Mercurial > repos > bigrna > gpsrna
comparison miRDeep_plant.pl @ 0:87fe81de0931 draft default tip
Uploaded
| author | bigrna |
|---|---|
| date | Sun, 04 Jan 2015 02:47:25 -0500 |
| parents | |
| children |
comparison
equal
deleted
inserted
replaced
| -1:000000000000 | 0:87fe81de0931 |
|---|---|
| 1 #!/usr/bin/perl | |
| 2 | |
| 3 use warnings; | |
| 4 use strict; | |
| 5 use Getopt::Std; | |
| 6 #use RNA; | |
| 7 | |
| 8 | |
| 9 ################################# MIRDEEP ################################################# | |
| 10 | |
| 11 ################################## USAGE ################################################## | |
| 12 | |
| 13 | |
| 14 my $usage= | |
| 15 "$0 file_signature file_structure temp_out_directory | |
| 16 | |
| 17 This is the core algorithm of miRDeep. It takes as input a file in blastparsed format with | |
| 18 information on the positions of reads aligned to potential precursor sequences (signature). | |
| 19 It also takes as input an RNAfold output file, giving information on the sequence, structure | |
| 20 and mimimum free energy of the potential precursor sequences. | |
| 21 | |
| 22 Extra arguments can be given. -s specifies a fastafile containing the known mature miRNA | |
| 23 sequences that should be considered for conservation purposes. -t prints out the potential | |
| 24 precursor sequences that do _not_ exceed the cut-off (default prints out the sequences that | |
| 25 exceeds the cut-off). -u gives limited output, that is only the ids of the potential precursors | |
| 26 that exceed the cut-off. -v varies the cut-off. -x is a sensitive option for Sanger sequences | |
| 27 obtained through conventional cloning. -z consider the number of base pairings in the lower | |
| 28 stems (this option is not well tested). | |
| 29 | |
| 30 -h print this usage | |
| 31 -s fasta file with known miRNAs | |
| 32 #-o temp directory ,maked befor running the program. | |
| 33 -t print filtered | |
| 34 -u limited output (only ids) | |
| 35 -v cut-off (default 1) | |
| 36 -x sensitive option for Sanger sequences | |
| 37 -y use Randfold | |
| 38 -z consider Drosha processing | |
| 39 "; | |
| 40 | |
| 41 | |
| 42 | |
| 43 | |
| 44 | |
| 45 ############################################################################################ | |
| 46 | |
| 47 ################################### INPUT ################################################## | |
| 48 | |
| 49 | |
| 50 #signature file in blast_parsed format | |
| 51 my $file_blast_parsed=shift or die $usage; | |
| 52 | |
| 53 #structure file outputted from RNAfold | |
| 54 my $file_struct=shift or die $usage; | |
| 55 | |
| 56 my $tmpdir=shift or die $usage; | |
| 57 #options | |
| 58 my %options=(); | |
| 59 getopts("hs:tuv:xyz",\%options); | |
| 60 | |
| 61 | |
| 62 | |
| 63 | |
| 64 | |
| 65 | |
| 66 ############################################################################################# | |
| 67 | |
| 68 ############################# GLOBAL VARIABLES ############################################## | |
| 69 | |
| 70 | |
| 71 #parameters | |
| 72 my $nucleus_lng=11; | |
| 73 | |
| 74 my $score_star=3.9; | |
| 75 my $score_star_not=-1.3; | |
| 76 my $score_nucleus=7.63; | |
| 77 my $score_nucleus_not=-1.17; | |
| 78 my $score_randfold=1.37; | |
| 79 my $score_randfold_not=-3.624; | |
| 80 my $score_intercept=0.3; | |
| 81 my @scores_stem=(-3.1,-2.3,-2.2,-1.6,-1.5,0.1,0.6,0.8,0.9,0.9,0); | |
| 82 my $score_min=1; | |
| 83 if($options{v}){$score_min=$options{v};} | |
| 84 if($options{x}){$score_min=-5;} | |
| 85 | |
| 86 my $e=2.718281828; | |
| 87 | |
| 88 #hashes | |
| 89 my %hash_desc; | |
| 90 my %hash_seq; | |
| 91 my %hash_struct; | |
| 92 my %hash_mfe; | |
| 93 my %hash_nuclei; | |
| 94 my %hash_mirs; | |
| 95 my %hash_query; | |
| 96 my %hash_comp; | |
| 97 my %hash_bp; | |
| 98 | |
| 99 #other variables | |
| 100 my $subject_old; | |
| 101 my $message_filter; | |
| 102 my $message_score; | |
| 103 my $lines; | |
| 104 my $out_of_bound; | |
| 105 | |
| 106 | |
| 107 | |
| 108 ############################################################################################## | |
| 109 | |
| 110 ################################ MAIN ###################################################### | |
| 111 | |
| 112 | |
| 113 #print help if that option is used | |
| 114 if($options{h}){die $usage;} | |
| 115 unless ($tmpdir=~/\/$/) {$tmpdir .="/";} | |
| 116 if(!(-s $tmpdir)){mkdir $tmpdir;} | |
| 117 $tmpdir .="TMP_DIR/"; | |
| 118 mkdir $tmpdir; | |
| 119 | |
| 120 #parse structure file outputted from RNAfold | |
| 121 parse_file_struct($file_struct); | |
| 122 | |
| 123 #if conservation is scored, the fasta file of known miRNA sequences is parsed | |
| 124 if($options{s}){create_hash_nuclei($options{s})}; | |
| 125 | |
| 126 #parse signature file in blast_parsed format and resolve each potential precursor | |
| 127 parse_file_blast_parsed($file_blast_parsed); | |
| 128 #`rm -rf $tmpdir`; | |
| 129 exit; | |
| 130 | |
| 131 | |
| 132 | |
| 133 | |
| 134 ############################################################################################## | |
| 135 | |
| 136 ############################## SUBROUTINES ################################################### | |
| 137 | |
| 138 | |
| 139 | |
| 140 sub parse_file_blast_parsed{ | |
| 141 | |
| 142 # read through the signature blastparsed file, fills up a hash with information on queries | |
| 143 # (deep sequences) mapping to the current subject (potential precursor) and resolve each | |
| 144 # potential precursor in turn | |
| 145 | |
| 146 my $file_blast_parsed=shift; | |
| 147 | |
| 148 open (FILE_BLAST_PARSED, "<$file_blast_parsed") or die "can not open $file_blast_parsed\n"; | |
| 149 while (my $line=<FILE_BLAST_PARSED>){ | |
| 150 if($line=~/^(\S+)\s+(\S+)\s+(\d+)\.+(\d+)\s+(\S+)\s+(\S+)\s+(\d+)\.+(\d+)\s+(\S+)\s+(\S+)\s+(\S+)\s+(.+)$/){ | |
| 151 my $query=$1; | |
| 152 my $query_lng=$2; | |
| 153 my $query_beg=$3; | |
| 154 my $query_end=$4; | |
| 155 my $subject=$5; | |
| 156 my $subject_lng=$6; | |
| 157 my $subject_beg=$7; | |
| 158 my $subject_end=$8; | |
| 159 my $e_value=$9; | |
| 160 my $pid=$10; | |
| 161 my $bitscore=$11; | |
| 162 my $other=$12; | |
| 163 | |
| 164 #if the new line concerns a new subject (potential precursor) then the old subject must be resolved | |
| 165 if($subject_old and $subject_old ne $subject){ | |
| 166 resolve_potential_precursor(); | |
| 167 } | |
| 168 | |
| 169 #resolve the strand | |
| 170 my $strand=find_strand($other); | |
| 171 | |
| 172 #resolve the number of reads that the deep sequence represents | |
| 173 my $freq=find_freq($query); | |
| 174 | |
| 175 #read information of the query (deep sequence) into hash | |
| 176 $hash_query{$query}{"subject_beg"}=$subject_beg; | |
| 177 $hash_query{$query}{"subject_end"}=$subject_end; | |
| 178 $hash_query{$query}{"strand"}=$strand; | |
| 179 $hash_query{$query}{"freq"}=$freq; | |
| 180 | |
| 181 #save the signature information | |
| 182 $lines.=$line; | |
| 183 | |
| 184 $subject_old=$subject; | |
| 185 } | |
| 186 } | |
| 187 resolve_potential_precursor(); | |
| 188 } | |
| 189 | |
| 190 sub resolve_potential_precursor{ | |
| 191 | |
| 192 # dissects the potential precursor in parts by filling hashes, and tests if it passes the | |
| 193 # initial filter and the scoring filter | |
| 194 | |
| 195 # binary variable whether the potential precursor is still viable | |
| 196 my $ret=1; | |
| 197 #print STDERR ">$subject_old\n"; | |
| 198 | |
| 199 fill_structure(); | |
| 200 #print STDERR "\%hash_bp",scalar keys %hash_bp,"\n"; | |
| 201 fill_pri(); | |
| 202 #print STDERR "\%hash_comp",scalar keys %hash_comp,"\n"; | |
| 203 | |
| 204 fill_mature(); | |
| 205 #print STDERR "\%hash_comp",scalar keys %hash_comp,"\n"; | |
| 206 | |
| 207 fill_star(); | |
| 208 #print STDERR "\%hash_comp",scalar keys %hash_comp,"\n"; | |
| 209 | |
| 210 fill_loop(); | |
| 211 #print STDERR "\%hash_comp",scalar keys %hash_comp,"\n"; | |
| 212 | |
| 213 fill_lower_flanks(); | |
| 214 #print STDERR "\%hash_comp",scalar keys %hash_comp,"\n"; | |
| 215 | |
| 216 # do_test_assemble(); | |
| 217 | |
| 218 # this is the actual classification | |
| 219 unless(pass_filtering_initial() and pass_threshold_score()){$ret=0;} | |
| 220 | |
| 221 print_results($ret); | |
| 222 | |
| 223 reset_variables(); | |
| 224 | |
| 225 return; | |
| 226 | |
| 227 } | |
| 228 | |
| 229 | |
| 230 | |
| 231 sub print_results{ | |
| 232 | |
| 233 my $ret=shift; | |
| 234 | |
| 235 # print out if the precursor is accepted and accepted precursors should be printed out | |
| 236 # or if the potential precursor is discarded and discarded potential precursors should | |
| 237 # be printed out | |
| 238 | |
| 239 if((!$options{t} and $ret) or ($options{t} and !$ret)){ | |
| 240 #full output | |
| 241 unless($options{u}){ | |
| 242 if($message_filter){print $message_filter;} | |
| 243 if($message_score){print $message_score;} | |
| 244 print_hash_comp(); | |
| 245 print $lines,"\n\n"; | |
| 246 return; | |
| 247 } | |
| 248 #limited output (only ids) | |
| 249 my $id=$hash_comp{"pri_id"}; | |
| 250 print "$id\n"; | |
| 251 } | |
| 252 } | |
| 253 | |
| 254 | |
| 255 | |
| 256 | |
| 257 | |
| 258 | |
| 259 | |
| 260 sub pass_threshold_score{ | |
| 261 | |
| 262 # this is the scoring | |
| 263 | |
| 264 #minimum free energy of the potential precursor | |
| 265 # my $score_mfe=score_mfe($hash_comp{"pri_mfe"}); | |
| 266 my $score_mfe=score_mfe($hash_comp{"pri_mfe"},$hash_comp{"pri_end"}); | |
| 267 | |
| 268 #count of reads that map in accordance with Dicer processing | |
| 269 my $score_freq=score_freq($hash_comp{"freq"}); | |
| 270 #print STDERR "score_mfe: $score_mfe\nscore_freq: $score_freq\n"; | |
| 271 | |
| 272 #basic score | |
| 273 my $score=$score_mfe+$score_freq; | |
| 274 | |
| 275 #scoring of conserved nucleus/seed (optional) | |
| 276 if($options{s}){ | |
| 277 | |
| 278 #if the nucleus is conserved | |
| 279 if(test_nucleus_conservation()){ | |
| 280 | |
| 281 #nucleus from position 2-8 | |
| 282 my $nucleus=substr($hash_comp{"mature_seq"},1,$nucleus_lng); | |
| 283 | |
| 284 #resolve DNA/RNA ambiguities | |
| 285 $nucleus=~tr/[T]/[U]/; | |
| 286 | |
| 287 #print score contribution | |
| 288 score_s("score_nucleus\t$score_nucleus"); | |
| 289 | |
| 290 #print the ids of known miRNAs with same nucleus | |
| 291 score_s("$hash_mirs{$nucleus}"); | |
| 292 #print STDERR "score_nucleus\t$score_nucleus\n"; | |
| 293 | |
| 294 #add to score | |
| 295 $score+=$score_nucleus; | |
| 296 | |
| 297 #if the nucleus is not conserved | |
| 298 }else{ | |
| 299 #print (negative) score contribution | |
| 300 score_s("score_nucleus\t$score_nucleus_not"); | |
| 301 | |
| 302 #add (negative) score contribution | |
| 303 $score+=$score_nucleus_not; | |
| 304 } | |
| 305 } | |
| 306 | |
| 307 #if the majority of potential star reads fall as expected from Dicer processing | |
| 308 if($hash_comp{"star_read"}){ | |
| 309 score_s("score_star\t$score_star"); | |
| 310 #print STDERR "score_star\t$score_star\n"; | |
| 311 $score+=$score_star; | |
| 312 }else{ | |
| 313 score_s("score_star\t$score_star_not"); | |
| 314 #print STDERR "score_star_not\t$score_star_not\n"; | |
| 315 $score+=$score_star_not; | |
| 316 } | |
| 317 | |
| 318 #score lower stems for potential for Drosha recognition (highly optional) | |
| 319 if($options{z}){ | |
| 320 my $stem_bp=$hash_comp{"stem_bp"}; | |
| 321 my $score_stem=$scores_stem[$stem_bp]; | |
| 322 $score+=$score_stem; | |
| 323 score_s("score_stem\t$score_stem"); | |
| 324 } | |
| 325 | |
| 326 #print STDERR "score_intercept\t$score_intercept\n"; | |
| 327 | |
| 328 $score+=$score_intercept; | |
| 329 | |
| 330 #score for randfold (optional) | |
| 331 if($options{y}){ | |
| 332 | |
| 333 # only calculate randfold value if it can make the difference between the potential precursor | |
| 334 # being accepted or discarded | |
| 335 if($score+$score_randfold>=$score_min and $score+$score_randfold_not<=$score_min){ | |
| 336 | |
| 337 #randfold value<0.05 | |
| 338 if(test_randfold()){$score+=$score_randfold;score_s("score_randfold\t$score_randfold");} | |
| 339 | |
| 340 #randfold value>0.05 | |
| 341 else{$score+=$score_randfold_not;score_s("score_randfold\t$score_randfold_not");} | |
| 342 } | |
| 343 } | |
| 344 | |
| 345 #round off values to one decimal | |
| 346 my $round_mfe=round($score_mfe*10)/10; | |
| 347 my $round_freq=round($score_freq*10)/10; | |
| 348 my $round=round($score*10)/10; | |
| 349 | |
| 350 #print scores | |
| 351 score_s("score_mfe\t$round_mfe\nscore_freq\t$round_freq\nscore\t$round"); | |
| 352 | |
| 353 #return 1 if the potential precursor is accepted, return 0 if discarded | |
| 354 unless($score>=$score_min){return 0;} | |
| 355 return 1; | |
| 356 } | |
| 357 | |
| 358 sub test_randfold{ | |
| 359 | |
| 360 #print sequence to temporary file, test randfold value, return 1 or 0 | |
| 361 | |
| 362 # print_file("pri_seq.fa",">pri_seq\n".$hash_comp{"pri_seq"}); | |
| 363 #my $tmpfile=$tmpdir.$hash_comp{"pri_id"}; | |
| 364 #open(FILE, ">$tmpfile"); | |
| 365 #print FILE ">pri_seq\n",$hash_comp{"pri_seq"}; | |
| 366 #close FILE; | |
| 367 | |
| 368 # my $p_value=`randfold -s $tmpfile 999 | cut -f 3`; | |
| 369 #my $p1=`randfold -s $tmpfile 999 | cut -f 3`; | |
| 370 #my $p2=`randfold -s $tmpfile 999 | cut -f 3`; | |
| 371 my $p1=&randfold_pvalue($hash_comp{"pri_seq"},999); | |
| 372 my $p2=&randfold_pvalue($hash_comp{"pri_seq"},999); | |
| 373 my $p_value=($p1+$p2)/2; | |
| 374 wait; | |
| 375 # system "rm $tmpfile"; | |
| 376 | |
| 377 if($p_value<=0.05){return 1;} | |
| 378 | |
| 379 return 0; | |
| 380 } | |
| 381 | |
| 382 sub randfold_pvalue{ | |
| 383 my $cpt_sup = 0; | |
| 384 my $cpt_inf = 0; | |
| 385 my $cpt_ega = 1; | |
| 386 | |
| 387 my ($seq,$number_of_randomizations)=@_; | |
| 388 #my $str =$seq; | |
| 389 #my $mfe = RNA::fold($seq,$str); | |
| 390 my $rnafold=`perl -e 'print "$seq"' | RNAfold --noPS`; | |
| 391 my @rawfolds=split/\s+/,$rnafold; | |
| 392 my $str=$rawfolds[1]; | |
| 393 my $mfe=$rawfolds[-1]; | |
| 394 $mfe=~s/\(//; | |
| 395 $mfe=~s/\)//; | |
| 396 | |
| 397 for (my $i=0;$i<$number_of_randomizations;$i++) { | |
| 398 $seq = shuffle_sequence_dinucleotide($seq); | |
| 399 #$str = $seq; | |
| 400 | |
| 401 #my $rand_mfe = RNA::fold($str,$str); | |
| 402 $rnafold=`perl -e 'print "$seq"' | RNAfold --noPS`; | |
| 403 my @rawfolds=split/\s+/,$rnafold; | |
| 404 my $str=$rawfolds[1]; | |
| 405 my $rand_mfe=$rawfolds[-1]; | |
| 406 $rand_mfe=~s/\(//; | |
| 407 $rand_mfe=~s/\)//; | |
| 408 | |
| 409 if ($rand_mfe < $mfe) { | |
| 410 $cpt_inf++; | |
| 411 } | |
| 412 if ($rand_mfe == $mfe) { | |
| 413 $cpt_ega++; | |
| 414 } | |
| 415 if ($rand_mfe > $mfe) { | |
| 416 $cpt_sup++; | |
| 417 } | |
| 418 } | |
| 419 | |
| 420 my $proba = ($cpt_ega + $cpt_inf) / ($number_of_randomizations + 1); | |
| 421 | |
| 422 #print "$name\t$mfe\t$proba\n"; | |
| 423 return $proba; | |
| 424 } | |
| 425 | |
| 426 sub shuffle_sequence_dinucleotide { | |
| 427 | |
| 428 my ($str) = @_; | |
| 429 | |
| 430 # upper case and convert to ATGC | |
| 431 $str = uc($str); | |
| 432 $str =~ s/U/T/g; | |
| 433 | |
| 434 my @nuc = ('A','T','G','C'); | |
| 435 my $count_swap = 0; | |
| 436 # set maximum number of permutations | |
| 437 my $stop = length($str) * 10; | |
| 438 | |
| 439 while($count_swap < $stop) { | |
| 440 | |
| 441 my @pos; | |
| 442 | |
| 443 # look start and end letters | |
| 444 my $firstnuc = $nuc[int(rand 4)]; | |
| 445 my $thirdnuc = $nuc[int(rand 4)]; | |
| 446 | |
| 447 # get positions for matching nucleotides | |
| 448 for (my $i=0;$i<(length($str)-2);$i++) { | |
| 449 if ((substr($str,$i,1) eq $firstnuc) && (substr($str,$i+2,1) eq $thirdnuc)) { | |
| 450 push (@pos,($i+1)); | |
| 451 $i++; | |
| 452 } | |
| 453 } | |
| 454 | |
| 455 # swap at random trinucleotides | |
| 456 my $max = scalar(@pos); | |
| 457 for (my $i=0;$i<$max;$i++) { | |
| 458 my $swap = int(rand($max)); | |
| 459 if ((abs($pos[$swap] - $pos[$i]) >= 3) && (substr($str,$pos[$i],1) ne substr($str,$pos[$swap],1))) { | |
| 460 $count_swap++; | |
| 461 my $w1 = substr($str,$pos[$i],1); | |
| 462 my $w2 = substr($str,$pos[$swap],1); | |
| 463 substr($str,$pos[$i],1,$w2); | |
| 464 substr($str,$pos[$swap],1,$w1); | |
| 465 } | |
| 466 } | |
| 467 } | |
| 468 return($str); | |
| 469 } | |
| 470 | |
| 471 sub test_nucleus_conservation{ | |
| 472 | |
| 473 #test if nucleus is identical to nucleus from known miRNA, return 1 or 0 | |
| 474 | |
| 475 my $nucleus=substr($hash_comp{"mature_seq"},1,$nucleus_lng); | |
| 476 $nucleus=~tr/[T]/[U]/; | |
| 477 if($hash_nuclei{$nucleus}){return 1;} | |
| 478 | |
| 479 return 0; | |
| 480 } | |
| 481 | |
| 482 | |
| 483 | |
| 484 sub pass_filtering_initial{ | |
| 485 | |
| 486 #test if the structure forms a plausible hairpin | |
| 487 unless(pass_filtering_structure()){filter_p("structure problem"); return 0;} | |
| 488 | |
| 489 #test if >90% of reads map to the hairpin in consistence with Dicer processing | |
| 490 unless(pass_filtering_signature()){filter_p("signature problem");return 0;} | |
| 491 | |
| 492 return 1; | |
| 493 | |
| 494 } | |
| 495 | |
| 496 | |
| 497 sub pass_filtering_signature{ | |
| 498 | |
| 499 #number of reads that map in consistence with Dicer processing | |
| 500 my $consistent=0; | |
| 501 | |
| 502 #number of reads that map inconsistent with Dicer processing | |
| 503 my $inconsistent=0; | |
| 504 | |
| 505 # number of potential star reads map in good consistence with Drosha/Dicer processing | |
| 506 # (3' overhangs relative to mature product) | |
| 507 my $star_perfect=0; | |
| 508 | |
| 509 # number of potential star reads that do not map in good consistence with 3' overhang | |
| 510 my $star_fuzzy=0; | |
| 511 | |
| 512 | |
| 513 #sort queries (deep sequences) by their position on the hairpin | |
| 514 my @queries=sort {$hash_query{$a}{"subject_beg"} <=> $hash_query{$b}{"subject_beg"}} keys %hash_query; | |
| 515 | |
| 516 foreach my $query(@queries){ | |
| 517 | |
| 518 #number of reads that the deep sequence represents | |
| 519 unless(defined($hash_query{$query}{"freq"})){next;} | |
| 520 my $query_freq=$hash_query{$query}{"freq"}; | |
| 521 | |
| 522 #test which Dicer product (if any) the deep sequence corresponds to | |
| 523 my $product=test_query($query); | |
| 524 | |
| 525 #if the deep sequence corresponds to a Dicer product, add to the 'consistent' variable | |
| 526 if($product){$consistent+=$query_freq;} | |
| 527 | |
| 528 #if the deep sequence do not correspond to a Dicer product, add to the 'inconsistent' variable | |
| 529 else{$inconsistent+=$query_freq;} | |
| 530 | |
| 531 #test a potential star sequence has good 3' overhang | |
| 532 if($product eq "star"){ | |
| 533 if(test_star($query)){$star_perfect+=$query_freq;} | |
| 534 else{$star_fuzzy+=$query_freq;} | |
| 535 } | |
| 536 } | |
| 537 | |
| 538 # if the majority of potential star sequences map in good accordance with 3' overhang | |
| 539 # score for the presence of star evidence | |
| 540 if($star_perfect>$star_fuzzy){$hash_comp{"star_read"}=1;} | |
| 541 | |
| 542 #total number of reads mapping to the hairpin | |
| 543 my $freq=$consistent+$inconsistent; | |
| 544 $hash_comp{"freq"}=$freq; | |
| 545 unless($freq>0){filter_s("read frequency too low"); return 0;} | |
| 546 | |
| 547 #unless >90% of the reads map in consistence with Dicer processing, the hairpin is discarded | |
| 548 my $inconsistent_fraction=$inconsistent/($inconsistent+$consistent); | |
| 549 unless($inconsistent_fraction<=0.1){filter_p("inconsistent\t$inconsistent\nconsistent\t$consistent"); return 0;} | |
| 550 | |
| 551 #the hairpin is retained | |
| 552 return 1; | |
| 553 } | |
| 554 | |
| 555 sub test_star{ | |
| 556 | |
| 557 #test if a deep sequence maps in good consistence with 3' overhang | |
| 558 | |
| 559 my $query=shift; | |
| 560 | |
| 561 #5' begin and 3' end positions | |
| 562 my $beg=$hash_query{$query}{"subject_beg"}; | |
| 563 my $end=$hash_query{$query}{"subject_end"}; | |
| 564 | |
| 565 #the difference between observed and expected begin positions must be 0 or 1 | |
| 566 my $offset=$beg-$hash_comp{"star_beg"}; | |
| 567 if($offset==0 or $offset==1 or $offset==-1){return 1;} | |
| 568 | |
| 569 return 0; | |
| 570 } | |
| 571 | |
| 572 | |
| 573 | |
| 574 sub test_query{ | |
| 575 | |
| 576 #test if deep sequence maps in consistence with Dicer processing | |
| 577 | |
| 578 my $query=shift; | |
| 579 | |
| 580 #begin, end, strand and read count | |
| 581 my $beg=$hash_query{$query}{"subject_beg"}; | |
| 582 my $end=$hash_query{$query}{"subject_end"}; | |
| 583 my $strand=$hash_query{$query}{"strand"}; | |
| 584 my $freq=$hash_query{$query}{"freq"}; | |
| 585 | |
| 586 #should not be on the minus strand (although this has in fact anecdotally been observed for known miRNAs) | |
| 587 if($strand eq '-'){return 0;} | |
| 588 | |
| 589 #the deep sequence is allowed to stretch 2 nt beyond the expected 5' end | |
| 590 my $fuzz_beg=2; | |
| 591 #the deep sequence is allowed to stretch 5 nt beyond the expected 3' end | |
| 592 my $fuzz_end=2; | |
| 593 | |
| 594 #if in accordance with Dicer processing, return the type of Dicer product | |
| 595 if(contained($beg,$end,$hash_comp{"mature_beg"}-$fuzz_beg,$hash_comp{"mature_end"}+$fuzz_end)){return "mature";} | |
| 596 if(contained($beg,$end,$hash_comp{"star_beg"}-$fuzz_beg,$hash_comp{"star_end"}+$fuzz_end)){return "star";} | |
| 597 if(contained($beg,$end,$hash_comp{"loop_beg"}-$fuzz_beg,$hash_comp{"loop_end"}+$fuzz_end)){return "loop";} | |
| 598 | |
| 599 #if not in accordance, return 0 | |
| 600 return 0; | |
| 601 } | |
| 602 | |
| 603 | |
| 604 sub pass_filtering_structure{ | |
| 605 | |
| 606 #The potential precursor must form a hairpin with miRNA precursor-like characteristics | |
| 607 | |
| 608 #return value | |
| 609 my $ret=1; | |
| 610 | |
| 611 #potential mature, star, loop and lower flank parts must be identifiable | |
| 612 unless(test_components()){return 0;} | |
| 613 | |
| 614 #no bifurcations | |
| 615 unless(no_bifurcations_precursor()){$ret=0;} | |
| 616 | |
| 617 #minimum 14 base pairings in duplex | |
| 618 unless(bp_duplex()>=15){$ret=0;filter_s("too few pairings in duplex");} | |
| 619 | |
| 620 #not more than 6 nt difference between mature and star length | |
| 621 unless(-6<diff_lng() and diff_lng()<6){$ret=0; filter_s("too big difference between mature and star length") } | |
| 622 | |
| 623 return $ret; | |
| 624 } | |
| 625 | |
| 626 | |
| 627 | |
| 628 | |
| 629 | |
| 630 | |
| 631 sub test_components{ | |
| 632 | |
| 633 #tests whether potential mature, star, loop and lower flank parts are identifiable | |
| 634 | |
| 635 unless($hash_comp{"mature_struct"}){ | |
| 636 filter_s("no mature"); | |
| 637 # print STDERR "no mature\n"; | |
| 638 return 0; | |
| 639 } | |
| 640 | |
| 641 unless($hash_comp{"star_struct"}){ | |
| 642 filter_s("no star"); | |
| 643 # print STDERR "no star\n"; | |
| 644 return 0; | |
| 645 } | |
| 646 | |
| 647 unless($hash_comp{"loop_struct"}){ | |
| 648 filter_s("no loop"); | |
| 649 # print STDERR "no loop\n"; | |
| 650 return 0; | |
| 651 } | |
| 652 | |
| 653 unless($hash_comp{"flank_first_struct"}){ | |
| 654 filter_s("no flanks"); | |
| 655 #print STDERR "no flanks_first_struct\n"; | |
| 656 return 0; | |
| 657 } | |
| 658 | |
| 659 unless($hash_comp{"flank_second_struct"}){ | |
| 660 filter_s("no flanks"); | |
| 661 # print STDERR "no flanks_second_struct\n"; | |
| 662 return 0; | |
| 663 } | |
| 664 return 1; | |
| 665 } | |
| 666 | |
| 667 | |
| 668 | |
| 669 | |
| 670 | |
| 671 sub no_bifurcations_precursor{ | |
| 672 | |
| 673 #tests whether there are bifurcations in the hairpin | |
| 674 | |
| 675 #assembles the potential precursor sequence and structure from the expected Dicer products | |
| 676 #this is the expected biological precursor, in contrast with 'pri_seq' that includes | |
| 677 #some genomic flanks on both sides | |
| 678 | |
| 679 my $pre_struct; | |
| 680 my $pre_seq; | |
| 681 if($hash_comp{"mature_arm"} eq "first"){ | |
| 682 $pre_struct.=$hash_comp{"mature_struct"}.$hash_comp{"loop_struct"}.$hash_comp{"star_struct"}; | |
| 683 $pre_seq.=$hash_comp{"mature_seq"}.$hash_comp{"loop_seq"}.$hash_comp{"star_seq"}; | |
| 684 }else{ | |
| 685 $pre_struct.=$hash_comp{"star_struct"}.$hash_comp{"loop_struct"}.$hash_comp{"mature_struct"}; | |
| 686 $pre_seq.=$hash_comp{"star_seq"}.$hash_comp{"loop_seq"}.$hash_comp{"mature_seq"}; | |
| 687 } | |
| 688 | |
| 689 #read into hash | |
| 690 $hash_comp{"pre_struct"}=$pre_struct; | |
| 691 $hash_comp{"pre_seq"}=$pre_seq; | |
| 692 | |
| 693 #simple pattern matching checks for bifurcations | |
| 694 unless($pre_struct=~/^((\.|\()+..(\.|\))+)$/){ | |
| 695 filter_s("bifurcation in precursor"); | |
| 696 # print STDERR "bifurcation in precursor\n"; | |
| 697 return 0; | |
| 698 } | |
| 699 | |
| 700 return 1; | |
| 701 } | |
| 702 | |
| 703 sub bp_precursor{ | |
| 704 | |
| 705 #total number of bps in the precursor | |
| 706 | |
| 707 my $pre_struct=$hash_comp{"pre_struct"}; | |
| 708 | |
| 709 #simple pattern matching | |
| 710 my $pre_bps=0; | |
| 711 while($pre_struct=~/\(/g){ | |
| 712 $pre_bps++; | |
| 713 } | |
| 714 return $pre_bps; | |
| 715 } | |
| 716 | |
| 717 | |
| 718 sub bp_duplex{ | |
| 719 | |
| 720 #total number of bps in the duplex | |
| 721 | |
| 722 my $duplex_bps=0; | |
| 723 my $mature_struct=$hash_comp{"mature_struct"}; | |
| 724 | |
| 725 #simple pattern matching | |
| 726 while($mature_struct=~/(\(|\))/g){ | |
| 727 $duplex_bps++; | |
| 728 } | |
| 729 return $duplex_bps; | |
| 730 } | |
| 731 | |
| 732 sub diff_lng{ | |
| 733 | |
| 734 #find difference between mature and star lengths | |
| 735 | |
| 736 my $mature_lng=length $hash_comp{"mature_struct"}; | |
| 737 my $star_lng=length $hash_comp{"star_struct"}; | |
| 738 my $diff_lng=$mature_lng-$star_lng; | |
| 739 return $diff_lng; | |
| 740 } | |
| 741 | |
| 742 | |
| 743 | |
| 744 sub do_test_assemble{ | |
| 745 | |
| 746 # not currently used, tests if the 'pri_struct' as assembled from the parts (Dicer products, lower flanks) | |
| 747 # is identical to 'pri_struct' before disassembly into parts | |
| 748 | |
| 749 my $assemble_struct; | |
| 750 | |
| 751 if($hash_comp{"flank_first_struct"} and $hash_comp{"mature_struct"} and $hash_comp{"loop_struct"} and $hash_comp{"star_struct"} and $hash_comp{"flank_second_struct"}){ | |
| 752 if($hash_comp{"mature_arm"} eq "first"){ | |
| 753 $assemble_struct.=$hash_comp{"flank_first_struct"}.$hash_comp{"mature_struct"}.$hash_comp{"loop_struct"}.$hash_comp{"star_struct"}.$hash_comp{"flank_second_struct"}; | |
| 754 }else{ | |
| 755 $assemble_struct.=$hash_comp{"flank_first_struct"}.$hash_comp{"star_struct"}.$hash_comp{"loop_struct"}.$hash_comp{"mature_struct"}.$hash_comp{"flank_second_struct"}; | |
| 756 } | |
| 757 unless($assemble_struct eq $hash_comp{"pri_struct"}){ | |
| 758 $hash_comp{"test_assemble"}=$assemble_struct; | |
| 759 print_hash_comp(); | |
| 760 } | |
| 761 } | |
| 762 return; | |
| 763 } | |
| 764 | |
| 765 | |
| 766 | |
| 767 sub fill_structure{ | |
| 768 | |
| 769 #reads the dot bracket structure into the 'bp' hash where each key and value are basepaired | |
| 770 | |
| 771 my $struct=$hash_struct{$subject_old}; | |
| 772 my $lng=length $struct; | |
| 773 | |
| 774 #local stack for keeping track of basepairings | |
| 775 my @bps; | |
| 776 | |
| 777 for(my $pos=1;$pos<=$lng;$pos++){ | |
| 778 my $struct_pos=excise_struct($struct,$pos,$pos,"+"); | |
| 779 | |
| 780 if($struct_pos eq "("){ | |
| 781 push(@bps,$pos); | |
| 782 } | |
| 783 | |
| 784 if($struct_pos eq ")"){ | |
| 785 my $pos_prev=pop(@bps); | |
| 786 $hash_bp{$pos_prev}=$pos; | |
| 787 $hash_bp{$pos}=$pos_prev; | |
| 788 } | |
| 789 } | |
| 790 return; | |
| 791 } | |
| 792 | |
| 793 | |
| 794 | |
| 795 sub fill_star{ | |
| 796 | |
| 797 #fills specifics on the expected star strand into 'comp' hash ('component' hash) | |
| 798 | |
| 799 #if the mature sequence is not plausible, don't look for the star arm | |
| 800 my $mature_arm=$hash_comp{"mature_arm"}; | |
| 801 unless($mature_arm){$hash_comp{"star_arm"}=0; return;} | |
| 802 | |
| 803 #if the star sequence is not plausible, don't fill into the hash | |
| 804 my($star_beg,$star_end)=find_star(); | |
| 805 my $star_arm=arm_star($star_beg,$star_end); | |
| 806 unless($star_arm){return;} | |
| 807 | |
| 808 #excise expected star sequence and structure | |
| 809 my $star_seq=excise_seq($hash_comp{"pri_seq"},$star_beg,$star_end,"+"); | |
| 810 my $star_struct=excise_seq($hash_comp{"pri_struct"},$star_beg,$star_end,"+"); | |
| 811 | |
| 812 #fill into hash | |
| 813 $hash_comp{"star_beg"}=$star_beg; | |
| 814 $hash_comp{"star_end"}=$star_end; | |
| 815 $hash_comp{"star_seq"}=$star_seq; | |
| 816 $hash_comp{"star_struct"}=$star_struct; | |
| 817 $hash_comp{"star_arm"}=$star_arm; | |
| 818 | |
| 819 return; | |
| 820 } | |
| 821 | |
| 822 | |
| 823 sub find_star{ | |
| 824 | |
| 825 #uses the 'bp' hash to find the expected star begin and end positions from the mature positions | |
| 826 | |
| 827 #the -2 is for the overhang | |
| 828 my $mature_beg=$hash_comp{"mature_beg"}; | |
| 829 my $mature_end=$hash_comp{"mature_end"}-2; | |
| 830 my $mature_lng=$mature_end-$mature_beg+1; | |
| 831 | |
| 832 #in some cases, the last nucleotide of the mature sequence does not form a base pair, | |
| 833 #and therefore does not basepair with the first nucleotide of the star sequence. | |
| 834 #In this case, the algorithm searches for the last nucleotide of the mature sequence | |
| 835 #to form a base pair. The offset is the number of nucleotides searched through. | |
| 836 my $offset_star_beg=0; | |
| 837 my $offset_beg=0; | |
| 838 | |
| 839 #the offset should not be longer than the length of the mature sequence, then it | |
| 840 #means that the mature sequence does not form any base pairs | |
| 841 while(!$offset_star_beg and $offset_beg<$mature_lng){ | |
| 842 if($hash_bp{$mature_end-$offset_beg}){ | |
| 843 $offset_star_beg=$hash_bp{$mature_end-$offset_beg}; | |
| 844 }else{ | |
| 845 $offset_beg++; | |
| 846 } | |
| 847 } | |
| 848 #when defining the beginning of the star sequence, compensate for the offset | |
| 849 my $star_beg=$offset_star_beg-$offset_beg; | |
| 850 | |
| 851 #same as above | |
| 852 my $offset_star_end=0; | |
| 853 my $offset_end=0; | |
| 854 while(!$offset_star_end and $offset_end<$mature_lng){ | |
| 855 if($hash_bp{$mature_beg+$offset_end}){ | |
| 856 $offset_star_end=$hash_bp{$mature_beg+$offset_end}; | |
| 857 }else{ | |
| 858 $offset_end++; | |
| 859 } | |
| 860 } | |
| 861 #the +2 is for the overhang | |
| 862 my $star_end=$offset_star_end+$offset_end+2; | |
| 863 | |
| 864 return($star_beg,$star_end); | |
| 865 } | |
| 866 | |
| 867 | |
| 868 sub fill_pri{ | |
| 869 | |
| 870 #fills basic specifics on the precursor into the 'comp' hash | |
| 871 | |
| 872 my $seq=$hash_seq{$subject_old}; | |
| 873 my $struct=$hash_struct{$subject_old}; | |
| 874 my $mfe=$hash_mfe{$subject_old}; | |
| 875 my $length=length $seq; | |
| 876 | |
| 877 $hash_comp{"pri_id"}=$subject_old; | |
| 878 $hash_comp{"pri_seq"}=$seq; | |
| 879 $hash_comp{"pri_struct"}=$struct; | |
| 880 $hash_comp{"pri_mfe"}=$mfe; | |
| 881 $hash_comp{"pri_beg"}=1; | |
| 882 $hash_comp{"pri_end"}=$length; | |
| 883 | |
| 884 return; | |
| 885 } | |
| 886 | |
| 887 | |
| 888 sub fill_mature{ | |
| 889 | |
| 890 #fills specifics on the mature sequence into the 'comp' hash | |
| 891 | |
| 892 my $mature_query=find_mature_query(); | |
| 893 my($mature_beg,$mature_end)=find_positions_query($mature_query); | |
| 894 my $mature_strand=find_strand_query($mature_query); | |
| 895 my $mature_seq=excise_seq($hash_comp{"pri_seq"},$mature_beg,$mature_end,$mature_strand); | |
| 896 my $mature_struct=excise_struct($hash_comp{"pri_struct"},$mature_beg,$mature_end,$mature_strand); | |
| 897 my $mature_arm=arm_mature($mature_beg,$mature_end,$mature_strand); | |
| 898 | |
| 899 $hash_comp{"mature_query"}=$mature_query; | |
| 900 $hash_comp{"mature_beg"}=$mature_beg; | |
| 901 $hash_comp{"mature_end"}=$mature_end; | |
| 902 $hash_comp{"mature_strand"}=$mature_strand; | |
| 903 $hash_comp{"mature_struct"}=$mature_struct; | |
| 904 $hash_comp{"mature_seq"}=$mature_seq; | |
| 905 $hash_comp{"mature_arm"}=$mature_arm; | |
| 906 | |
| 907 return; | |
| 908 } | |
| 909 | |
| 910 | |
| 911 | |
| 912 sub fill_loop{ | |
| 913 | |
| 914 #fills specifics on the loop sequence into the 'comp' hash | |
| 915 | |
| 916 #unless both mature and star sequences are plausible, do not look for the loop | |
| 917 unless($hash_comp{"mature_arm"} and $hash_comp{"star_arm"}){return;} | |
| 918 | |
| 919 my $loop_beg; | |
| 920 my $loop_end; | |
| 921 | |
| 922 #defining the begin and end positions of the loop from the mature and star positions | |
| 923 #excision depends on whether the mature or star sequence is 5' of the loop ('first') | |
| 924 if($hash_comp{"mature_arm"} eq "first"){ | |
| 925 $loop_beg=$hash_comp{"mature_end"}+1; | |
| 926 }else{ | |
| 927 $loop_end=$hash_comp{"mature_beg"}-1; | |
| 928 } | |
| 929 | |
| 930 if($hash_comp{"star_arm"} eq "first"){ | |
| 931 $loop_beg=$hash_comp{"star_end"}+1; | |
| 932 }else{ | |
| 933 $loop_end=$hash_comp{"star_beg"}-1; | |
| 934 } | |
| 935 | |
| 936 #unless the positions are plausible, do not fill into hash | |
| 937 unless(test_loop($loop_beg,$loop_end)){return;} | |
| 938 | |
| 939 my $loop_seq=excise_seq($hash_comp{"pri_seq"},$loop_beg,$loop_end,"+"); | |
| 940 my $loop_struct=excise_struct($hash_comp{"pri_struct"},$loop_beg,$loop_end,"+"); | |
| 941 | |
| 942 $hash_comp{"loop_beg"}=$loop_beg; | |
| 943 $hash_comp{"loop_end"}=$loop_end; | |
| 944 $hash_comp{"loop_seq"}=$loop_seq; | |
| 945 $hash_comp{"loop_struct"}=$loop_struct; | |
| 946 | |
| 947 return; | |
| 948 } | |
| 949 | |
| 950 | |
| 951 sub fill_lower_flanks{ | |
| 952 | |
| 953 #fills specifics on the lower flanks and unpaired strands into the 'comp' hash | |
| 954 | |
| 955 #unless both mature and star sequences are plausible, do not look for the flanks | |
| 956 unless($hash_comp{"mature_arm"} and $hash_comp{"star_arm"}){return;} | |
| 957 | |
| 958 my $flank_first_end; | |
| 959 my $flank_second_beg; | |
| 960 | |
| 961 #defining the begin and end positions of the flanks from the mature and star positions | |
| 962 #excision depends on whether the mature or star sequence is 5' in the potenitial precursor ('first') | |
| 963 if($hash_comp{"mature_arm"} eq "first"){ | |
| 964 $flank_first_end=$hash_comp{"mature_beg"}-1; | |
| 965 }else{ | |
| 966 $flank_second_beg=$hash_comp{"mature_end"}+1; | |
| 967 } | |
| 968 | |
| 969 if($hash_comp{"star_arm"} eq "first"){ | |
| 970 $flank_first_end=$hash_comp{"star_beg"}-1; | |
| 971 }else{ | |
| 972 $flank_second_beg=$hash_comp{"star_end"}+1; | |
| 973 } | |
| 974 | |
| 975 #unless the positions are plausible, do not fill into hash | |
| 976 unless(test_flanks($flank_first_end,$flank_second_beg)){return;} | |
| 977 | |
| 978 $hash_comp{"flank_first_end"}=$flank_first_end; | |
| 979 $hash_comp{"flank_second_beg"}=$flank_second_beg; | |
| 980 $hash_comp{"flank_first_seq"}=excise_seq($hash_comp{"pri_seq"},$hash_comp{"pri_beg"},$hash_comp{"flank_first_end"},"+"); | |
| 981 $hash_comp{"flank_second_seq"}=excise_seq($hash_comp{"pri_seq"},$hash_comp{"flank_second_beg"},$hash_comp{"pri_end"},"+"); | |
| 982 $hash_comp{"flank_first_struct"}=excise_struct($hash_comp{"pri_struct"},$hash_comp{"pri_beg"},$hash_comp{"flank_first_end"},"+"); | |
| 983 $hash_comp{"flank_second_struct"}=excise_struct($hash_comp{"pri_struct"},$hash_comp{"flank_second_beg"},$hash_comp{"pri_end"},"+"); | |
| 984 | |
| 985 if($options{z}){ | |
| 986 fill_stems_drosha(); | |
| 987 } | |
| 988 | |
| 989 return; | |
| 990 } | |
| 991 | |
| 992 | |
| 993 sub fill_stems_drosha{ | |
| 994 | |
| 995 #scores the number of base pairings formed by the first ten nt of the lower stems | |
| 996 #in general, the more stems, the higher the score contribution | |
| 997 #warning: this options has not been thoroughly tested | |
| 998 | |
| 999 my $flank_first_struct=$hash_comp{"flank_first_struct"}; | |
| 1000 my $flank_second_struct=$hash_comp{"flank_second_struct"}; | |
| 1001 | |
| 1002 my $stem_first=substr($flank_first_struct,-10); | |
| 1003 my $stem_second=substr($flank_second_struct,0,10); | |
| 1004 | |
| 1005 my $stem_bp_first=0; | |
| 1006 my $stem_bp_second=0; | |
| 1007 | |
| 1008 #find base pairings by simple pattern matching | |
| 1009 while($stem_first=~/\(/g){ | |
| 1010 $stem_bp_first++; | |
| 1011 } | |
| 1012 | |
| 1013 while($stem_second=~/\)/g){ | |
| 1014 $stem_bp_second++; | |
| 1015 } | |
| 1016 | |
| 1017 my $stem_bp=min2($stem_bp_first,$stem_bp_second); | |
| 1018 | |
| 1019 $hash_comp{"stem_first"}=$stem_first; | |
| 1020 $hash_comp{"stem_second"}=$stem_second; | |
| 1021 $hash_comp{"stem_bp_first"}=$stem_bp_first; | |
| 1022 $hash_comp{"stem_bp_second"}=$stem_bp_second; | |
| 1023 $hash_comp{"stem_bp"}=$stem_bp; | |
| 1024 | |
| 1025 return; | |
| 1026 } | |
| 1027 | |
| 1028 | |
| 1029 | |
| 1030 | |
| 1031 sub arm_mature{ | |
| 1032 | |
| 1033 #tests whether the mature sequence is in the 5' ('first') or 3' ('second') arm of the potential precursor | |
| 1034 | |
| 1035 my ($beg,$end,$strand)=@_; | |
| 1036 | |
| 1037 #mature and star sequences should alway be on plus strand | |
| 1038 if($strand eq "-"){return 0;} | |
| 1039 | |
| 1040 #there should be no bifurcations and minimum one base pairing | |
| 1041 my $struct=excise_seq($hash_comp{"pri_struct"},$beg,$end,$strand); | |
| 1042 if(defined($struct) and $struct=~/^(\(|\.)+$/ and $struct=~/\(/){ | |
| 1043 return "first"; | |
| 1044 }elsif(defined($struct) and $struct=~/^(\)|\.)+$/ and $struct=~/\)/){ | |
| 1045 return "second"; | |
| 1046 } | |
| 1047 return 0; | |
| 1048 } | |
| 1049 | |
| 1050 | |
| 1051 sub arm_star{ | |
| 1052 | |
| 1053 #tests whether the star sequence is in the 5' ('first') or 3' ('second') arm of the potential precursor | |
| 1054 | |
| 1055 my ($beg,$end)=@_; | |
| 1056 | |
| 1057 #unless the begin and end positions are plausible, test negative | |
| 1058 unless($beg>0 and $beg<=$hash_comp{"pri_end"} and $end>0 and $end<=$hash_comp{"pri_end"} and $beg<=$end){return 0;} | |
| 1059 | |
| 1060 #no overlap between the mature and the star sequence | |
| 1061 if($hash_comp{"mature_arm"} eq "first"){ | |
| 1062 ($hash_comp{"mature_end"}<$beg) or return 0; | |
| 1063 }elsif($hash_comp{"mature_arm"} eq "second"){ | |
| 1064 ($end<$hash_comp{"mature_beg"}) or return 0; | |
| 1065 } | |
| 1066 | |
| 1067 #there should be no bifurcations and minimum one base pairing | |
| 1068 my $struct=excise_seq($hash_comp{"pri_struct"},$beg,$end,"+"); | |
| 1069 if($struct=~/^(\(|\.)+$/ and $struct=~/\(/){ | |
| 1070 return "first"; | |
| 1071 }elsif($struct=~/^(\)|\.)+$/ and $struct=~/\)/){ | |
| 1072 return "second"; | |
| 1073 } | |
| 1074 return 0; | |
| 1075 } | |
| 1076 | |
| 1077 | |
| 1078 sub test_loop{ | |
| 1079 | |
| 1080 #tests the loop positions | |
| 1081 | |
| 1082 my ($beg,$end)=@_; | |
| 1083 | |
| 1084 #unless the begin and end positions are plausible, test negative | |
| 1085 unless($beg>0 and $beg<=$hash_comp{"pri_end"} and $end>0 and $end<=$hash_comp{"pri_end"} and $beg<=$end){return 0;} | |
| 1086 | |
| 1087 return 1; | |
| 1088 } | |
| 1089 | |
| 1090 | |
| 1091 sub test_flanks{ | |
| 1092 | |
| 1093 #tests the positions of the lower flanks | |
| 1094 | |
| 1095 my ($beg,$end)=@_; | |
| 1096 | |
| 1097 #unless the begin and end positions are plausible, test negative | |
| 1098 unless($beg>0 and $beg<=$hash_comp{"pri_end"} and $end>0 and $end<=$hash_comp{"pri_end"} and $beg<=$end){return 0;} | |
| 1099 | |
| 1100 return 1; | |
| 1101 } | |
| 1102 | |
| 1103 | |
| 1104 sub comp{ | |
| 1105 | |
| 1106 #subroutine to retrive from the 'comp' hash | |
| 1107 | |
| 1108 my $type=shift; | |
| 1109 my $component=$hash_comp{$type}; | |
| 1110 return $component; | |
| 1111 } | |
| 1112 | |
| 1113 | |
| 1114 sub find_strand_query{ | |
| 1115 | |
| 1116 #subroutine to find the strand for a given query | |
| 1117 | |
| 1118 my $query=shift; | |
| 1119 my $strand=$hash_query{$query}{"strand"}; | |
| 1120 return $strand; | |
| 1121 } | |
| 1122 | |
| 1123 | |
| 1124 sub find_positions_query{ | |
| 1125 | |
| 1126 #subroutine to find the begin and end positions for a given query | |
| 1127 | |
| 1128 my $query=shift; | |
| 1129 my $beg=$hash_query{$query}{"subject_beg"}; | |
| 1130 my $end=$hash_query{$query}{"subject_end"}; | |
| 1131 return ($beg,$end); | |
| 1132 } | |
| 1133 | |
| 1134 | |
| 1135 | |
| 1136 sub find_mature_query{ | |
| 1137 | |
| 1138 #finds the query with the highest frequency of reads and returns it | |
| 1139 #is used to determine the positions of the potential mature sequence | |
| 1140 | |
| 1141 my @queries=sort {$hash_query{$b}{"freq"} <=> $hash_query{$a}{"freq"}} keys %hash_query; | |
| 1142 my $mature_query=$queries[0]; | |
| 1143 return $mature_query; | |
| 1144 } | |
| 1145 | |
| 1146 | |
| 1147 | |
| 1148 | |
| 1149 sub reset_variables{ | |
| 1150 | |
| 1151 #resets the hashes for the next potential precursor | |
| 1152 | |
| 1153 # %hash_query=(); | |
| 1154 # %hash_comp=(); | |
| 1155 # %hash_bp=(); | |
| 1156 foreach my $key (keys %hash_query) {delete($hash_query{$key});} | |
| 1157 foreach my $key (keys %hash_comp) {delete($hash_comp{$key});} | |
| 1158 foreach my $key (keys %hash_bp) {delete($hash_bp{$key});} | |
| 1159 | |
| 1160 # $message_filter=(); | |
| 1161 # $message_score=(); | |
| 1162 # $lines=(); | |
| 1163 undef($message_filter); | |
| 1164 undef($message_score); | |
| 1165 undef($lines); | |
| 1166 return; | |
| 1167 } | |
| 1168 | |
| 1169 | |
| 1170 | |
| 1171 sub excise_seq{ | |
| 1172 | |
| 1173 #excise sub sequence from the potential precursor | |
| 1174 | |
| 1175 my($seq,$beg,$end,$strand)=@_; | |
| 1176 | |
| 1177 #begin can be equal to end if only one nucleotide is excised | |
| 1178 unless($beg<=$end){print STDERR "begin can not be smaller than end for $subject_old\n";exit;} | |
| 1179 | |
| 1180 #rarely, permuted combinations of signature and structure cause out of bound excision errors. | |
| 1181 #this happens once appr. every two thousand combinations | |
| 1182 unless($beg<=length($seq)){$out_of_bound++;return 0;} | |
| 1183 | |
| 1184 #if on the minus strand, the reverse complement should be excised | |
| 1185 if($strand eq "-"){$seq=revcom($seq);} | |
| 1186 | |
| 1187 #the blast parsed format is 1-indexed, substr is 0-indexed | |
| 1188 my $sub_seq=substr($seq,$beg-1,$end-$beg+1); | |
| 1189 | |
| 1190 return $sub_seq; | |
| 1191 | |
| 1192 } | |
| 1193 | |
| 1194 sub excise_struct{ | |
| 1195 | |
| 1196 #excise sub structure | |
| 1197 | |
| 1198 my($struct,$beg,$end,$strand)=@_; | |
| 1199 my $lng=length $struct; | |
| 1200 | |
| 1201 #begin can be equal to end if only one nucleotide is excised | |
| 1202 unless($beg<=$end){print STDERR "begin can not be smaller than end for $subject_old\n";exit;} | |
| 1203 | |
| 1204 #rarely, permuted combinations of signature and structure cause out of bound excision errors. | |
| 1205 #this happens once appr. every two thousand combinations | |
| 1206 unless($beg<=length($struct)){return 0;} | |
| 1207 | |
| 1208 #if excising relative to minus strand, positions are reversed | |
| 1209 if($strand eq "-"){($beg,$end)=rev_pos($beg,$end,$lng);} | |
| 1210 | |
| 1211 #the blast parsed format is 1-indexed, substr is 0-indexed | |
| 1212 my $sub_struct=substr($struct,$beg-1,$end-$beg+1); | |
| 1213 | |
| 1214 return $sub_struct; | |
| 1215 } | |
| 1216 | |
| 1217 | |
| 1218 sub create_hash_nuclei{ | |
| 1219 #parses a fasta file with sequences of known miRNAs considered for conservation purposes | |
| 1220 #reads the nuclei into a hash | |
| 1221 | |
| 1222 my ($file) = @_; | |
| 1223 my ($id, $desc, $sequence, $nucleus) = (); | |
| 1224 | |
| 1225 open (FASTA, "<$file") or die "can not open $file\n"; | |
| 1226 while (<FASTA>) | |
| 1227 { | |
| 1228 chomp; | |
| 1229 if (/^>(\S+)(.*)/) | |
| 1230 { | |
| 1231 $id = $1; | |
| 1232 $desc = $2; | |
| 1233 $sequence = ""; | |
| 1234 $nucleus = ""; | |
| 1235 while (<FASTA>){ | |
| 1236 chomp; | |
| 1237 if (/^>(\S+)(.*)/){ | |
| 1238 $nucleus = substr($sequence,1,$nucleus_lng); | |
| 1239 $nucleus =~ tr/[T]/[U]/; | |
| 1240 $hash_mirs{$nucleus} .="$id\t"; | |
| 1241 $hash_nuclei{$nucleus} += 1; | |
| 1242 | |
| 1243 $id = $1; | |
| 1244 $desc = $2; | |
| 1245 $sequence = ""; | |
| 1246 $nucleus = ""; | |
| 1247 next; | |
| 1248 } | |
| 1249 $sequence .= $_; | |
| 1250 } | |
| 1251 } | |
| 1252 } | |
| 1253 $nucleus = substr($sequence,1,$nucleus_lng); | |
| 1254 $nucleus =~ tr/[T]/[U]/; | |
| 1255 $hash_mirs{$nucleus} .="$id\t"; | |
| 1256 $hash_nuclei{$nucleus} += 1; | |
| 1257 close FASTA; | |
| 1258 } | |
| 1259 | |
| 1260 | |
| 1261 sub parse_file_struct{ | |
| 1262 #parses the output from RNAfoldand reads it into hashes | |
| 1263 my($file) = @_; | |
| 1264 my($id,$desc,$seq,$struct,$mfe) = (); | |
| 1265 open (FILE_STRUCT, "<$file") or die "can not open $file\n"; | |
| 1266 while (<FILE_STRUCT>){ | |
| 1267 chomp; | |
| 1268 if (/^>(\S+)\s*(.*)/){ | |
| 1269 $id= $1; | |
| 1270 $desc= $2; | |
| 1271 $seq= ""; | |
| 1272 $struct= ""; | |
| 1273 $mfe= ""; | |
| 1274 while (<FILE_STRUCT>){ | |
| 1275 chomp; | |
| 1276 if (/^>(\S+)\s*(.*)/){ | |
| 1277 $hash_desc{$id} = $desc; | |
| 1278 $hash_seq{$id} = $seq; | |
| 1279 $hash_struct{$id} = $struct; | |
| 1280 $hash_mfe{$id} = $mfe; | |
| 1281 $id = $1; | |
| 1282 $desc = $2; | |
| 1283 $seq = ""; | |
| 1284 $struct = ""; | |
| 1285 $mfe = ""; | |
| 1286 next; | |
| 1287 } | |
| 1288 if(/^\w/){ | |
| 1289 tr/uU/tT/; | |
| 1290 $seq .= $_; | |
| 1291 next; | |
| 1292 } | |
| 1293 if(/((\.|\(|\))+)/){$struct .=$1;} | |
| 1294 if(/\((\s*-\d+\.\d+)\)/){$mfe = $1;} | |
| 1295 } | |
| 1296 } | |
| 1297 } | |
| 1298 $hash_desc{$id} = $desc; | |
| 1299 $hash_seq{$id} = $seq; | |
| 1300 $hash_struct{$id} = $struct; | |
| 1301 $hash_mfe{$id} = $mfe; | |
| 1302 close FILE_STRUCT; | |
| 1303 return; | |
| 1304 } | |
| 1305 | |
| 1306 | |
| 1307 sub score_s{ | |
| 1308 | |
| 1309 #this score message is appended to the end of the string of score messages outputted for the potential precursor | |
| 1310 | |
| 1311 my $message=shift; | |
| 1312 $message_score.=$message."\n";; | |
| 1313 return; | |
| 1314 } | |
| 1315 | |
| 1316 | |
| 1317 | |
| 1318 sub score_p{ | |
| 1319 | |
| 1320 #this score message is appended to the beginning of the string of score messages outputted for the potential precursor | |
| 1321 | |
| 1322 my $message=shift; | |
| 1323 $message_score=$message."\n".$message_score; | |
| 1324 return; | |
| 1325 } | |
| 1326 | |
| 1327 | |
| 1328 | |
| 1329 sub filter_s{ | |
| 1330 | |
| 1331 #this filtering message is appended to the end of the string of filtering messages outputted for the potential precursor | |
| 1332 | |
| 1333 my $message=shift; | |
| 1334 $message_filter.=$message."\n"; | |
| 1335 return; | |
| 1336 } | |
| 1337 | |
| 1338 | |
| 1339 sub filter_p{ | |
| 1340 | |
| 1341 #this filtering message is appended to the beginning of the string of filtering messages outputted for the potential precursor | |
| 1342 | |
| 1343 my $message=shift; | |
| 1344 if(defined $message_filter){$message_filter=$message."\n".$message_filter;} | |
| 1345 else{$message_filter=$message."\n";} | |
| 1346 return; | |
| 1347 } | |
| 1348 | |
| 1349 | |
| 1350 sub find_freq{ | |
| 1351 | |
| 1352 #finds the frequency of a given read query from its id. | |
| 1353 | |
| 1354 my($query)=@_; | |
| 1355 | |
| 1356 if($query=~/x(\d+)/i){ | |
| 1357 my $freq=$1; | |
| 1358 return $freq; | |
| 1359 }else{ | |
| 1360 #print STDERR "Problem with read format\n"; | |
| 1361 return 0; | |
| 1362 } | |
| 1363 } | |
| 1364 | |
| 1365 | |
| 1366 sub print_hash_comp{ | |
| 1367 | |
| 1368 #prints the 'comp' hash | |
| 1369 | |
| 1370 my @keys=sort keys %hash_comp; | |
| 1371 foreach my $key(@keys){ | |
| 1372 my $value=$hash_comp{$key}; | |
| 1373 print "$key \t$value\n"; | |
| 1374 } | |
| 1375 } | |
| 1376 | |
| 1377 | |
| 1378 | |
| 1379 sub print_hash_bp{ | |
| 1380 | |
| 1381 #prints the 'bp' hash | |
| 1382 | |
| 1383 my @keys=sort {$a<=>$b} keys %hash_bp; | |
| 1384 foreach my $key(@keys){ | |
| 1385 my $value=$hash_bp{$key}; | |
| 1386 print "$key\t$value\n"; | |
| 1387 } | |
| 1388 print "\n"; | |
| 1389 } | |
| 1390 | |
| 1391 | |
| 1392 | |
| 1393 sub find_strand{ | |
| 1394 | |
| 1395 #A subroutine to find the strand, parsing different blast formats | |
| 1396 | |
| 1397 my($other)=@_; | |
| 1398 | |
| 1399 my $strand="+"; | |
| 1400 | |
| 1401 if($other=~/-/){ | |
| 1402 $strand="-"; | |
| 1403 } | |
| 1404 | |
| 1405 if($other=~/minus/i){ | |
| 1406 $strand="-"; | |
| 1407 } | |
| 1408 return($strand); | |
| 1409 } | |
| 1410 | |
| 1411 | |
| 1412 sub contained{ | |
| 1413 | |
| 1414 #Is the stretch defined by the first positions contained in the stretch defined by the second? | |
| 1415 | |
| 1416 my($beg1,$end1,$beg2,$end2)=@_; | |
| 1417 | |
| 1418 testbeginend($beg1,$end1,$beg2,$end2); | |
| 1419 | |
| 1420 if($beg2<=$beg1 and $end1<=$end2){ | |
| 1421 return 1; | |
| 1422 }else{ | |
| 1423 return 0; | |
| 1424 } | |
| 1425 } | |
| 1426 | |
| 1427 | |
| 1428 sub testbeginend{ | |
| 1429 | |
| 1430 #Are the beginposition numerically smaller than the endposition for each pair? | |
| 1431 | |
| 1432 my($begin1,$end1,$begin2,$end2)=@_; | |
| 1433 | |
| 1434 unless($begin1<=$end1 and $begin2<=$end2){ | |
| 1435 print STDERR "beg can not be larger than end for $subject_old\n"; | |
| 1436 exit; | |
| 1437 } | |
| 1438 } | |
| 1439 | |
| 1440 | |
| 1441 sub rev_pos{ | |
| 1442 | |
| 1443 # The blast_parsed format always uses positions that are relative to the 5' of the given strand | |
| 1444 # This means that for a sequence of length n, the first nucleotide on the minus strand base pairs with | |
| 1445 # the n't nucleotide on the plus strand | |
| 1446 | |
| 1447 # This subroutine reverses the begin and end positions of positions of the minus strand so that they | |
| 1448 # are relative to the 5' end of the plus strand | |
| 1449 | |
| 1450 my($beg,$end,$lng)=@_; | |
| 1451 | |
| 1452 my $new_end=$lng-$beg+1; | |
| 1453 my $new_beg=$lng-$end+1; | |
| 1454 | |
| 1455 return($new_beg,$new_end); | |
| 1456 } | |
| 1457 | |
| 1458 sub round { | |
| 1459 | |
| 1460 #rounds to nearest integer | |
| 1461 | |
| 1462 my($number) = shift; | |
| 1463 return int($number + .5); | |
| 1464 | |
| 1465 } | |
| 1466 | |
| 1467 | |
| 1468 sub rev{ | |
| 1469 | |
| 1470 #reverses the order of nucleotides in a sequence | |
| 1471 | |
| 1472 my($sequence)=@_; | |
| 1473 | |
| 1474 my $rev=reverse $sequence; | |
| 1475 | |
| 1476 return $rev; | |
| 1477 } | |
| 1478 | |
| 1479 sub com{ | |
| 1480 | |
| 1481 #the complementary of a sequence | |
| 1482 | |
| 1483 my($sequence)=@_; | |
| 1484 | |
| 1485 $sequence=~tr/acgtuACGTU/TGCAATGCAA/; | |
| 1486 | |
| 1487 return $sequence; | |
| 1488 } | |
| 1489 | |
| 1490 sub revcom{ | |
| 1491 | |
| 1492 #reverse complement | |
| 1493 | |
| 1494 my($sequence)=@_; | |
| 1495 | |
| 1496 my $revcom=rev(com($sequence)); | |
| 1497 | |
| 1498 return $revcom; | |
| 1499 } | |
| 1500 | |
| 1501 | |
| 1502 sub max2 { | |
| 1503 | |
| 1504 #max of two numbers | |
| 1505 | |
| 1506 my($a, $b) = @_; | |
| 1507 return ($a>$b ? $a : $b); | |
| 1508 } | |
| 1509 | |
| 1510 sub min2 { | |
| 1511 | |
| 1512 #min of two numbers | |
| 1513 | |
| 1514 my($a, $b) = @_; | |
| 1515 return ($a<$b ? $a : $b); | |
| 1516 } | |
| 1517 | |
| 1518 | |
| 1519 | |
| 1520 sub score_freq{ | |
| 1521 | |
| 1522 # scores the count of reads that map to the potential precursor | |
| 1523 # Assumes geometric distribution as described in methods section of manuscript | |
| 1524 | |
| 1525 my $freq=shift; | |
| 1526 | |
| 1527 #parameters of known precursors and background hairpins | |
| 1528 my $parameter_test=0.999; | |
| 1529 my $parameter_control=0.6; | |
| 1530 | |
| 1531 #log_odds calculated directly to avoid underflow | |
| 1532 my $intercept=log((1-$parameter_test)/(1-$parameter_control)); | |
| 1533 my $slope=log($parameter_test/$parameter_control); | |
| 1534 my $log_odds=$slope*$freq+$intercept; | |
| 1535 | |
| 1536 #if no strong evidence for 3' overhangs, limit the score contribution to 0 | |
| 1537 unless($options{x} or $hash_comp{"star_read"}){$log_odds=min2($log_odds,0);} | |
| 1538 | |
| 1539 return $log_odds; | |
| 1540 } | |
| 1541 | |
| 1542 | |
| 1543 | |
| 1544 ##sub score_mfe{ | |
| 1545 | |
| 1546 # scores the minimum free energy in kCal/mol of the potential precursor | |
| 1547 # Assumes Gumbel distribution as described in methods section of manuscript | |
| 1548 | |
| 1549 ## my $mfe=shift; | |
| 1550 | |
| 1551 #numerical value, minimum 1 | |
| 1552 ## my $mfe_adj=max2(1,-$mfe); | |
| 1553 | |
| 1554 #parameters of known precursors and background hairpins, scale and location | |
| 1555 ## my $prob_test=prob_gumbel_discretized($mfe_adj,5.5,32); | |
| 1556 ## my $prob_background=prob_gumbel_discretized($mfe_adj,4.8,23); | |
| 1557 | |
| 1558 ## my $odds=$prob_test/$prob_background; | |
| 1559 ## my $log_odds=log($odds); | |
| 1560 | |
| 1561 ## return $log_odds; | |
| 1562 ##} | |
| 1563 | |
| 1564 sub score_mfe{ | |
| 1565 # use bignum; | |
| 1566 | |
| 1567 # scores the minimum free energy in kCal/mol of the potential precursor | |
| 1568 # Assumes Gumbel distribution as described in methods section of manuscript | |
| 1569 | |
| 1570 my ($mfe,$mlng)=@_; | |
| 1571 | |
| 1572 #numerical value, minimum 1 | |
| 1573 my $mfe_adj=max2(1,-$mfe); | |
| 1574 my $mfe_adj1=$mfe/$mlng; | |
| 1575 #parameters of known precursors and background hairpins, scale and location | |
| 1576 my $a=1.339e-12;my $b=2.778e-13;my $c=45.834; | |
| 1577 my $ev=$e**($mfe_adj1*$c); | |
| 1578 #print STDERR "\n***",$ev,"**\t",$ev+$b,"\t"; | |
| 1579 my $log_odds=($a/($b+$ev)); | |
| 1580 | |
| 1581 | |
| 1582 my $prob_test=prob_gumbel_discretized($mfe_adj,5.5,32); | |
| 1583 my $prob_background=prob_gumbel_discretized($mfe_adj,4.8,23); | |
| 1584 | |
| 1585 my $odds=$prob_test/$prob_background; | |
| 1586 my $log_odds_2=log($odds); | |
| 1587 #print STDERR "log_odds :",$log_odds,"\t",$log_odds_2,"\n"; | |
| 1588 return $log_odds; | |
| 1589 } | |
| 1590 | |
| 1591 | |
| 1592 | |
| 1593 sub prob_gumbel_discretized{ | |
| 1594 | |
| 1595 # discretized Gumbel distribution, probabilities within windows of 1 kCal/mol | |
| 1596 # uses the subroutine that calculates the cdf to find the probabilities | |
| 1597 | |
| 1598 my ($var,$scale,$location)=@_; | |
| 1599 | |
| 1600 my $bound_lower=$var-0.5; | |
| 1601 my $bound_upper=$var+0.5; | |
| 1602 | |
| 1603 my $cdf_lower=cdf_gumbel($bound_lower,$scale,$location); | |
| 1604 my $cdf_upper=cdf_gumbel($bound_upper,$scale,$location); | |
| 1605 | |
| 1606 my $prob=$cdf_upper-$cdf_lower; | |
| 1607 | |
| 1608 return $prob; | |
| 1609 } | |
| 1610 | |
| 1611 | |
| 1612 sub cdf_gumbel{ | |
| 1613 | |
| 1614 # calculates the cumulative distribution function of the Gumbel distribution | |
| 1615 | |
| 1616 my ($var,$scale,$location)=@_; | |
| 1617 | |
| 1618 my $cdf=$e**(-($e**(-($var-$location)/$scale))); | |
| 1619 | |
| 1620 return $cdf; | |
| 1621 } | |
| 1622 |