
  65    -seq => 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac',
  66 );
  67 $reverse = Bio::SeqFeature::Primer->new(
  68    -seq => 'ACGGGCGGTGTGTAC',
  69 );
  70 ok $search = Bio::Tools::AmpliconSearch->new(
  71    -template       => $seq,
  72    -rev_primer     => $reverse,
  73    -attach_primers => 0,
  74 ), 'Reverse primer only';
  75 is $search->fwd_primer, undef;
  76 is $search->rev_primer->seq->seq, 'ACGGGCGGTGTGTAC';
  77 is $search->template->seq, 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac';
  78 ok $amplicon = $search->next_amplicon;
  79 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
  80 is $amplicon->start, 1;
  81 is $amplicon->end, 50;
  82 is $amplicon->strand, 1;
  83 is $amplicon->seq->seq, 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGT';
  84 is $amplicon->fwd_primer, undef;
  85 is $amplicon->rev_primer, undef;
  86 is $search->next_amplicon, undef;
  87
  88
  89 # Forward and reverse primers, no amplicon
  90
  91 $seq = Bio::PrimarySeq->new(
  92    -seq => 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac',
  93 );
  94 $forward = Bio::PrimarySeq->new(
  95    -seq => 'AAACTTAAAGGAATTGACGG',
  96 );
  97 $reverse = Bio::PrimarySeq->new(
  98    -seq => 'CCCCCCCCCC',
  99 );
 100 ok $search = Bio::Tools::AmpliconSearch->new(
 101    -template   => $seq,
 102    -fwd_primer => $forward,
 103    -rev_primer => $reverse,
 104 ), 'Two primers, no match';
 105 is $search->fwd_primer->seq, 'AAACTTAAAGGAATTGACGG';
 106 is $search->rev_primer->seq, 'CCCCCCCCCC';
 107 is $search->template->seq, 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac';
 108 is $search->next_amplicon, undef;
 109
 110
 111 # Degenerate forward and reverse primers from file, single amplicon
 112
 113 ok $search = Bio::Tools::AmpliconSearch->new(
 114    -template    => $seq,
 115    -primer_file => test_input_file('forward_reverse_primers.fa'),
 116 ), 'Two degenerate primers from a file';
 117 is $search->fwd_primer->seq, 'AAACTYAAAKGAATTGRCGG';
 118 is $search->rev_primer->seq, 'ACGGGCGGTGTGTRC';
 119 is $search->template->seq, 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac';
 120 ok $amplicon = $search->next_amplicon;
 121 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 122 is $amplicon->start, 4;
 123 is $amplicon->end, 50;
 124 is $amplicon->strand, 1;
 125 is $amplicon->seq->seq, 'AAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGT';
 126 is $search->next_amplicon, undef;
 127
 128
 129 # Same thing but with RNA template
 130
 131 ok $search = Bio::Tools::AmpliconSearch->new(
 132    -template    => $rna,
 133    -primer_file => test_input_file('forward_reverse_primers.fa'),
 134 ), 'RNA template';
 135 is $search->fwd_primer->seq, 'AAACTYAAAKGAATTGRCGG';
 136 is $search->rev_primer->seq, 'ACGGGCGGTGTGTRC';
 137 is $search->template->seq, 'acgAAACUUAAAGGAAUUGACGGacguacguacguGUACACACCGCCCGUacguac';
 138 ok $amplicon = $search->next_amplicon;
 139 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 140 is $amplicon->start, 4;
 141 is $amplicon->end, 50;
 142 is $amplicon->strand, 1;
 143 is $amplicon->seq->seq, 'AAACUUAAAGGAAUUGACGGacguacguacguGUACACACCGCCCGU';
 144 is $search->next_amplicon, undef;
 145
 146
 147 # Forward primer only, in sequence file
 148
 149 ok $search = Bio::Tools::AmpliconSearch->new(
 150    -primer_file => test_input_file('forward_primer.fa'),
 151 ), 'Forward primer from file';
 152 ok $search->template($seq);
 153 is $search->fwd_primer->seq, 'AAACTTAAAGGAATTGACGG';
 154 is $search->rev_primer, undef;
 155 ok $template_seq = $search->template;
 156 isa_ok $template_seq, 'Bio::Seq';
 157 is $template_seq->seq, 'acgAAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac';
 158 ok $amplicon = $search->next_amplicon;
 159 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 160 is $amplicon->start, 4;
 161 is $amplicon->end, 56;
 162 is $amplicon->strand, 1;
 163 is $amplicon->seq->seq, 'AAACTTAAAGGAATTGACGGacgtacgtacgtGTACACACCGCCCGTacgtac';
 164 is $amplicon = $search->next_amplicon, undef;
 165
 166
 167 # Multiple amplicons
 168
 169 $seq = Bio::PrimarySeq->new(
 170    -seq => 'acgtaCCCCacgtacgtacTTTTTTacgtaCCCCacgtaTTTTTTacgtacgtac',
 171 );
 172 $forward = Bio::PrimarySeq->new(
 173    -seq => 'CCCC',
 174 );
 175 $reverse = Bio::PrimarySeq->new(
 176    -seq => 'AAAAAA',
 177 );
 178 ok $search = Bio::Tools::AmpliconSearch->new(
 179    -template   => $seq,
 180    -fwd_primer => $forward,
 181    -rev_primer => $reverse,
 182 ), 'Multiple amplicons';
 183 ok $amplicon = $search->next_amplicon;
 184 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 185 is $amplicon->start, 6;
 186 is $amplicon->end, 25;
 187 is $amplicon->strand, 1;
 188 is $amplicon->seq->seq, 'CCCCacgtacgtacTTTTTT';
 189 ok $amplicon = $search->next_amplicon;
 190 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 191 is $amplicon->start, 31;
 192 is $amplicon->end, 45;
 193 is $amplicon->strand, 1;
 194 is $amplicon->seq->seq, 'CCCCacgtaTTTTTT';
 195 is $search->next_amplicon, undef;
 196
 197
 198 # Amplicon on reverse strand
 199
 200 $seq = Bio::PrimarySeq->new(
 201    # Reverse-complement of previous sequence... should have same amplicons
 202    -seq => 'gtacgtacgtAAAAAAtacgtGGGGtacgtAAAAAAgtacgtacgtGGGGtacgt',
 203 );
 204 $forward = Bio::PrimarySeq->new(
 205    -seq => 'CCCC',
 206 );
 207 $reverse = Bio::PrimarySeq->new(
 208    -seq => 'AAAAAA',
 209 );
 210 ok $search = Bio::Tools::AmpliconSearch->new(
 211    -template   => $seq,
 212    -fwd_primer => $forward,
 213    -rev_primer => $reverse,
 214 ), 'Amplicon on reverse strand';
 215 ok $amplicon = $search->next_amplicon;
 216 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 217 is $amplicon->start, 11;
 218 is $amplicon->end, 25;
 219 is $amplicon->strand, -1;
 220 is $amplicon->seq->seq, 'CCCCacgtaTTTTTT';
 221 ok $amplicon = $search->next_amplicon;
 222 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 223 is $amplicon->start, 31;
 224 is $amplicon->end, 50;
 225 is $amplicon->strand, -1;
 226 is $amplicon->seq->seq, 'CCCCacgtacgtacTTTTTT';
 227 is $search->next_amplicon, undef;
 228
 229
 230 # Overlapping amplicons (1)
 231
 232 $seq = Bio::PrimarySeq->new(
 233    -seq => 'acgtaCCCCacgtacgtacgtacgCCCCacgtaTTTTTTacgtacgtac',
 234 );
 235 $forward = Bio::PrimarySeq->new(
 236    -seq => 'CCCC',
 237 );
 238 $reverse = Bio::PrimarySeq->new(
 239    -seq => 'AAAAAA',
 240 );
 241 ok $search = Bio::Tools::AmpliconSearch->new(
 242    -template   => $seq,
 243    -fwd_primer => $forward,
 244    -rev_primer => $reverse,
 245 ), 'Overlapping amplicons (1)';
 246 ok $amplicon = $search->next_amplicon;
 247 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 248 is $amplicon->start, 25;
 249 is $amplicon->end, 39;
 250 is $amplicon->strand, 1;
 251 is $amplicon->seq->seq, 'CCCCacgtaTTTTTT';
 252 is $search->next_amplicon, undef;
 253
 254
 255 # Overlapping amplicons (2)
 256
 257 $seq = Bio::PrimarySeq->new(
 258    -seq => 'acgtaCCCCacgtacgtacgtacgTTTTTTacgtaTTTTTTacgtacgtac',
 259 );
 260 $forward = Bio::PrimarySeq->new(
 261    -seq => 'CCCC',
 262 );
 263 $reverse = Bio::PrimarySeq->new(
 264    -seq => 'AAAAAA',
 265 );
 266 ok $search = Bio::Tools::AmpliconSearch->new(
 267    -template   => $seq,
 268    -fwd_primer => $forward,
 269    -rev_primer => $reverse,
 270 ), 'Overlapping amplicons (2)';
 271 ok $amplicon = $search->next_amplicon;
 272 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 273 is $amplicon->start, 6;
 274 is $amplicon->end, 30;
 275 is $amplicon->strand, 1;
 276 is $amplicon->seq->seq, 'CCCCacgtacgtacgtacgTTTTTT';
 277 is $search->next_amplicon, undef;
 278
 279
 280 # Overlapping amplicons (3)
 281
 282 $seq = Bio::PrimarySeq->new(
 283    -seq => 'acgtaCCCCacgtacgCCCCacgtacgaTTTTTTacgtaTTTTTTaaaaaaaaaa',
 284 );
 285 $forward = Bio::PrimarySeq->new(
 286    -seq => 'CCCC',
 287 );
 288 $reverse = Bio::PrimarySeq->new(
 289    -seq => 'AAAAAA',
 290 );
 291 ok $search = Bio::Tools::AmpliconSearch->new(
 292    -template   => $seq,
 293    -fwd_primer => $forward,
 294    -rev_primer => $reverse,
 295 ), 'Overlapping amplicons (3)';
 296 ok $amplicon = $search->next_amplicon;
 297 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 298 is $amplicon->start, 17;
 299 is $amplicon->end, 34;
 300 is $amplicon->strand, 1;
 301 is $amplicon->seq->seq, 'CCCCacgtacgaTTTTTT';
 302 is $search->next_amplicon, undef;
 303
 304
 305 # Amplicons on both strands
 306
 307 $seq = Bio::PrimarySeq->new(
 308    -seq => 'acgtaCCCCacgtaacgtaTTTTTTacAAAAAAgtGGGGacgtaacgta',
 309 );
 310 $forward = Bio::PrimarySeq->new(
 311    -seq => 'CCCC',
 312 );
 313 $reverse = Bio::PrimarySeq->new(
 314    -seq => 'AAAAAA',
 315 );
 316 ok $search = Bio::Tools::AmpliconSearch->new(
 317    -template   => $seq,
 318    -fwd_primer => $forward,
 319    -rev_primer => $reverse,
 320 ), 'Amplicons on both strands';
 321 ok $amplicon = $search->next_amplicon;
 322 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 323 is $amplicon->start, 6;
 324 is $amplicon->end, 25;
 325 is $amplicon->strand, 1;
 326 is $amplicon->seq->seq, 'CCCCacgtaacgtaTTTTTT';
 327 ok $amplicon = $search->next_amplicon;
 328 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 329 is $amplicon->start, 28;
 330 is $amplicon->end, 39;
 331 is $amplicon->strand, -1;
 332 is $amplicon->seq->seq, 'CCCCacTTTTTT';
 333 is $search->next_amplicon, undef;
 334
 335
 336 # Overlapping amplicons on both strands
 337
 338 $seq = Bio::PrimarySeq->new(
 339    -seq => 'acgtaCCCCacgtacgtacagAAAAAATTTTTTacgtGGGGacgtacgtac',
 340 );
 341 $forward = Bio::PrimarySeq->new(
 342    -seq => 'CCCC',
 343 );
 344 $reverse = Bio::PrimarySeq->new(
 345    -seq => 'AAAAAA',
 346 );
 347 ok $search = Bio::Tools::AmpliconSearch->new(
 348    -template   => $seq,
 349    -fwd_primer => $forward,
 350    -rev_primer => $reverse,
 351 ), 'Overlapping amplicons on both strands';
 352 ok $amplicon = $search->next_amplicon;
 353 isa_ok $amplicon, 'Bio::SeqFeature::Amplicon';
 354 is $amplicon->start, 22;
 355 is $amplicon->end, 41;
 356 is $amplicon->strand, -1;
 357 is $amplicon->seq->seq, 'CCCCacgtAAAAAATTTTTT';
 358 is $search->next_amplicon, undef;
 359
 360
 361 # Annotate template
 362
 363 $seq = Bio::PrimarySeq->new(
 364    -seq => 'acgtaCCCCacgtacgtacTTTTTTacAAAAAAgtGGGGacgtacgtac',
 365 );
 366 $forward = Bio::PrimarySeq->new(
 367    -seq => 'CCCC',
 368 );
 369 $reverse = Bio::PrimarySeq->new(
 370    -seq => 'AAAAAA',
 371 );
 372 ok $search = Bio::Tools::AmpliconSearch->new(
 373    -template   => $seq,
 374    -fwd_primer => $forward,
 375    -rev_primer => $reverse,
 376 ), 'Annotated template';
 377 ok $annotated = $search->annotate_template;
 378 isa_ok $annotated, 'Bio::Seq';
 379 $num_feats = 0;
 380 for my $feat ( $annotated->get_SeqFeatures ) {
 381    $num_feats++;
 382    isa_ok $feat, 'Bio::SeqFeature::Amplicon';
 383 }
 384 is $num_feats, 2;
 385 is $search->next_amplicon, undef;
 386
 387
 388 # Update primers
 389
 390 $seq = Bio::PrimarySeq->new(
 391    -seq => 'acgtaCCCCacgtacgtacTTTTTTacCTCTCTgtTGTGTGacgtacgtac',
 392 );
 393 $forward = Bio::PrimarySeq->new(
 394    -seq => 'CTCTCT',
 395 );
 396 $reverse = Bio::PrimarySeq->new(
 397    -seq => 'CACACA',
 398 );
 399 $forward2 = Bio::PrimarySeq->new(
 400    -seq => 'CCCC',
 401 );
 402 $reverse2 = Bio::PrimarySeq->new(
 403    -seq => 'AAAAAA',
 404 );
 405 ok $search = Bio::Tools::AmpliconSearch->new(
 406    -template   => $seq,
 407 ), 'Update primers';
 408 ok $search->fwd_primer($forward);
 409 is $search->fwd_primer->seq, 'CTCTCT';
 410 ok $search->rev_primer($reverse);
 411 is $search->rev_primer->seq, 'CACACA';
 412 ok $amplicon = $search->next_amplicon;
 413 is $amplicon->seq->seq, 'CTCTCTgtTGTGTG';
 414 is $search->next_amplicon, undef;
 415 ok $search->fwd_primer($forward2);
 416 ok $search->rev_primer($reverse2);
 417 is $search->fwd_primer->seq, 'CCCC';
 418 is $search->rev_primer->seq, 'AAAAAA';
 419 ok $amplicon = $search->next_amplicon;
 420 is $amplicon->seq->seq, 'CCCCacgtacgtacTTTTTT';
 421 is $search->next_amplicon, undef;
 422
 423
 424 # Update template
 425
 426 $seq = Bio::PrimarySeq->new(
 427    -seq => 'acgtaCCCCacgtacgtacTTTTTTa',
 428 );
 429 $seq2 = Bio::PrimarySeq->new(
 430    -seq => 'aCCCCgaTTTTTTgacgtacgtac',
 431 );
 432 $forward = Bio::PrimarySeq->new(
 433    -seq => 'CCCC',
 434 );
 435 $reverse = Bio::PrimarySeq->new(
 436    -seq => 'AAAAAA',
 437 );
 438 ok $search = Bio::Tools::AmpliconSearch->new(
 439    -fwd_primer => $forward,
 440    -rev_primer => $reverse,
 441 ), 'Update template';
 442 ok $search->template($seq);
 443 is $search->template->seq, 'acgtaCCCCacgtacgtacTTTTTTa';
 444 ok $amplicon = $search->next_amplicon;
 445 is $amplicon->seq->seq, 'CCCCacgtacgtacTTTTTT';
 446 is $search->next_amplicon, undef;
 447 ok $search->template($seq2);
 448 is $search->template->seq, 'aCCCCgaTTTTTTgacgtacgtac';
 449 ok $amplicon = $search->next_amplicon;
 450 is $amplicon->seq->seq, 'CCCCgaTTTTTT';
 451 is $search->next_amplicon, undef;
 452