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comparison PGAP-1.2.1/inparanoid.pl @ 0:83e62a1aeeeb draft

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1 #! /usr/bin/perl
2 ###############################################################################
3 # InParanoid version 4.1
4 # Copyright (C) Erik Sonnhammer, Kristoffer Forslund, Isabella Pekkari,
5 # Ann-Charlotte Berglund, Maido Remm, 2007
6 #
7 # This program is provided under the terms of a personal license to the recipient and may only
8 # be used for the recipient's own research at an academic insititution.
9 #
10 # Distribution of the results of this program must be discussed with the authors.
11 # For using this program in a company or for commercial purposes, a commercial license is required.
12 # Contact Erik.Sonnhammer@sbc.su.se in both cases
13 #
14 # Make sure that Perl XML libraries are installed!
15 #
16 # NOTE: This script requires blastall (NCBI BLAST) version 2.2.16 or higher, that supports
17 # compositional score matrix adjustment (-C2 flag).
18
19 my $usage =" Usage: inparanoid.pl <FASTAFILE with sequences of species A> <FASTAFILE with sequences of species B> [FASTAFILE with sequences of species C]
20
21 ";
22
23 ###############################################################################
24 # The program calculates orthologs between 2 datasets of proteins
25 # called A and B. Both datasets should be in multi-fasta file
26 # - Additionally, it tries to assign paralogous sequences (in-paralogs) to each
27 # thus forming paralogous clusters.
28 # - Confidence of in-paralogs is calculated in relative scale between 0-100%.
29 # This confidence value is dependent on how far is given sequence from the
30 # seed ortholog of given group
31 # - Confidence of groups can be calculated with bootstrapping. This is related
32 # to score difference between best hit and second best hit.
33 # - Optionally it can use a species C as outgroup.
34
35 ###############################################################################
36 # You may need to run the following command manually to increase your
37 # default datasize limit: 'limit datasize 500000 kb'
38
39 ###############################################################################
40 # Set following variables: #
41 ###############################################################################
42
43 # What do you want the program to do? #
44 $run_blast = 1; # Set to 1 if you don't have the 4 BLAST output files #
45 # Requires 'blastall', 'formatdb' (NCBI BLAST2) #
46 # and parser 'blast_parser.pl' #
47 $blast_two_passes = 1; # Set to 1 to run 2-pass strategy #
48 # (strongly recommended, but slower) #
49 $run_inparanoid = 1;
50 $use_bootstrap = 1;# Use bootstrapping to estimate the confidence of orthologs#
51 # Needs additional programs 'seqstat.jar' and 'blast2faa.pl'
52 $use_outgroup = 0; # Use proteins from the third genome as an outgroup #
53 # Reject best-best hit if outgroup sequence is MORE #
54 # similar to one of the sequences #
55 # (by more than $outgroup_cutoff bits) #
56
57 # Define location of files and programs:
58 #$blastall = "blastall -VT"; #Remove -VT for blast version 2.2.12 or earlier
59 $blastall = "$ARGV[0] -a $ARGV[1]"; #Add -aN to use N processors
60 $formatdb = "$ARGV[2]";
61 $seqstat = "seqstat.jar";
62 $blastParser = "blast_parser.pl";
63
64 #$matrix = "BLOSUM62"; # Reasonable default for comparison of eukaryotes.
65 $matrix = "BLOSUM45"; #(for prokaryotes),
66 #$matrix = "BLOSUM80"; #(orthologs within metazoa),
67 #$matrix = "PAM70";
68 #$matrix = "PAM30";
69
70 # Output options: #
71 $output = 0; # table_stats-format output #
72 $table = 0; # Print tab-delimited table of orthologs to file "table.txt" #
73 # Each orthologous group with all inparalogs is on one line #
74 $mysql_table = 1; # Print out sql tables for the web server #
75 # Each inparalog is on separate line #
76 $html = 0; # HTML-format output #
77
78 # Algorithm parameters:
79 # Default values should work without problems.
80 # MAKE SURE, however, that the score cutoff here matches what you used for BLAST!
81 $score_cutoff = $ARGV[3]; # In bits. Any match below this is ignored #
82 $outgroup_cutoff = 50; # In bits. Outgroup sequence hit must be this many bits#
83 # stronger to reject best-best hit between A and B #
84 $conf_cutoff = 0.05; # Include in-paralogs with this confidence or better #
85 $group_overlap_cutoff = 0.5; # Merge groups if ortholog in one group has more #
86 # than this confidence in other group #
87 $grey_zone = 0; # This many bits signifies the difference between 2 scores #
88 $show_times = 0; # Show times spent for execution of each part of the program #
89 # (This does not work properly) #
90 $debug = 0; # Print debugging messages or not. Levels 0,1,2 and 4 exist #
91
92 my $seq_overlap_cutoff = $ARGV[4]; # Match area should cover at least this much of longer sequence. Match area is defined as area from start of
93 # first segment to end of last segment, i.e segments 1-10 and 90-100 gives a match length of 100.
94 my $segment_coverage_cutoff = $ARGV[5]; # Actually matching segments must cover this much of longer sequence.
95 # For example, segments 1-10 and 90-100 gives a total length of 20.
96
97 splice(@ARGV,0,6);
98
99 ###############################################################################
100 # No changes should be required below this line #
101 ###############################################################################
102 $ENV{CLASSPATH} = "./$seqstat" if ($use_bootstrap);
103
104 if (!@ARGV){
105 print STDERR $usage;
106 exit 1;
107 }
108
109 if ((@ARGV < 2) and ($run_inparanoid)){
110 print STDERR "\n When \$run_inparanoid=1, at least two distinct FASTA files have to be specified.\n";
111
112 print STDERR $usage;
113 exit 1;
114 }
115
116 if ((!$run_blast) and (!$run_inparanoid)){
117 print STDERR "run_blast or run_inparanoid has to be set!\n";
118 exit 1;
119 }
120
121 # Input files:
122 $fasta_seq_fileA = "$ARGV[0]";
123 $fasta_seq_fileB = "$ARGV[1]";
124 $fasta_seq_fileC = "$ARGV[2]" if ($use_outgroup); # This is outgroup file
125
126 my $blast_outputAB = $fasta_seq_fileA . "-" . $fasta_seq_fileB;
127 my $blast_outputBA = $fasta_seq_fileB . "-" . $fasta_seq_fileA;
128 my $blast_outputAA = $fasta_seq_fileA . "-" . $fasta_seq_fileA;
129 my $blast_outputBB = $fasta_seq_fileB . "-" . $fasta_seq_fileB;
130
131 if ($use_outgroup){
132 $blast_outputAC = $fasta_seq_fileA . "-" . $fasta_seq_fileC;
133 $blast_outputBC = $fasta_seq_fileB . "-" . $fasta_seq_fileC;
134 }
135 my %idA; # Name -> ID combinations for species 1
136 my %idB; # Name -> ID combinations for species 2
137 my @nameA; # ID -> Name combinations for species 1
138 my @nameB; # ID -> Name combinations for species 2
139 my @nameC;
140 my %scoreAB; # Hashes with pairwise BLAST scores (in bits)
141 my %scoreBA;
142 my %scoreAA;
143 my %scoreBB;
144 my @hitnAB; # 1-D arrays that keep the number of pairwise hits
145 my @hitnBA;
146 my @hitnAA;
147 my @hitnBB;
148 my @hitAB; # 2-D arrays that keep the actual matching IDs
149 my @hitBA;
150 my @hitAA;
151 my @hitBB;
152 my @besthitAB; # IDs of best hits in other species (may contain more than one ID)
153 my @besthitBA; # IDs of best hits in other species (may contain more than one ID)
154 my @bestscoreAB; # best match A -> B
155 my @bestscoreBA; # best match B -> A
156 my @ortoA; # IDs of ortholog candidates from species A
157 my @ortoB; # IDs of ortholog candidates from species B
158 my @ortoS; # Scores between ortoA and ortoB pairs
159 my @paralogsA; # List of paralog IDs in given cluster
160 my @paralogsB; # List of paralog IDs in given cluster
161 my @confPA; # Confidence values for A paralogs
162 my @confPB; # Confidence values for B paralogs
163 my @confA; # Confidence values for orthologous groups
164 my @confB; # Confidence values for orthologous groups
165 my $prev_time = 0;
166
167 $outputfile = "Output." . $ARGV[0] . "-" . $ARGV[1];
168 if ($output){
169 open OUTPUT, ">$outputfile" or warn "Could not write to OUTPUT file $filename\n";
170 }
171
172 #################################################
173 # Assign ID numbers for species A
174 #################################################
175 open A, "$fasta_seq_fileA" or die "File A with sequences in FASTA format is missing
176 Usage $0 <FASTAFILE with sequences of species A> <FASTAFILE with sequences of species B> <FASTAFILE with sequences of species C>\n";
177 $id = 0;
178 while (<A>){
179 if(/^\>/){
180 ++$id;
181 chomp;
182 s/\>//;
183 @tmp = split(/\s+/);
184 #$name = substr($tmp[0],0,25);
185 $name = $tmp[0];
186 $idA{$name} = int($id);
187 $nameA[$id] = $name;
188 }
189 }
190 close A;
191 $A = $id;
192 print "$A sequences in file $fasta_seq_fileA\n";
193
194 if ($output){
195 print OUTPUT "$A sequences in file $fasta_seq_fileA\n";
196 }
197
198 if (@ARGV >= 2) {
199 #################################################
200 # Assign ID numbers for species B
201 #################################################
202 open B, "$fasta_seq_fileB" or die "File B with sequences in FASTA format is missing
203 Usage $0 <FASTAFILE with sequences of species A> <FASTAFILE with sequences of species B> <FASTAFILE with sequences of species C>\n";
204 $id = 0;
205 while (<B>){
206 if(/^\>/){
207 ++$id;
208 chomp;
209 s/\>//;
210 @tmp = split(/\s+/);
211 #$name = substr($tmp[0],0,25);
212 $name = $tmp[0];
213 $idB{$name} = int($id);
214 $nameB[$id] = $name;
215 }
216 }
217 $B = $id;
218 print "$B sequences in file $fasta_seq_fileB\n";
219 close B;
220
221 if ($output){
222 print OUTPUT "$B sequences in file $fasta_seq_fileB\n";
223 }
224 }
225 #################################################
226 # Assign ID numbers for species C (outgroup)
227 #################################################
228 if ($use_outgroup){
229 open C, "$fasta_seq_fileC" or die "File C with sequences in FASTA format is missing
230 Usage $0 <FASTAFILE with sequences of species A> <FASTAFILE with sequences of species B> <FASTAFILE with sequences of species C>\n";
231 $id = 0;
232 while (<C>){
233 if(/^\>/){
234 ++$id;
235 chomp;
236 s/\>//;
237 @tmp = split(/\s+/);
238 #$name = substr($tmp[0],0,25);
239 $name = $tmp[0];
240 $idC{$name} = int($id);
241 $nameC[$id] = $name;
242 }
243 }
244 $C = $id;
245 print "$C sequences in file $fasta_seq_fileC\n";
246 close C;
247 if ($output){
248 print OUTPUT "$C sequences in file $fasta_seq_fileC\n";
249 }
250 }
251 if ($show_times){
252 ($user_time,,,) = times;
253 printf ("Indexing sequences took %.2f seconds\n", ($user_time - $prev_time));
254 $prev_time = $user_time;
255 }
256
257 #################################################
258 # Run BLAST if not done already
259 #################################################
260 if ($run_blast){
261 print "Trying to run BLAST now - this may take several hours ... or days in worst case!\n";
262 print STDERR "Formatting BLAST databases\n";
263 system ("$formatdb -i $fasta_seq_fileA");
264 system ("$formatdb -i $fasta_seq_fileB") if (@ARGV >= 2);
265 system ("$formatdb -i $fasta_seq_fileC") if ($use_outgroup);
266 print STDERR "Done formatting\nStarting BLAST searches...\n";
267
268 # Run blast only if the files do not already exist is not default.
269 # NOTE: you should have done this beforehand, because you probably
270 # want two-pass blasting anyway which is not implemented here
271 # this is also not adapted to use specific compositional adjustment settings
272 # and might not use the proper blast parser...
273
274 do_blast ($fasta_seq_fileA, $fasta_seq_fileA, $A, $A, $blast_outputAA);
275
276 if (@ARGV >= 2) {
277 do_blast ($fasta_seq_fileA, $fasta_seq_fileB, $B, $B, $blast_outputAB);
278 do_blast ($fasta_seq_fileB, $fasta_seq_fileA, $A, $A, $blast_outputBA);
279 do_blast ($fasta_seq_fileB, $fasta_seq_fileB, $B, $B, $blast_outputBB);
280 }
281
282 if ($use_outgroup){
283
284 do_blast ($fasta_seq_fileA, $fasta_seq_fileC, $A, $C, $blast_outputAC);
285 do_blast ($fasta_seq_fileB, $fasta_seq_fileC, $B, $C, $blast_outputBC);
286 }
287
288 if ($show_times){
289 ($user_time,,,) = times;
290 printf ("BLAST searches took %.2f seconds\n", ($user_time - $prev_time));
291 $prev_time = $user_time;
292 }
293 print STDERR "Done BLAST searches. ";
294
295 } else {
296 print STDERR "Skipping blast! \n";
297 }
298
299 if ($run_inparanoid){
300 print STDERR "Starting ortholog detection...\n";
301 #################################################
302 # Read in best hits from blast output file AB
303 #################################################
304 $count = 0;
305 open AB, "$blast_outputAB" or die "Blast output file A->B is missing\n";
306 $old_idQ = 0;
307 while (<AB>){
308 chomp;
309 @Fld = split(/\s+/); # Get query, match and score
310
311 if( scalar @Fld < 9 ){
312 if($Fld[0]=~/done/){
313 print STDERR "AB ok\n";
314 }
315 next;
316 }
317
318 $q = $Fld[0];
319 $m = $Fld[1];
320 $idQ = $idA{$q}; # ID of query sequence
321 $idM = $idB{$m}; # ID of match sequence
322 $score = $Fld[2];
323
324 next if (!overlap_test(@Fld));
325
326 # Score must be equal to or above cut-off
327 next if ($score < $score_cutoff);
328
329 if(!$count || $q ne $oldq){
330 print "Match $m, score $score, ID for $q is missing\n" if ($debug == 2 and !(exists($idA{$q})));
331 $hitnAB[$idA{$oldq}] = $hit if($count); # Record number of hits for previous query
332 $hit = 0;
333 ++$count;
334 $oldq = $q;
335 }
336 ++$hit;
337 $hitAB[$idQ][$hit] = int($idM);
338 # printf ("hitAB[%d][%d] = %d\n",$idQ,$hit,$idM);
339 $scoreAB{"$idQ:$idM"} = $score;
340 $scoreBA{"$idM:$idQ"} = $score_cutoff; # Initialize mutual hit score - sometimes this is below score_cutoff
341 $old_idQ = $idQ;
342 # }
343 }
344 $hitnAB[$idQ] = $hit; # For the last query
345 #printf ("hitnAB[1] = %d\n",$hitnAB[1]);
346 #printf ("hitnAB[%d] = %d\n",$idQ,$hit);
347 close AB;
348 if ($output){
349 print OUTPUT "$count sequences $fasta_seq_fileA have homologs in dataset $fasta_seq_fileB\n";
350 }
351 #################################################
352 # Read in best hits from blast output file BA
353 #################################################
354 $count = 0;
355 open BA, "$blast_outputBA" or die "Blast output file B->A is missing\n";
356 $old_idQ = 0;
357 while (<BA>){
358 chomp;
359 @Fld = split(/\s+/); # Get query, match and score
360
361 if( scalar @Fld < 9 ){
362 if($Fld[0]=~/done/){
363 print STDERR "BA ok\n";
364 }
365 next;
366 }
367
368 $q = $Fld[0];
369 $m = $Fld[1];
370 $idQ = $idB{$q};
371 $idM = $idA{$m};
372 $score = $Fld[2];
373
374 next if (!overlap_test(@Fld));
375
376 next if ($score < $score_cutoff);
377
378 if(!$count || $q ne $oldq){
379 print "ID for $q is missing\n" if ($debug == 2 and (!exists($idB{$q})));
380 $hitnBA[$idB{$oldq}] = $hit if($count); # Record number of hits for previous query
381 $hit = 0;
382 ++$count;
383 $oldq = $q;
384 }
385 ++$hit;
386 $hitBA[$idQ][$hit] = int($idM);
387 # printf ("hitBA[%d][%d] = %d\n",$idQ,$hit,$idM);
388 $scoreBA{"$idQ:$idM"} = $score;
389 $scoreAB{"$idM:$idQ"} = $score_cutoff if ($scoreAB{"$idM:$idQ"} < $score_cutoff); # Initialize missing scores
390 $old_idQ = $idQ;
391 # }
392 }
393 $hitnBA[$idQ] = $hit; # For the last query
394 #printf ("hitnBA[%d] = %d\n",$idQ,$hit);
395 close BA;
396 if ($output){
397 print OUTPUT "$count sequences $fasta_seq_fileB have homologs in dataset $fasta_seq_fileA\n";
398 }
399 ##################### Equalize AB scores and BA scores ##########################
400
401 ###################################################################################################################################### Modification by Isabella 1
402
403 # I removed the time consuming all vs all search and equalize scores for all pairs where there was a hit
404
405 foreach my $key (keys %scoreAB) {
406
407 my ($a, $b) = split(':', $key);
408 my $key2 = $b . ':' . $a;
409
410 # If debugg mod is 5 and the scores A-B and B-A are unequal
411 # the names of the two sequences and their scores are printed
412 if ($scoreAB{$key} != $scoreBA{$key2}){
413 printf ("%-20s\t%-20s\t%d\t%d\n",$nameA[$a], $nameB[$b], $scoreAB{$key}, $scoreBA{$key2}) if ($debug == 5);
414 }
415
416 # Set score AB and score BA to the mean of scores AB and BA.
417 # The final score is saved as an integer so .5 needs to be added to avoid rounding errors
418 $scoreAB{$key} = $scoreBA{$key2} = int(($scoreAB{$key} + $scoreBA{$key2})/2.0 +.5);
419 }
420
421 # For all ids for sequences from organism A
422 #for $a(1..$A){
423 #For all ids for sequences from organism B
424 #for $b(1..$B){
425
426 # No need to equalize score if there was no match between sequence with id $a from species A
427 # and sequence with id $b from species B
428 # next if (!$scoreAB{"$a:$b"});
429
430 # If debugg mod is 5 and the scores A-B and B-A are unequal
431 # the names of the two sequences and their scores are printed
432 # if ($scoreAB{"$a:$b"} != $scoreBA{"$b:$a"}){
433 # printf ("%-20s\t%-20s\t%d\t%d\n",$nameA[$a], $nameB[$b], $scoreAB{"$a:$b"}, $scoreBA{"$b:$a"}) if ($debug == 5);
434 # }
435
436 # Set score AB and score BA to the mean of scores AB and BA.
437 # The final score is saved as an integer so .5 needs to be added to avoid rounding errors
438 # $scoreAB{"$a:$b"} = $scoreBA{"$b:$a"} = int(($scoreAB{"$a:$b"} + $scoreBA{"$b:$a"})/2.0 +.5);
439
440 # printf ("scoreAB{%d: %d} = %d\n", $a, $b, $scoreAB{"$a:$b"});
441 # printf ("scoreBA{%d: %d} = %d\n", $b, $a, $scoreBA{"$a:$b"});
442 #}
443 # }
444
445 ####################################################################################################################################### End modification by Isabella 1
446
447 ##################### Re-sort hits, besthits and bestscore #######################
448 for $idA(1..$A){
449 # print "Loop index = $idA\n";
450 # printf ("hitnAB[%d] = %d\n",$idA, $hitnAB[$idA]);
451 next if (!($hitnAB[$idA]));
452 for $hit (1..($hitnAB[$idA]-1)){ # Sort hits by score
453 while($scoreAB{"$idA:$hitAB[$idA][$hit]"} < $scoreAB{"$idA:$hitAB[$idA][$hit+1]"}){
454 $tmp = $hitAB[$idA][$hit];
455 $hitAB[$idA][$hit] = $hitAB[$idA][$hit+1];
456 $hitAB[$idA][$hit+1] = $tmp;
457 --$hit if ($hit > 1);
458 }
459 }
460 $bestscore = $bestscoreAB[$idA] = $scoreAB{"$idA:$hitAB[$idA][1]"};
461 $besthitAB[$idA] = $hitAB[$idA][1];
462 for $hit (2..$hitnAB[$idA]){
463 if ($bestscore - $scoreAB{"$idA:$hitAB[$idA][$hit]"} <= $grey_zone){
464 $besthitAB[$idA] .= " $hitAB[$idA][$hit]";
465 }
466 else {
467 last;
468 }
469 }
470 undef $is_besthitAB[$idA]; # Create index that we can check later
471 grep (vec($is_besthitAB[$idA],$_,1) = 1, split(/ /,$besthitAB[$idA]));
472 # printf ("besthitAB[%d] = hitAB[%d][%d] = %d\n",$idA,$idA,$hit,$besthitAB[$idA]);
473
474 }
475
476 for $idB(1..$B){
477 # print "Loop index = $idB\n";
478 next if (!($hitnBA[$idB]));
479 for $hit (1..($hitnBA[$idB]-1)){
480 # Sort hits by score
481 while($scoreBA{"$idB:$hitBA[$idB][$hit]"} < $scoreBA{"$idB:$hitBA[$idB][$hit+1]"}){
482 $tmp = $hitBA[$idB][$hit];
483 $hitBA[$idB][$hit] = $hitBA[$idB][$hit+1];
484 $hitBA[$idB][$hit+1] = $tmp;
485 --$hit if ($hit > 1);
486 }
487 }
488 $bestscore = $bestscoreBA[$idB] = $scoreBA{"$idB:$hitBA[$idB][1]"};
489 $besthitBA[$idB] = $hitBA[$idB][1];
490 for $hit (2..$hitnBA[$idB]){
491 if ($bestscore - $scoreBA{"$idB:$hitBA[$idB][$hit]"} <= $grey_zone){
492 $besthitBA[$idB] .= " $hitBA[$idB][$hit]";
493 }
494 else {last;}
495 }
496 undef $is_besthitBA[$idB]; # Create index that we can check later
497 grep (vec($is_besthitBA[$idB],$_,1) = 1, split(/ /,$besthitBA[$idB]));
498 # printf ("besthitBA[%d] = %d\n",$idA,$besthitAB[$idA]);
499 }
500
501 if ($show_times){
502 ($user_time,,,) = times;
503 printf ("Reading and sorting homologs took %.2f seconds\n", ($user_time - $prev_time));
504 $prev_time = $user_time;
505 }
506
507 ######################################################
508 # Now find orthologs:
509 ######################################################
510 $o = 0;
511
512 for $i(1..$A){ # For each ID in file A
513 if (defined $besthitAB[$i]){
514 @besthits = split(/ /,$besthitAB[$i]);
515 for $hit (@besthits){
516 if (vec($is_besthitBA[$hit],$i,1)){
517 ++$o;
518 $ortoA[$o] = $i;
519 $ortoB[$o] = $hit;
520 $ortoS[$o] = $scoreAB{"$i:$hit"}; # Should be equal both ways
521 # --$o if ($ortoS[$o] == $score_cutoff); # Ignore orthologs that are exactly at score_cutoff
522 print "Accept! " if ($debug == 2);
523 }
524 else {print " " if ($debug == 2);}
525 printf ("%-20s\t%d\t%-20s\t", $nameA[$i], $bestscoreAB[$i], $nameB[$hit]) if ($debug == 2);
526 print "$bestscoreBA[$hit]\t$besthitBA[$hit]\n" if ($debug == 2);
527 }
528 }
529 }
530 print "$o ortholog candidates detected\n" if ($debug);
531 #####################################################
532 # Sort orthologs by ID and then by score:
533 #####################################################
534
535 ####################################################################################################### Modification by Isabella 2
536
537 # Removed time consuiming bubble sort. Created an index array and sort that according to id and score.
538 # The put all clusters on the right place.
539
540 # Create an array used to store the position each element shall have in the final array
541 # The elements are initialized with the position numbers
542 my @position_index_array = (1..$o);
543
544 # Sort the position list according to id
545 my @id_sorted_position_list = sort { ($ortoA[$a]+$ortoB[$a]) <=> ($ortoA[$b] + $ortoB[$b]) } @position_index_array;
546
547 # Sort the list according to score
548 my @score_id_sorted_position_list = sort { $ortoS[$b] <=> $ortoS[$a] } @id_sorted_position_list;
549
550 # Create new arrays for the sorted information
551 my @new_ortoA;
552 my @new_ortoB;
553 my @new_orthoS;
554
555 # Add the information to the new arrays in the orer specifeid by the index array
556 for (my $index_in_list = 0; $index_in_list < scalar @score_id_sorted_position_list; $index_in_list++) {
557
558
559 my $old_index = $score_id_sorted_position_list[$index_in_list];
560 $new_ortoA[$index_in_list + 1] = $ortoA[$old_index];
561 $new_ortoB[$index_in_list + 1] = $ortoB[$old_index];
562 $new_ortoS[$index_in_list + 1] = $ortoS[$old_index];
563 }
564
565 @ortoA = @new_ortoA;
566 @ortoB = @new_ortoB;
567 @ortoS = @new_ortoS;
568
569 # Use bubblesort to sort ortholog pairs by id
570 # for $i(1..($o-1)){
571 # while(($ortoA[$i]+$ortoB[$i]) > ($ortoA[$i+1] + $ortoB[$i+1])){
572 # $tempA = $ortoA[$i];
573 # $tempB = $ortoB[$i];
574 # $tempS = $ortoS[$i];
575 #
576 # $ortoA[$i] = $ortoA[$i+1];
577 # $ortoB[$i] = $ortoB[$i+1];
578 # $ortoS[$i] = $ortoS[$i+1];
579 #
580 # $ortoA[$i+1] = $tempA;
581 # $ortoB[$i+1] = $tempB;
582 # $ortoS[$i+1] = $tempS;
583 #
584 # --$i if ($i > 1);
585 # }
586 # }
587 #
588 # # Use bubblesort to sort ortholog pairs by score
589 # for $i(1..($o-1)){
590 # while($ortoS[$i] < $ortoS[$i+1]){
591 # # Swap places:
592 # $tempA = $ortoA[$i];
593 # $tempB = $ortoB[$i];
594 # $tempS = $ortoS[$i];
595 #
596 # $ortoA[$i] = $ortoA[$i+1];
597 # $ortoB[$i] = $ortoB[$i+1];
598 # $ortoS[$i] = $ortoS[$i+1];
599 #
600 # $ortoA[$i+1] = $tempA;
601 # $ortoB[$i+1] = $tempB;
602 # $ortoS[$i+1] = $tempS;
603 #
604 # --$i if ($i > 1);
605 # }
606 # }
607
608 ###################################################################################################### End modification bt Isabella 2
609
610 @all_ortologsA = ();
611 @all_ortologsB = ();
612 for $i(1..$o){
613 push(@all_ortologsA,$ortoA[$i]); # List of all orthologs
614 push(@all_ortologsB,$ortoB[$i]);
615 }
616 undef $is_ortologA; # Create index that we can check later
617 undef $is_ortologB;
618 grep (vec($is_ortologA,$_,1) = 1, @all_ortologsA);
619 grep (vec($is_ortologB,$_,1) = 1, @all_ortologsB);
620
621 if ($show_times){
622 ($user_time,,,) = times;
623 printf ("Finding and sorting orthologs took %.2f seconds\n", ($user_time - $prev_time));
624 $prev_time = $user_time;
625 }
626 #################################################
627 # Read in best hits from blast output file AC
628 #################################################
629 if ($use_outgroup){
630 $count = 0;
631 open AC, "$blast_outputAC" or die "Blast output file A->C is missing\n";
632 while (<AC>){
633 chomp;
634 @Fld = split(/\s+/); # Get query, match and score
635
636 if( scalar @Fld < 9 ){
637 if($Fld[0]=~/done/){
638 print STDERR "AC ok\n";
639 }
640 next;
641 }
642
643 $q = $Fld[0];
644 $m = $Fld[1];
645 $idQ = $idA{$q};
646 $idM = $idC{$m};
647 $score = $Fld[2];
648 next unless (vec($is_ortologA,$idQ,1));
649
650 next if (!overlap_test(@Fld));
651
652 next if ($score < $score_cutoff);
653
654 next if ($count and ($q eq $oldq));
655 # Only comes here if this is the best hit:
656 $besthitAC[$idQ] = $idM;
657 $bestscoreAC[$idQ] = $score;
658 $oldq = $q;
659 ++$count;
660 }
661 close AC;
662 #################################################
663 # Read in best hits from blast output file BC
664 #################################################
665 $count = 0;
666 open BC, "$blast_outputBC" or die "Blast output file B->C is missing\n";
667 while (<BC>){
668 chomp;
669 @Fld = split(/\s+/); # Get query, match and score
670
671 if( scalar @Fld < 9 ){
672 if($Fld[0]=~/done/){
673 print STDERR "BC ok\n";
674 }
675 next;
676 }
677
678 $q = $Fld[0];
679 $m = $Fld[1];
680 $idQ = $idB{$q};
681 $idM = $idC{$m};
682 $score = $Fld[2];
683 next unless (vec($is_ortologB,$idQ,1));
684
685 next if (!overlap_test(@Fld));
686
687 next if ($score < $score_cutoff);
688
689 next if ($count and ($q eq $oldq));
690 # Only comes here if this is the best hit:
691 $besthitBC[$idQ] = $idM;
692 $bestscoreBC[$idQ] = $score;
693 $oldq = $q;
694 ++$count;
695 }
696 close BC;
697 ################################
698 # Detect rooting problems
699 ################################
700 $rejected = 0;
701 @del = ();
702 $file = "rejected_sequences." . $fasta_seq_fileC;
703 open OUTGR, ">$file";
704 for $i (1..$o){
705 $diff1 = $diff2 = 0;
706 $idA = $ortoA[$i];
707 $idB = $ortoB[$i];
708 $diff1 = $bestscoreAC[$idA] - $ortoS[$i];
709 $diff2 = $bestscoreBC[$idB] - $ortoS[$i];
710 if ($diff1 > $outgroup_cutoff){
711 print OUTGR "Ortholog pair $i ($nameA[$idA]-$nameB[$idB]).
712 $nameA[$idA] from $fasta_seq_fileA is closer to $nameC[$besthitAC[$idA]] than to $nameB[$idB]\n";
713 print OUTGR " $ortoS[$i] < $bestscoreAC[$idA] by $diff1\n";
714 }
715 if ($diff2 > $outgroup_cutoff){
716 print OUTGR "Ortholog pair $i ($nameA[$idA]-$nameB[$idB]).
717 $nameB[$idB] from $fasta_seq_fileB is closer to $nameC[$besthitBC[$idB]] than to $nameA[$idA]\n";
718 print OUTGR " $ortoS[$i] < $bestscoreBC[$idB] by $diff2\n";
719 }
720 if (($diff1 > $outgroup_cutoff) or ($diff2 > $outgroup_cutoff)){
721 ++$rejected;
722 $del[$i] = 1;
723 }
724 }
725 print "Number of rejected groups: $rejected (outgroup sequence was closer by more than $outgroup_cutoff bits)\n";
726 close OUTGR;
727 } # End of $use_outgroup
728 ################################
729 # Read inside scores from AA
730 ################################
731 $count = 0;
732 $max_hit = 0;
733 open AA, "$blast_outputAA" or die "Blast output file A->A is missing\n";
734 while (<AA>) {
735 chomp; # strip newline
736
737 @Fld = split(/\s+/); # Get query and match names
738
739 if( scalar @Fld < 9 ){
740 if($Fld[0]=~/done/){
741 print STDERR "AA ok\n";
742 }
743 next;
744 }
745
746 $q = $Fld[0];
747 $m = $Fld[1];
748 $score = $Fld[2];
749 next unless (vec($is_ortologA,$idA{$q},1));
750
751 next if (!overlap_test(@Fld));
752
753 next if ($score < $score_cutoff);
754
755 if(!$count || $q ne $oldq){ # New query
756 $max_hit = $hit if ($hit > $max_hit);
757 $hit = 0;
758 $oldq = $q;
759 }
760 ++$hit;
761 ++$count;
762 $scoreAA{"$idA{$q}:$idA{$m}"} = int($score + 0.5);
763 $hitAA[$idA{$q}][$hit] = int($idA{$m});
764 $hitnAA[$idA{$q}] = $hit;
765 }
766 close AA;
767 if ($output){
768 print OUTPUT "$count $fasta_seq_fileA-$fasta_seq_fileA matches\n";
769 }
770 ################################
771 # Read inside scores from BB
772 ################################
773 $count = 0;
774 open BB, "$blast_outputBB" or die "Blast output file B->B is missing\n";
775 while (<BB>) {
776 chomp; # strip newline
777
778 @Fld = split(/\s+/); # Get query and match names
779
780 if( scalar @Fld < 9 ){
781 if($Fld[0]=~/done/){
782 print STDERR "BB ok\n";
783 }
784 next;
785 }
786
787 $q = $Fld[0];
788 $m = $Fld[1];
789 $score = $Fld[2];
790 next unless (vec($is_ortologB,$idB{$q},1));
791
792 next if (!overlap_test(@Fld));
793
794 next if ($score < $score_cutoff);
795
796 if(!$count || $q ne $oldq){ # New query
797 $max_hit = $hit if ($hit > $max_hit);
798 $oldq = $q;
799 $hit = 0;
800 }
801 ++$count;
802 ++$hit;
803 $scoreBB{"$idB{$q}:$idB{$m}"} = int($score + 0.5);
804 $hitBB[$idB{$q}][$hit] = int($idB{$m});
805 $hitnBB[$idB{$q}] = $hit;
806 }
807 close BB;
808 if ($output){
809 print OUTPUT "$count $fasta_seq_fileB-$fasta_seq_fileB matches\n";
810 }
811 if ($show_times){
812 ($user_time,,,) = times;
813 printf ("Reading paralogous hits took %.2f seconds\n", ($user_time - $prev_time));
814 $prev_time = $user_time;
815 }
816 print "Maximum number of hits per sequence was $max_hit\n" if ($debug);
817 #####################################################
818 # Find paralogs:
819 #####################################################
820 for $i(1..$o){
821 $merge[$i] = 0;
822 next if($del[$i]); # If outgroup species was closer to one of the seed orthologs
823 $idA = $ortoA[$i];
824 $idB = $ortoB[$i];
825 local @membersA = ();
826 local @membersB = ();
827
828 undef $is_paralogA[$i];
829 undef $is_paralogB[$i];
830
831 print "$i: Ortholog pair $nameA[$idA] and $nameB[$idB]. $hitnAA[$idA] hits for A and $hitnBB[$idB] hits for B\n" if ($debug);
832 # Check if current ortholog is already clustered:
833 for $j(1..($i-1)){
834 # Overlap type 1: Both orthologs already clustered here -> merge
835 if ((vec($is_paralogA[$j],$idA,1)) and (vec($is_paralogB[$j],$idB,1))){
836 $merge[$i] = $j;
837 print "Merge CASE 1: group $i ($nameB[$idB]-$nameA[$idA]) and $j ($nameB[$ortoB[$j]]-$nameA[$ortoA[$j]])\n" if ($debug);
838 last;
839 }
840 # Overlap type 2: 2 competing ortholog pairs -> merge
841 elsif (($ortoS[$j] - $ortoS[$i] <= $grey_zone)
842 and (($ortoA[$j] == $ortoA[$i]) or ($ortoB[$j] == $ortoB[$i]))
843 # and ($paralogsA[$j])
844 ){ # The last condition is false if the previous cluster has been already deleted
845 $merge[$i] = $j;
846 print "Merge CASE 2: group $i ($nameA[$ortoA[$i]]-$nameB[$ortoB[$i]]) and $j ($nameA[$ortoA[$j]]-$nameB[$ortoB[$j]])\n" if ($debug);
847 last;
848 }
849 # Overlap type 3: DELETE One of the orthologs belongs to some much stronger cluster -> delete
850 elsif (((vec($is_paralogA[$j],$idA,1)) or (vec($is_paralogB[$j],$idB,1))) and
851 ($ortoS[$j] - $ortoS[$i] > $score_cutoff)){
852 print "Delete CASE 3: Cluster $i -> $j, score $ortoS[$i] -> $ortoS[$j], ($nameA[$ortoA[$j]]-$nameB[$ortoB[$j]])\n" if ($debug);
853 $merge[$i]= -1; # Means - do not add sequences to this cluster
854 $paralogsA[$i] = "";
855 $paralogsB[$i] = "";
856 last;
857 }
858 # Overlap type 4: One of the orthologs is close to the center of other cluster
859 elsif (((vec($is_paralogA[$j],$idA,1)) and ($confPA[$idA] > $group_overlap_cutoff)) or
860 ((vec($is_paralogB[$j],$idB,1)) and ($confPB[$idB] > $group_overlap_cutoff))){
861 print "Merge CASE 4: Cluster $i -> $j, score $ortoS[$i] -> $ortoS[$j], ($nameA[$ortoA[$j]]-$nameB[$ortoB[$j]])\n" if ($debug);
862 $merge[$i] = $j;
863 last;
864 }
865 # Overlap type 5:
866 # All clusters that were overlapping, but not catched by previous "if" statements will be DIVIDED!
867 }
868 next if ($merge[$i] < 0); # This cluster should be deleted
869 ##### Check for paralogs in A
870 $N = $hitnAA[$idA];
871 for $j(1..$N){
872 $hitID = $hitAA[$idA][$j]; # hit of idA
873 # print "Working with $nameA[$hitID]\n" if ($debug == 2);
874 # Decide whether this hit is inside the paralog circle:
875 if ( ($idA == $hitID) or ($scoreAA{"$idA:$hitID"} >= $bestscoreAB[$idA]) and
876 ($scoreAA{"$idA:$hitID"} >= $bestscoreAB[$hitID])){
877 if ($debug == 2){
878 print " Paralog candidates: ";
879 printf ("%-20s: %-20s", $nameA[$idA], $nameA[$hitID]);
880 print "\t$scoreAA{\"$idA:$hitID\"} : $bestscoreAB[$idA] : $bestscoreAB[$hitID]\n";
881 }
882 $paralogs = 1;
883 if ($scoreAA{"$idA:$idA"} == $ortoS[$i]){
884 if ($scoreAA{"$idA:$hitID"} == $scoreAA{"$idA:$idA"}){
885 $conf_here = 1.0; # In the center
886 }
887 else{
888 $conf_here = 0.0; # On the border
889 }
890 }
891 else {
892 $conf_here = ($scoreAA{"$idA:$hitID"} - $ortoS[$i]) /
893 ($scoreAA{"$idA:$idA"} - $ortoS[$i]);
894 }
895 # Check if this paralog candidate is already clustered in other clusters
896 for $k(1..($i-1)){
897 if (vec($is_paralogA[$k],$hitID,1)){ # Yes, found in cluster $k
898 if($debug == 2){
899 print " $nameA[$hitID] is already in cluster $k, together with:";
900 print " $nameA[$ortoA[$k]] and $nameB[$ortoB[$k]] ";
901 print "($scoreAA{\"$ortoA[$k]:$hitID\"})";
902 }
903 if (($confPA[$hitID] >= $conf_here) and
904 ($j != 1)){ # The seed ortholog CAN NOT remain there
905 print " and remains there.\n" if ($debug == 2);
906 $paralogs = 0; # No action
907 }
908 else { # Ortholog of THIS cluster is closer than ortholog of competing cluster $k
909 print " and should be moved here!\n" if ($debug == 2); # Remove from other cluster, add to this cluster
910 @membersAK = split(/ /, $paralogsA[$k]); # This array contains IDs
911 $paralogsA[$k] = "";# Remove all paralogs from cluster $k
912 @tmp = ();
913 for $m(@membersAK){
914 push(@tmp,$m) if ($m != $hitID); # Put other members back
915 }
916 $paralogsA[$k] = join(' ',@tmp);
917 undef $is_paralogA[$k]; # Create index that we can check later
918 grep (vec($is_paralogA[$k],$_,1) = 1, @tmp);
919 }
920 last;
921 }
922 }
923 next if (! $paralogs); # Skip that paralog - it is already in cluster $k
924 push (@membersA,$hitID); # Add this hit to paralogs of A
925 }
926 }
927 # Calculate confidence values now:
928 @tmp = ();
929 for $idP (@membersA){ # For each paralog calculate conf value
930 if($scoreAA{"$idA:$idA"} == $ortoS[$i]){
931 if ($scoreAA{"$idA:$idP"} == $scoreAA{"$idA:$idA"}){
932 $confPA[$idP] = 1.00;
933 }
934 else{
935 $confPA[$idP] = 0.00;
936 }
937 }
938 else{
939 $confPA[$idP] = ($scoreAA{"$idA:$idP"} - $ortoS[$i]) /
940 ($scoreAA{"$idA:$idA"} - $ortoS[$i]);
941 }
942 push (@tmp, $idP) if ($confPA[$idP] >= $conf_cutoff); # If one wishes to use only significant paralogs
943 }
944 @membersA = @tmp;
945 ########### Merge if necessary:
946 if ($merge[$i] > 0){ # Merge existing cluster with overlapping cluster
947 @tmp = split(/ /,$paralogsA[$merge[$i]]);
948 for $m (@membersA){
949 push (@tmp, $m) unless (vec($is_paralogA[$merge[$i]],$m,1));
950 }
951 $paralogsA[$merge[$i]] = join(' ',@tmp);
952 undef $is_paralogA[$merge[$i]];
953 grep (vec($is_paralogA[$merge[$i]],$_,1) = 1, @tmp); # Refresh index of paralog array
954 }
955 ######### Typical new cluster:
956 else{ # Create a new cluster
957 $paralogsA[$i] = join(' ',@membersA);
958 undef $is_paralogA; # Create index that we can check later
959 grep (vec($is_paralogA[$i],$_,1) = 1, @membersA);
960 }
961 ##### The same procedure for species B:
962 $N = $hitnBB[$idB];
963 for $j(1..$N){
964 $hitID = $hitBB[$idB][$j];
965 # print "Working with $nameB[$hitID]\n" if ($debug == 2);
966 if ( ($idB == $hitID) or ($scoreBB{"$idB:$hitID"} >= $bestscoreBA[$idB]) and
967 ($scoreBB{"$idB:$hitID"} >= $bestscoreBA[$hitID])){
968 if ($debug == 2){
969 print " Paralog candidates: ";
970 printf ("%-20s: %-20s", $nameB[$idB], $nameB[$hitID]);
971 print "\t$scoreBB{\"$idB:$hitID\"} : ";
972 print "$bestscoreBA[$idB] : $bestscoreBA[$hitID]\n";
973 }
974 $paralogs = 1;
975 if ($scoreBB{"$idB:$idB"} == $ortoS[$i]){
976 if ($scoreBB{"$idB:$hitID"} == $scoreBB{"$idB:$idB"}){
977 $conf_here = 1.0;
978 }
979 else{
980 $conf_here = 0.0;
981 }
982 }
983 else{
984 $conf_here = ($scoreBB{"$idB:$hitID"} - $ortoS[$i]) /
985 ($scoreBB{"$idB:$idB"} - $ortoS[$i]);
986 }
987
988 # Check if this paralog candidate is already clustered in other clusters
989 for $k(1..($i-1)){
990 if (vec($is_paralogB[$k],$hitID,1)){ # Yes, found in cluster $k
991 if($debug == 2){
992 print " $nameB[$hitID] is already in cluster $k, together with:";
993 print " $nameB[$ortoB[$k]] and $nameA[$ortoA[$k]] ";
994 print "($scoreBB{\"$ortoB[$k]:$hitID\"})";
995 }
996 if (($confPB[$hitID] >= $conf_here) and
997 ($j != 1)){ # The seed ortholog CAN NOT remain there
998 print " and remains there.\n" if ($debug == 2);
999 $paralogs = 0; # No action
1000 }
1001 else { # Ortholog of THIS cluster is closer than ortholog of competing cluster $k
1002 print " and should be moved here!\n" if ($debug == 2); # Remove from other cluster, add to this cluster
1003 @membersBK = split(/ /, $paralogsB[$k]); # This array contains names, not IDs
1004 $paralogsB[$k] = "";
1005 @tmp = ();
1006 for $m(@membersBK){
1007 push(@tmp,$m) if ($m != $hitID); # Put other members back
1008 }
1009 $paralogsB[$k] = join(' ',@tmp);
1010 undef $is_paralogB[$k]; # Create index that we can check later
1011 grep (vec($is_paralogB[$k],$_,1) = 1, @tmp);
1012 }
1013 last; # Don't search in other clusters
1014 }
1015 }
1016 next if (! $paralogs); # Skip that paralog - it is already in cluster $k
1017 push (@membersB,$hitID);
1018 }
1019 }
1020 # Calculate confidence values now:
1021 @tmp = ();
1022 for $idP (@membersB){ # For each paralog calculate conf value
1023 if($scoreBB{"$idB:$idB"} == $ortoS[$i]){
1024 if ($scoreBB{"$idB:$idP"} == $scoreBB{"$idB:$idB"}){
1025 $confPB[$idP] = 1.0;
1026 }
1027 else{
1028 $confPB[$idP] = 0.0;
1029 }
1030 }
1031 else{
1032 $confPB[$idP] = ($scoreBB{"$idB:$idP"} - $ortoS[$i]) /
1033 ($scoreBB{"$idB:$idB"} - $ortoS[$i]);
1034 }
1035 push (@tmp, $idP) if ($confPB[$idP] >= $conf_cutoff); # If one wishes to use only significant paralogs
1036 }
1037 @membersB = @tmp;
1038 ########### Merge if necessary:
1039 if ($merge[$i] > 0){ # Merge existing cluster with overlapping cluster
1040 @tmp = split(/ /,$paralogsB[$merge[$i]]);
1041 for $m (@membersB){
1042 push (@tmp, $m) unless (vec($is_paralogB[$merge[$i]],$m,1));
1043 }
1044 $paralogsB[$merge[$i]] = join(' ',@tmp);
1045 undef $is_paralogB[$merge[$i]];
1046 grep (vec($is_paralogB[$merge[$i]],$_,1) = 1, @tmp); # Refresh index of paralog array
1047 }
1048 ######### Typical new cluster:
1049 else{ # Create a new cluster
1050 $paralogsB[$i] = join(' ',@membersB);
1051 undef $is_paralogB; # Create index that we can check later
1052 grep (vec($is_paralogB[$i],$_,1) = 1, @membersB);
1053 }
1054 }
1055 if ($show_times){
1056 ($user_time,,,) = times;
1057 printf ("Finding in-paralogs took %.2f seconds\n", ($user_time - $prev_time));
1058 $prev_time = $user_time;
1059 }
1060 #####################################################
1061 &clean_up(1);
1062 ####################################################
1063 # Find group for orphans. If cluster contains only one member, find where it should go:
1064 for $i (1..$o){
1065 @membersA = split(/ /, $paralogsA[$i]);
1066 @membersB = split(/ /, $paralogsB[$i]);
1067 $na = @membersA;
1068 $nb = @membersB;
1069 if (($na == 0) and $nb){
1070 print "Warning: empty A cluster $i\n";
1071 for $m (@membersB){
1072 $bestscore = 0;
1073 $bestgroup = 0;
1074 $bestmatch = 0;
1075 for $j (1..$o) {
1076 next if ($i == $j); # Really need to check against all 100% members of the group.
1077 @membersBJ = split(/ /, $paralogsB[$j]);
1078 for $k (@membersBJ){
1079 next if ($confPB[$k] != 1); # For all 100% in-paralogs
1080 $score = $scoreBB{"$m:$k"};
1081 if ($score > $bestscore){
1082 $bestscore = $score;
1083 $bestgroup = $j;
1084 $bestmatch = $k;
1085 }
1086 }
1087 }
1088 print "Orphan $nameB[$m] goes to group $bestgroup with $nameB[$bestmatch]\n" ;
1089 @members = split(/ /, $paralogsB[$bestgroup]);
1090 push (@members, $m);
1091 $paralogsB[$bestgroup] = join(' ',@members);
1092 $paralogsB[$i] = "";
1093 undef $is_paralogB[$bestgroup];
1094 undef $is_paralogB[$i];
1095 grep (vec($is_paralogB[$bestgroup],$_,1) = 1, @members); # Refresh index of paralog array
1096 # grep (vec($is_paralogB[$i],$_,1) = 1, ());
1097 }
1098 }
1099 if ($na and ($nb == 0)){
1100 print "Warning: empty B cluster $i\n";
1101 for $m (@membersA){
1102 $bestscore = 0;
1103 $bestgroup = 0;
1104 $bestmatch = 0;
1105 for $j (1..$o) {
1106 next if ($i == $j);
1107 @membersAJ = split(/ /, $paralogsA[$j]);
1108 for $k (@membersAJ){
1109 next if ($confPA[$k] != 1); # For all 100% in-paralogs
1110 $score = $scoreAA{"$m:$k"};
1111 if ($score > $bestscore){
1112 $bestscore = $score;
1113 $bestgroup = $j;
1114 $bestmatch = $k;
1115 }
1116 }
1117 }
1118 print "Orphan $nameA[$m] goes to group $bestgroup with $nameA[$bestmatch]\n";
1119 @members = split(/ /, $paralogsA[$bestgroup]);
1120 push (@members, $m);
1121 $paralogsA[$bestgroup] = join(' ',@members);
1122 $paralogsA[$i] = "";
1123 undef $is_paralogA[$bestgroup];
1124 undef $is_paralogA[$i];
1125 grep (vec($is_paralogA[$bestgroup],$_,1) = 1, @members); # Refresh index of paralog array
1126 # grep (vec($is_paralogA[$i],$_,1) = 1, ());
1127 }
1128 }
1129 }
1130
1131 &clean_up(1);
1132 ###################
1133 $htmlfile = "orthologs." . $ARGV[0] . "-" . $ARGV[1] . ".html";
1134 if ($html){
1135 open HTML, ">$htmlfile" or warn "Could not write to HTML file $filename\n";
1136 }
1137
1138
1139 if ($output){
1140 print OUTPUT "\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\n";
1141 print OUTPUT "$o groups of orthologs\n";
1142 print OUTPUT "$totalA in-paralogs from $fasta_seq_fileA\n";
1143 print OUTPUT "$totalB in-paralogs from $fasta_seq_fileB\n";
1144 print OUTPUT "Grey zone $grey_zone bits\n";
1145 print OUTPUT "Score cutoff $score_cutoff bits\n";
1146 print OUTPUT "In-paralogs with confidence less than $conf_cutoff not shown\n";
1147 print OUTPUT "Sequence overlap cutoff $seq_overlap_cutoff\n";
1148 print OUTPUT "Group merging cutoff $group_overlap_cutoff\n";
1149 print OUTPUT "Scoring matrix $matrix\n";
1150 print OUTPUT "\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\n";
1151 }
1152 if ($html){
1153 print HTML "<pre>\n";
1154 print HTML "\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\n";
1155 print HTML "$o groups of orthologs\n";
1156 print HTML "$totalA in-paralogs from $fasta_seq_fileA\n";
1157 print HTML "$totalB in-paralogs from $fasta_seq_fileB\n";
1158 print HTML "Grey zone $grey_zone bits\n";
1159 print HTML "Score cutoff $score_cutoff bits\n";
1160 print HTML "In-paralogs with confidence less than $conf_cutoff not shown\n";
1161 print HTML "Sequence overlap cutoff $seq_overlap_cutoff\n";
1162 print HTML "Group merging cutoff $group_overlap_cutoff\n";
1163 print HTML "Scoring matrix $matrix\n";
1164 print HTML "\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\n";
1165 }
1166 # ##############################################################################
1167 # Check for alternative orthologs, sort paralogs by confidence and print results
1168 # ##############################################################################
1169 if ($use_bootstrap and $debug){
1170 open FF, ">BS_vs_bits" or warn "Could not write to file BS_vs_bits\n";
1171 }
1172 for $i(1..$o){
1173 @membersA = split(/ /, $paralogsA[$i]);
1174 @membersB = split(/ /, $paralogsB[$i]);
1175 $message = "";
1176 $htmlmessage = "";
1177
1178 $idB = $ortoB[$i];
1179 $nB = $hitnBA[$idB];
1180 for $idA(@membersA){
1181 next if ($confPA[$idA] != 1.0);
1182 $nA = $hitnAB[$idA];
1183 $confA[$i] = $ortoS[$i]; # default
1184 $bsA[$idA] = 1.0;
1185 ##############
1186 for $j(1..$nB){
1187 $idH = $hitBA[$idB][$j];
1188 ################ Some checks for alternative orthologs:
1189 # 1. Don't consider sequences that are already in this cluster
1190 next if (vec($is_paralogA[$i],$idH,1));
1191 next if ($confPA[$idH] > 0); # If $conf_cutoff > 0 idH might be incide circle, but not paralog
1192
1193 # 2. Check if candidate for alternative ortholog is already clustered in stronger clusters
1194 $in_other_cluster = 0;
1195 for $k(1..($i-1)){ # Check if current ortholog is already clustered
1196 if (vec($is_paralogA[$k],$idH,1)){
1197 $in_other_cluster = $k;
1198 last;
1199 }
1200 }
1201 # next if ($in_other_cluster); # This hit is clustered in cluster $k. It cannot be alternative ortholog
1202
1203 # 3. The best hit of candidate ortholog should be ortoA or at least to belong into this cluster
1204 @besthits = split (/ /,$besthitAB[$idH]);
1205 $this_family = 0;
1206 for $bh (@besthits){
1207 $this_family = 1 if ($idB == $bh);
1208 }
1209 # next unless ($this_family); # There was an alternative BA match but it's best match did not belong here
1210 ################# Done with checks - if sequence passed, then it could be an alternative ortholog
1211 $confA[$i] = $ortoS[$i] - $scoreBA{"$idB:$idH"};
1212 if ($use_bootstrap){
1213 if ($confA[$i] < $ortoS[$i]){ # Danger zone - check for bootstrap
1214 $bsA[$idA] = &bootstrap($fasta_seq_fileB,$idB,$idA,$idH);
1215 }
1216 else {
1217 $bsA[$idA] = 1.0;
1218 }
1219 }
1220 last;
1221 }
1222 $message .= sprintf("Bootstrap support for %s as seed ortholog is %d%%.", $nameA[$idA], 100*$bsA[$idA]);
1223 $message .= sprintf(" Alternative seed ortholog is %s (%d bits away from this cluster)", $nameA[$idH], $confA[$i]) if ($bsA[$idA] < 0.75);
1224 $message .= sprintf("\n");
1225 if ($html){
1226 if ($bsA[$idA] < 0.75){
1227 $htmlmessage .= sprintf("<font color=\"red\">");
1228 }
1229 elsif ($bsA[$idA] < 0.95){
1230 $htmlmessage .= sprintf("<font color=\"\#FFCC00\">");
1231 }
1232 else {
1233 $htmlmessage .= sprintf("<font color=\"green\">");
1234 }
1235 $htmlmessage .= sprintf("Bootstrap support for %s as seed ortholog is %d%%.\n", $nameA[$idA], 100*$bsA[$idA]);
1236 $htmlmessage .= sprintf("Alternative seed ortholog is %s (%d bits away from this cluster)\n", $nameA[$idH], $confA[$i]) if ($bsA[$idA] < 0.75);
1237 $htmlmessage .= sprintf("</font>");
1238 }
1239 printf (FF "%s\t%d\t%d\n", $nameA[$idA], $confA[$i], 100*$bsA[$idA]) if ($use_bootstrap and $debug);
1240 }
1241 ########
1242 $idA = $ortoA[$i];
1243 $nA = $hitnAB[$idA];
1244 for $idB(@membersB){
1245 next if ($confPB[$idB] != 1.0);
1246 $nB = $hitnBA[$idB];
1247 $confB[$i] = $ortoS[$i]; # default
1248 $bsB[$idB] = 1.0;
1249
1250 for $j(1..$nA){ # For all AB hits of given ortholog
1251 $idH = $hitAB[$idA][$j];
1252 # ############### Some checks for alternative orthologs:
1253 # 1. Don't consider sequences that are already in this cluster
1254 next if (vec($is_paralogB[$i],$idH,1));
1255 next if ($confPB[$idH] > 0); # If $conf_cutoff > 0 idH might be incide circle, but not paralog
1256
1257 # 2. Check if candidate for alternative ortholog is already clustered in stronger clusters
1258 $in_other_cluster = 0;
1259 for $k(1..($i-1)){
1260 if (vec($is_paralogB[$k],$idH,1)){
1261 $in_other_cluster = $k;
1262 last; # out from this cycle
1263 }
1264 }
1265 # next if ($in_other_cluster); # This hit is clustered in cluster $k. It cannot be alternative ortholog
1266
1267 # 3. The best hit of candidate ortholog should be ortoA
1268 @besthits = split (/ /,$besthitBA[$idH]);
1269 $this_family = 0;
1270 for $bh (@besthits){
1271 $this_family = 1 if ($idA == $bh);
1272 }
1273 # next unless ($this_family); # There was an alternative BA match but it's best match did not belong here
1274 # ################ Done with checks - if sequence passed, then it could be an alternative ortholog
1275 $confB[$i] = $ortoS[$i] - $scoreAB{"$idA:$idH"};
1276 if ($use_bootstrap){
1277 if ($confB[$i] < $ortoS[$i]){
1278 $bsB[$idB] = &bootstrap($fasta_seq_fileA,$idA,$idB,$idH);
1279 }
1280 else {
1281 $bsB[$idB] = 1.0;
1282 }
1283 }
1284 last;
1285 }
1286 $message .= sprintf("Bootstrap support for %s as seed ortholog is %d%%.", $nameB[$idB], 100*$bsB[$idB]);
1287 $message .= sprintf(" Alternative seed ortholog is %s (%d bits away from this cluster)", $nameB[$idH],$confB[$i]) if ($bsB[$idB] < 0.75);
1288 $message .= sprintf("\n");
1289 if ($html){
1290 if ($bsB[$idB] < 0.75){
1291 $htmlmessage .= sprintf("<font color=\"red\">");
1292 }
1293 elsif ($bsB[$idB] < 0.95){
1294 $htmlmessage .= sprintf("<font color=\"\#FFCC00\">");
1295 }
1296 else {
1297 $htmlmessage .= sprintf("<font color=\"green\">");
1298 }
1299 $htmlmessage .= sprintf("Bootstrap support for %s as seed ortholog is %d%%.\n", $nameB[$idB], 100*$bsB[$idB]);
1300 $htmlmessage .= sprintf("Alternative seed ortholog is %s (%d bits away from this cluster)\n", $nameB[$idH],$confB[$i]) if ($bsB[$idB] < 0.75);
1301 $htmlmessage .= sprintf("</font>");
1302 }
1303 printf (FF "%s\t%d\t%d\n", $nameB[$idB], $confB[$i], 100*$bsB[$idB]) if ($use_bootstrap and $debug);
1304 }
1305 close FF;
1306 ########### Print header ###############
1307 if ($output){
1308 print OUTPUT "___________________________________________________________________________________\n";
1309 print OUTPUT "Group of orthologs #" . $i .". Best score $ortoS[$i] bits\n";
1310 print OUTPUT "Score difference with first non-orthologous sequence - ";
1311 printf (OUTPUT "%s:%d %s:%d\n", $fasta_seq_fileA,$confA[$i],$fasta_seq_fileB,$confB[$i]);
1312 }
1313
1314 if ($html){
1315 print HTML "</pre>\n";
1316 print HTML "<hr WIDTH=\"100%\">";
1317 print HTML "<h3>";
1318 print HTML "Group of orthologs #" . $i .". Best score $ortoS[$i] bits<br>\n";
1319 print HTML "Score difference with first non-orthologous sequence - ";
1320 printf (HTML "%s:%d %s:%d</h3><pre>\n", $fasta_seq_fileA,$confA[$i],$fasta_seq_fileB,$confB[$i]);
1321 }
1322 ########### Sort and print members of A ############
1323 $nA = @membersA;
1324 $nB = @membersB;
1325 $nMAX = ($nA > $nB) ? $nA : $nB;
1326 # Sort membersA inside the cluster by confidence:
1327 for $m (0..($nA-1)){
1328 while($confPA[$membersA[$m]] < $confPA[$membersA[$m+1]]){
1329 $temp = $membersA[$m];
1330 $membersA[$m] = $membersA[$m+1];
1331 $membersA[$m+1] = $temp;
1332 --$m if ($m > 1);
1333 }
1334 }
1335 $paralogsA[$i] = join(' ',@membersA); # Put them back together
1336 # Sort membersB inside the cluster by confidence:
1337 for $m (0..($nB-1)){
1338 while($confPB[$membersB[$m]] < $confPB[$membersB[$m+1]]){
1339 $temp = $membersB[$m];
1340 $membersB[$m] = $membersB[$m+1];
1341 $membersB[$m+1] = $temp;
1342 --$m if ($m > 1);
1343 }
1344 }
1345 $paralogsB[$i] = join(' ',@membersB); # Put them back together
1346 # Print to text file and to HTML file
1347 for $m (0..($nMAX-1)){
1348 if ($m < $nA){
1349 if ($output){
1350 printf (OUTPUT "%-20s\t%.2f%%\t\t", $nameA[$membersA[$m]], (100*$confPA[$membersA[$m]]));
1351 }
1352 if ($html){
1353 print HTML "<B>" if ($confPA[$membersA[$m]] == 1);
1354 printf (HTML "%-20s\t%.2f%%\t\t", $nameA[$membersA[$m]], (100*$confPA[$membersA[$m]]));
1355 print HTML "</B>" if ($confPA[$membersA[$m]] == 1);
1356 }
1357 }
1358 else {
1359 printf (OUTPUT "%-20s\t%-7s\t\t", " ", " ");
1360 printf (HTML "%-20s\t%-7s\t\t", " ", " ") if ($html);
1361 }
1362 if ($m < $nB){
1363 if ($output){
1364 printf (OUTPUT "%-20s\t%.2f%%\n", $nameB[$membersB[$m]], (100*$confPB[$membersB[$m]]));
1365 }
1366 if ($html){
1367 print HTML "<B>" if ($confPB[$membersB[$m]] == 1);
1368 printf (HTML "%-20s\t%.2f%%", $nameB[$membersB[$m]], (100*$confPB[$membersB[$m]]));
1369 print HTML "</B>" if ($confPB[$membersB[$m]] == 1);
1370 print HTML "\n";
1371 }
1372 }
1373 else {
1374 printf (OUTPUT "%-20s\t%-7s\n", " ", " ") if($output);
1375 print HTML "\n" if ($html);
1376 }
1377 }
1378 print OUTPUT $message if ($use_bootstrap and $output);
1379 print HTML "$htmlmessage" if ($use_bootstrap and $html);
1380 }
1381 if ($output) {
1382 close OUTPUT;
1383 print "Output saved to file $outputfile\n";
1384 }
1385 if ($html){
1386 close HTML;
1387 print "HTML output saved to $htmlfile\n";
1388 }
1389
1390 if ($table){
1391 $filename = "table." . $ARGV[0] . "-" . $ARGV[1];
1392 open F, ">$filename" or die;
1393 print F "OrtoID\tScore\tOrtoA\tOrtoB\n";
1394 for $i(1..$o){
1395 print F "$i\t$ortoS[$i]\t";
1396 @members = split(/ /, $paralogsA[$i]);
1397 for $m (@members){
1398 $m =~ s/://g;
1399 printf (F "%s %.3f ", $nameA[$m], $confPA[$m]);
1400 }
1401 print F "\t";
1402 @members = split(/ /, $paralogsB[$i]);
1403 for $m (@members){
1404 $m =~ s/://g;
1405 printf (F "%s %.3f ", $nameB[$m], $confPB[$m]);
1406 }
1407 print F "\n";
1408 }
1409 close F;
1410 print "Table output saved to $filename\n";
1411 }
1412 if ($mysql_table){
1413 $filename2 = "sqltable." . $ARGV[0] . "-" . $ARGV[1];
1414 open F2, ">$filename2" or die;
1415 for $i(1..$o){
1416 @membersA = split(/ /, $paralogsA[$i]);
1417 for $m (@membersA){
1418 # $m =~ s/://g;
1419 if ($use_bootstrap && $bsA[$m]) {
1420 printf (F2 "%d\t%d\t%s\t%.3f\t%s\t%d%\n", $i, $ortoS[$i], $ARGV[0], $confPA[$m], $nameA[$m], 100*$bsA[$m]);
1421 } else {
1422 printf (F2 "%d\t%d\t%s\t%.3f\t%s\n", $i, $ortoS[$i], $ARGV[0], $confPA[$m], $nameA[$m]);
1423 }
1424 }
1425 @membersB = split(/ /, $paralogsB[$i]);
1426 for $m (@membersB){
1427 # $m =~ s/://g;
1428 if ($use_bootstrap && $bsB[$m]) {
1429 printf (F2 "%d\t%d\t%s\t%.3f\t%s\t%d%\n", $i, $ortoS[$i], $ARGV[1], $confPB[$m], $nameB[$m], 100*$bsB[$m]);
1430 }else {
1431 printf (F2 "%d\t%d\t%s\t%.3f\t%s\n", $i, $ortoS[$i], $ARGV[1], $confPB[$m], $nameB[$m]);
1432 }
1433 }
1434 }
1435 close F2;
1436 print "mysql output saved to $filename2\n";
1437 }
1438 if ($show_times){
1439 ($user_time,,,) = times;
1440 printf ("Finding bootstrap values and printing took %.2f seconds\n", ($user_time - $prev_time));
1441 printf ("The overall execution time: %.2f seconds\n", $user_time);
1442 }
1443 if ($run_blast) {
1444 unlink "formatdb.log";
1445 unlink "$fasta_seq_fileA.phr", "$fasta_seq_fileA.pin", "$fasta_seq_fileA.psq";
1446 unlink "$fasta_seq_fileB.phr", "$fasta_seq_fileB.pin", "$fasta_seq_fileB.psq" if (@ARGV >= 2);
1447 unlink "$fasta_seq_fileC.phr", "$fasta_seq_fileC.pin", "$fasta_seq_fileC.psq" if ($use_outgroup);
1448 }
1449 }
1450
1451 ##############################################################
1452 # Functions:
1453 ##############################################################
1454 sub clean_up { # Sort members within cluster and clusters by size
1455 ############################################################################################### Modification by Isabella 3
1456
1457 # Sort on index arrays with perl's built in sort instead of using bubble sort.
1458
1459 $var = shift;
1460 $totalA = $totalB = 0;
1461 # First pass: count members within each cluster
1462 foreach $i (1..$o) {
1463 @membersA = split(/ /, $paralogsA[$i]);
1464 $clusnA[$i] = @membersA; # Number of members in this cluster
1465 $totalA += $clusnA[$i];
1466 $paralogsA[$i] = join(' ',@membersA);
1467
1468 @membersB = split(/ /, $paralogsB[$i]);
1469 $clusnB[$i] = @membersB; # Number of members in this cluster
1470 $totalB += $clusnB[$i];
1471 $paralogsB[$i] = join(' ',@membersB);
1472
1473 $clusn[$i] = $clusnB[$i] + $clusnA[$i]; # Number of members in given group
1474 }
1475
1476 # Create an array used to store the position each element shall have in the final array
1477 # The elements are initialized with the position numbers
1478 my @position_index_array = (1..$o);
1479
1480 # Sort the position list according to cluster size
1481 my @cluster_sorted_position_list = sort { $clusn[$b] <=> $clusn[$a]} @position_index_array;
1482
1483 # Create new arrays for the sorted information
1484 my @new_paralogsA;
1485 my @new_paralogsB;
1486 my @new_is_paralogA;
1487 my @new_is_paralogB;
1488 my @new_clusn;
1489 my @new_ortoS;
1490 my @new_ortoA;
1491 my @new_ortoB;
1492
1493
1494 # Add the information to the new arrays in the orer specifeid by the index array
1495 for (my $index_in_list = 0; $index_in_list < scalar @cluster_sorted_position_list; $index_in_list++) {
1496
1497 my $old_index = $cluster_sorted_position_list[$index_in_list];
1498
1499 if (!$clusn[$old_index]) {
1500 $o = (scalar @new_ortoS) - 1;
1501 last;
1502 }
1503
1504 $new_paralogsA[$index_in_list + 1] = $paralogsA[$old_index];
1505 $new_paralogsB[$index_in_list + 1] = $paralogsB[$old_index];
1506 $new_is_paralogA[$index_in_list + 1] = $is_paralogA[$old_index];
1507 $new_is_paralogB[$index_in_list + 1] = $is_paralogB[$old_index];
1508 $new_clusn[$index_in_list + 1] = $clusn[$old_index];
1509 $new_ortoA[$index_in_list + 1] = $ortoA[$old_index];
1510 $new_ortoB[$index_in_list + 1] = $ortoB[$old_index];
1511 $new_ortoS[$index_in_list + 1] = $ortoS[$old_index];
1512 }
1513
1514 @paralogsA = @new_paralogsA;
1515 @paralogsB = @new_paralogsB;
1516 @is_paralogA = @new_is_paralogA;
1517 @is_paralogB = @new_is_paralogB;
1518 @clusn = @new_clusn;
1519 @ortoS = @new_ortoS;
1520 @ortoA = @new_ortoA;
1521 @ortoB = @new_ortoB;
1522
1523 # Create an array used to store the position each element shall have in the final array
1524 # The elements are initialized with the position numbers
1525 @position_index_array = (1..$o);
1526
1527 # Sort the position list according to score
1528 @score_sorted_position_list = sort { $ortoS[$b] <=> $ortoS[$a] } @position_index_array;
1529
1530 # Create new arrays for the sorted information
1531 my @new_paralogsA2 = ();
1532 my @new_paralogsB2 = ();
1533 my @new_is_paralogA2 = ();
1534 my @new_is_paralogB2 = ();
1535 my @new_clusn2 = ();
1536 my @new_ortoS2 = ();
1537 my @new_ortoA2 = ();
1538 my @new_ortoB2 = ();
1539
1540 # Add the information to the new arrays in the orer specifeid by the index array
1541 for (my $index_in_list = 0; $index_in_list < scalar @score_sorted_position_list; $index_in_list++) {
1542
1543 my $old_index = $score_sorted_position_list[$index_in_list];
1544 $new_paralogsA2[$index_in_list + 1] = $paralogsA[$old_index];
1545 $new_paralogsB2[$index_in_list + 1] = $paralogsB[$old_index];
1546 $new_is_paralogA2[$index_in_list + 1] = $is_paralogA[$old_index];
1547 $new_is_paralogB2[$index_in_list + 1] = $is_paralogB[$old_index];
1548 $new_clusn2[$index_in_list + 1] = $clusn[$old_index];
1549 $new_ortoA2[$index_in_list + 1] = $ortoA[$old_index];
1550 $new_ortoB2[$index_in_list + 1] = $ortoB[$old_index];
1551 $new_ortoS2[$index_in_list + 1] = $ortoS[$old_index];
1552 }
1553
1554 @paralogsA = @new_paralogsA2;
1555 @paralogsB = @new_paralogsB2;
1556 @is_paralogA = @new_is_paralogA2;
1557 @is_paralogB = @new_is_paralogB2;
1558 @clusn = @new_clusn2;
1559 @ortoS = @new_ortoS2;
1560 @ortoA = @new_ortoA2;
1561 @ortoB = @new_ortoB2;
1562
1563 #################################################################################### End modification by Isabella 3
1564
1565 }
1566 sub bootstrap{
1567 my $species = shift;
1568 my $seq_id1 = shift; # Query ID from $species
1569 my $seq_id2 = shift; # Best hit ID from other species
1570 my $seq_id3 = shift; # Second best hit
1571 # Retrieve sequence 1 from $species and sequence 2 from opposite species
1572 my $significance = 0.0;
1573
1574 if ($species eq $fasta_seq_fileA){
1575 $file1 = $fasta_seq_fileA;
1576 $file2 = $fasta_seq_fileB;
1577 }
1578 elsif ($species eq $fasta_seq_fileB){
1579 $file1 = $fasta_seq_fileB;
1580 $file2 = $fasta_seq_fileA;
1581 }
1582 else {
1583 print "Bootstrap values for ortholog groups are not calculated\n";
1584 return 0;
1585 }
1586
1587 open A, $file1 or die;
1588 $id = 0;
1589 $print_this_seq = 0;
1590 $seq1 = "";
1591 $seq2 = "";
1592
1593 $query_file = $seq_id1 . ".faq";
1594 open Q, ">$query_file" or die;
1595
1596 while (<A>){
1597 if(/^\>/){
1598 ++$id;
1599 $print_this_seq = ($id == $seq_id1)?1:0;
1600 }
1601 print Q if ($print_this_seq);
1602 }
1603 close A;
1604 close Q;
1605 ###
1606 open B, $file2 or die;
1607 $db_file = $seq_id2 . ".fas";
1608 open DB, ">$db_file" or die;
1609 $id = 0;
1610 $print_this_seq = 0;
1611
1612 while (<B>){
1613 if(/^\>/){
1614 ++$id;
1615 $print_this_seq = (($id == $seq_id2) or ($id == $seq_id3))?1:0;
1616 }
1617 print DB if ($print_this_seq);
1618 }
1619 close B;
1620 close DB;
1621
1622 system "$formatdb -i $db_file";
1623 # Use soft masking in 1-pass mode for simplicity.
1624 system "$blastall -F\"m S\" -i $query_file -z 5000000 -d $db_file -p blastp -M $matrix -m7 | ./$blastParser 0 -a > $seq_id2.faa";
1625
1626 # Note: Changed score cutoff 50 to 0 for blast2faa.pl (060402).
1627 # Reason: after a cluster merger a score can be less than the cutoff (50)
1628 # which will remove the sequence in blast2faa.pl. The bootstrapping will
1629 # then fail.
1630 # AGAIN, updaye
1631
1632 if (-s("$seq_id2.faa")){
1633
1634 system("java -jar $seqstat -m $matrix -n 1000 -i $seq_id2.faa > $seq_id2.bs"); # Can handle U, u
1635
1636 if (-s("$seq_id2.bs")){
1637 open BS, "$seq_id2.bs" or die "pac failed\n";
1638 $_ = <BS>;
1639 ($dummy1,$dummy2,$dummy3,$dummy4,$significance) = split(/\s+/);
1640 close BS;
1641 }
1642 else{
1643 print STDERR "pac failed\n"; # if ($debug);
1644 $significance = -0.01;
1645 }
1646 }
1647 else{
1648 print STDERR "blast2faa for $query_file / $db_file failed\n"; # if ($debug);
1649 $significance = 0.0;
1650 }
1651
1652 unlink "$seq_id2.fas", "$seq_id2.faa", "$seq_id2.bs", "$seq_id1.faq";
1653 unlink "formatdb.log", "$seq_id2.fas.psq", "$seq_id2.fas.pin", "$seq_id2.fas.phr";
1654
1655 return $significance;
1656 }
1657
1658 sub overlap_test{
1659 my @Fld = @_;
1660
1661 # Filter out fragmentary hits by:
1662 # Ignore hit if aggregate matching area covers less than $seq_overlap_cutoff of sequence.
1663 # Ignore hit if local matching segments cover less than $segment_coverage_cutoff of sequence.
1664 #
1665 # $Fld[3] and $Fld[4] are query and subject lengths.
1666 # $Fld[5] and $Fld[6] are lengths of the aggregate matching region on query and subject. (From start of first matching segment to end of last matching segment).
1667 # $Fld[7] and $Fld[8] are local matching length on query and subject (Sum of all segments length's on query).
1668
1669 $retval = 1;
1670 # if ($Fld[3] >= $Fld[4]) {
1671 if ($Fld[5] < ($seq_overlap_cutoff * $Fld[3])) {$retval = 0};
1672 if ($Fld[7] < ($segment_coverage_cutoff * $Fld[3])) {$retval = 0};
1673 # }
1674
1675 # if ($Fld[4] >= $Fld[3]) {
1676 if ($Fld[6] < ($seq_overlap_cutoff * $Fld[4])) {$retval = 0};
1677 if ($Fld[8] < ($segment_coverage_cutoff * $Fld[4])) {$retval = 0};
1678 # }
1679
1680 # print "$Fld[3] $Fld[5] $Fld[7]; $Fld[4] $Fld[6] $Fld[8]; retval=$retval\n";
1681
1682 return $retval;
1683 }
1684
1685 sub do_blast
1686 {
1687 my @parameter=@_;
1688 my $resultfile=@parameter[@parameter-1];
1689 my $go_to_blast=1;
1690 my $resultfilesize;
1691 if (-e $resultfile)
1692 {
1693 $resultfilesize= -s "$resultfile";
1694 if ($resultfilesize >10240)
1695 {
1696 $go_to_blast=0;
1697 }
1698 }
1699 if ($go_to_blast)
1700 {
1701 if ($blast_two_passes)
1702 {
1703 do_blast_2pass(@parameter);
1704 } else
1705 {
1706 do_blast_1pass(@parameter);
1707 }
1708 }
1709 }
1710
1711 sub do_blast_1pass {
1712 my @Fld = @_;
1713
1714 # $Fld [0] is query
1715 # $Fld [1] is database
1716 # $Fld [2] is query size
1717 # $Fld [3] is database size
1718 # $Fld [4] is output name
1719
1720 # Use soft masking (low complexity masking by SEG in search phase, not in alignment phase).
1721 system ("$blastall -F\"m S\" -i $Fld[0] -d $Fld[1] -p blastp -v $Fld[3] -b $Fld[3] -M $matrix -z 5000000 -m7 | ./$blastParser $score_cutoff > $Fld[4]");
1722 }
1723
1724 sub do_blast_2pass {
1725
1726 my @Fld = @_;
1727
1728 # $Fld [0] is query
1729 # $Fld [1] is database
1730 # $Fld [2] is query size
1731 # $Fld [3] is database size
1732 # $Fld [4] is output name
1733
1734 # assume the script has already formatted the database
1735 # we will now do 2-pass approach
1736
1737 # load sequences
1738
1739 %sequencesA = ();
1740 %sequencesB = ();
1741
1742 open (FHA, $Fld [0]);
1743 while (<FHA>) {
1744
1745 $aLine = $_;
1746 chomp ($aLine);
1747
1748 $seq = "";
1749
1750 if ($aLine =~ />/) {
1751 @words = split (/\s/, $aLine);
1752 $seqID = $words[0];
1753 $sequencesA {$seqID} = "";
1754 }
1755 else {
1756 $sequencesA {$seqID} = $sequencesA {$seqID}.$aLine;
1757 }
1758 }
1759 close (FHA);
1760
1761 open (FHB, $Fld [1]);
1762 while (<FHB>) {
1763 $aLine = $_;
1764 chomp ($aLine);
1765
1766 $seq = "";
1767
1768 if ($aLine =~ />/) {
1769 @words = split (/\s/, $aLine);
1770 $seqID = $words[0];
1771 $sequencesB {$seqID} = "";
1772 }
1773 else {
1774 $sequencesB {$seqID} = $sequencesB {$seqID}.$aLine;
1775 }
1776 }
1777 close (FHB);
1778
1779 # Do first pass with compositional adjustment on and soft masking.
1780 # This efficiently removes low complexity matches but truncates alignments,
1781 # making a second pass necessary.
1782 print STDERR "\nStarting first BLAST pass for $Fld[0] - $Fld[1] on ";
1783 system("date");
1784 open FHR, "$blastall -C3 -F\"m S\" -i $Fld[0] -d $Fld[1] -p blastp -v $Fld[3] -b $Fld[3] -M $matrix -z 5000000 -m7 | ./$blastParser $score_cutoff|";
1785
1786 %theHits = ();
1787 while (<FHR>) {
1788 $aLine = $_;
1789 chomp ($aLine);
1790 @words = split (/\s+/, $aLine);
1791
1792 if (exists ($theHits {$words [0]})) {
1793 $theHits {$words [0]} = $theHits {$words [0]}." ".$words [1];
1794 }
1795 else {
1796 $theHits {$words [0]} = $words [1];
1797 }
1798
1799 }
1800 close (FHR);
1801
1802 $tmpdir = "."; # May be slightly (5%) faster using the RAM disk "/dev/shm".
1803 $tmpi = "$tmpdir/tmpi";
1804 $tmpd = "$tmpdir/tmpd";
1805
1806 # Do second pass with compositional adjustment off to get full-length alignments.
1807 print STDERR "\nStarting second BLAST pass for $Fld[0] - $Fld[1] on ";
1808 system("date");
1809 unlink "$Fld[4]";
1810 foreach $aQuery (keys % theHits) {
1811
1812 # Create single-query file
1813 open (FHT, ">$tmpi");
1814 print FHT ">$aQuery\n".$sequencesA {">$aQuery"}."\n";
1815 close (FHT);
1816
1817 # Create mini-database of hit sequences
1818 open (FHT, ">$tmpd");
1819 foreach $aHit (split (/\s/, $theHits {$aQuery})) {
1820 print FHT ">$aHit\n".$sequencesB {">$aHit"}."\n";
1821 }
1822 close (FHT);
1823
1824 # Run Blast and add to output
1825 system ("$formatdb -i $tmpd");
1826 system ("$blastall -C0 -FF -i $tmpi -d $tmpd -p blastp -v $Fld[3] -b $Fld[3] -M $matrix -z 5000000 -m7 | ./$blastParser $score_cutoff >> $Fld[4]");
1827 }
1828
1829 unlink "$tmpi", "$tmpd", "formatdb.log", "$tmpd.phr", "$tmpd.pin", "$tmpd.psq";
1830 }
1831
1832
1833 # Date Modification
1834 # -------- ---------------------------------------------------
1835 #
1836 # 2006-04-02 [1.36] - Changed score cutoff 50 to 0 for blast2faa.pl.
1837 # Reason: after a cluster merger a score can be less than the cutoff (50)
1838 # which will remove the sequence in blast2faa.pl. The bootstrapping will
1839 # then fail.
1840 # - Fixed bug with index variable in bootstrap routine.
1841 #
1842 # 2006-06-01 [2.0] - Fixed bug in blast_parser.pl: fields 7 and 8 were swapped,
1843 # it was supposed to print match_area before HSP_length.
1844 # - Fixed bug in blastall call: -v param was wrong for the A-B
1845 # and B-A comparisons.
1846 # -
1847 # - Changed "cluster" to "group" consistently in output.
1848 # - Changed "main ortholog" to "seed ortholog" in output.
1849 # - Replace U -> X before running seqstat.jar, otherwise it crashes.
1850 # 2006-08-04 [2.0] - In bootstrap subroutine, replace U with X, otherwise seqstat
1851 # will crash as this is not in the matrix (should fix this in seqstat)
1852 # 2006-08-04 [2.1] - Changed to writing default output to file.
1853 # - Added options to run blast only.
1854 # - Fixed some file closing bugs.
1855 # 2007-12-14 [3.0] - Sped up sorting routines (by Isabella).
1856 # - New XML-based blast_parser.
1857 # - New seqstat.jar to handle u and U.
1858 # - Modified overlap criterion for rejecting matches. Now it agrees with the paper.
1859 # 2009-04-01 [4.0] - Further modification of overlap criteria (require that they are met for both query and subject).
1860 # - Changed bit score cutoff to 40, which is suitable for compositionally adjusted BLAST.
1861 # - Added in 2-pass algorithm.
1862 # 2009-06-11 [4.0] - Moved blasting out to subroutine.
1863 # - Changed blasting in bootstrap subroutine to use unconditional score matrix adjustment and SEG hard masking,
1864 # to be the same as first step of 2-pass blast.
1865 # 2009-06-17 [4.0] - Compensated a Blast "bug" that sometimes gives a self-match lower score than a non-identical match.
1866 # This can happen with score matrix adjustment and can lead to missed orthologs.
1867 # 2009-08-18 [4.0] - Consolidated Blast filtering parameters for 2-pass (-C3 -F\"m S\"; -C0 -FF)
1868 # 2009-10-09 [4.1] - Fixed bug that caused failure if Fasta header lines had more than one word.