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get_homologues-est.pl
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get_homologues-est.pl
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#!/usr/bin/env perl
# 2021 Bruno Contreras-Moreira (1) and Pablo Vinuesa (2):
# 1: http://www.eead.csic.es/compbio (Estacion Experimental Aula Dei-CSIC/Fundacion ARAID, Spain)
# 2: http://www.ccg.unam.mx/~vinuesa (Center for Genomic Sciences, UNAM, Mexico)
# This program uses BLASTN to define clusters of 'orthologous' gene/transcript DNA sequences
# and pan-transcriptome & pan-genome sets. Several algorithms are available and search parameters
# are customizable. It is designed to process (in a multicore computer or HPC cluster) files contained in a
# directory (-d), so that new .fna files can be added while conserving previous BLASTN/Pfam results.
# In general the program tries to re-use previous results when run with the same input directory.
$|=1;
use strict;
use warnings;
use Getopt::Std;
use Fcntl qw(:flock);
use File::Temp qw(tempfile);
use File::Basename;
use Benchmark;
use Cwd;
use FindBin '$Bin';
use lib "$Bin/lib";
use lib "$Bin/lib/bioperl-1.5.2_102/";
use HPCluster;
use phyTools; #also imports constants SEQ,NAME used as array subindices
use marfil_homology; # includes $FASTAEXTENSION and other global variables set there such as @taxa
my $VERSION = '3.x';
## settings for local batch blast,hmmscan jobs
my $BATCHSIZE = 1000; # after HS_BLASTN bench
## global variables that control some algorithmic choices
my $MINSEQLENGTH = 20; # min length for input sequences to be considered (~ primer or miRNA)
my $MAXSEQLENGTH = 25000; # max length for input sequences, warning issued: long seqs often cause downstream problems
# In our barley tests with de novo Trinity assemblies, max length ~ 17Kb
# HQ Haruna Nijo barley flcDNAs reach 23Kb
my $NOFSAMPLESREPORT = 20; # number of genome samples used for the generation of pan/core genomes
my $MAXEVALUEBLASTSEARCH = 0.01; # required to reduce size of blast output files
my $MAXPFAMSEQS = 250; # default for -m cluster jobs, it is updated to 1000 with -m local
my $PRINTCLUSTERSSCREEN = 0; # whether cluster names should be printed to screen
my $FULLENGTHFLAG = 'flcdna'; # input sequences in files with names containing this flag are considered full length, instead of fragments
my $MINREDOVERLAP = 40; # as in tgicl, min overlap to handle possibly redundant isoforms
my $TRIMULTIHSP = 1; # correct overlaps when calculating cover of multi-hsp hits (alternative = 0)
my $NOCLOUDINCORE = 1; # when calling -M -c -t X initial core/pan size excludes cloud genes, those with occup < X 8alternative 0)
my $INCLUDEORDER = 0; # use implicit -I taxon order for -c composition analyses
my $KEEPSCNDHSPS = 1; # by default only the first BLAST hsp is kept for further calculations
my $COMPRESSBLAST = 1; # save disk space by compressing BLASTN results, requires gzip
## list of features/binaries required by this program (do not edit)
my @FEATURES2CHECK = ('EXE_BLASTN','EXE_FORMATDB','EXE_MCL','EXE_HMMPFAM',
'EXE_INPARA','EXE_ORTHO','EXE_HOMOL','EXE_ISOFORM','EXE_SPLITBLAST','EXE_SPLITHMMPFAM');
################################################################
my ($newDIR,$output_mask,$pancore_mask,$include_file,%included_input_files,%opts) = ('','','',0);
my ($exclude_inparalogues,$doMCL,$do_PFAM,$reference_proteome_string) = (0,0,0,0);
my ($isoform_overlap,$onlyblast,$inputDIR,$cluster_list_file) = ($MINREDOVERLAP,0);
my ($isoform_best_hit,$n_of_cpus,$do_minimal_BDBHs,$add_rd_isoforms,$do_soft) = (0,$BLAST_NOCPU,0,0,0);
my ($do_ANIb_matrix,$do_POCP_matrix) = (0,0);
my ($min_cluster_size,$runmode,$do_genome_composition,$saveRAM,$ANIb_matrix_file,$POCP_matrix_file);
my ($evalue_cutoff,$pi_cutoff,$pmatch_cutoff) =
($BLAST_PVALUE_CUTOFF_DEFAULT,$PERCENT_IDENTITY_CUTOFF_EST_DEFAULT,$PERCENT_MATCH_CUTOFF_DEFAULT);
my $MCLinflation = $MCL_INFLATION_DEFAULT;
my $random_number_generator_seed = 0;
my $pwd = getcwd(); $pwd .= '/';
my $start_time = new Benchmark();
getopts('hvbcesPDMoALzi:n:m:d:r:t:I:E:S:C:F:R:', \%opts);
if(($opts{'h'})||(scalar(keys(%opts))==0))
{
print "\nusage: $0 [options]\n\n";
print "-h this message\n";
print "-v print version, credits and checks installation\n";
print "-d directory with input FASTA files (.fna , optionally .faa), (use of pre-clustered sequences\n";
print " 1 per sample, or subdirectories (subdir.clusters/subdir_) ignores -c)\n";
print " with pre-clustered sequences (.faa/.fna ). Files matching\n";
print " tag '$FULLENGTHFLAG' are handled as full-length transcripts.\n";
print " Allows for files to be added later.\n";
print " Creates output folder named 'directory_est_homologues'\n";
print "\nOptional parameters:\n";
print "-o only run BLASTN/Pfam searches and exit ".
"(useful to pre-compute searches)\n";
print "-i cluster redundant isoforms, including those that can be ".
"(min overlap, default: -i $isoform_overlap,\n";
print " concatenated with no overhangs, and perform ".
" use -i 0 to disable)\n".
" calculations with longest\n";
#print "-H group sequences matching the same best hit as isoforms (optional, requires -i,)\n"; #not tested yet
print "-c report transcriptome composition analysis ".
"(follows order in -I file if enforced,\n".
" ".
" with -t N skips clusters occup<N [OMCL],\n".
" ".
" ignores -r,-e)\n";
print "-R set random seed for genome composition analysis ".
"(optional, requires -c, example -R 1234)\n";
if(eval{ require DB_File } ) # show only if DB_File is available (it should as it is core)
{
print "-s save memory by using BerkeleyDB; default parsing stores\n".
" sequence hits in RAM\n";
}
print "-m runmode [local|cluster|dryrun] ".
"(default: -m local)\n";
print "-n nb of threads for BLASTN/HMMER/MCL in 'local' runmode (default=$n_of_cpus)\n";
print "-I file with .fna files in -d to be included ".
"(takes all by default, requires -d)\n";
print "\nAlgorithms instead of default bidirectional best-hits (BDBH):\n";
print "-M use orthoMCL algorithm (OMCL, PubMed=12952885)\n";
print "\nOptions that control sequence similarity searches:\n";
print "-C min \%coverage of shortest sequence in BLAST alignments ";
print "(range [1-100],default: -C $PERCENT_MATCH_CUTOFF_DEFAULT)\n";
print "-E max E-value ".
"(default: -E $BLAST_PVALUE_CUTOFF_DEFAULT , max=$MAXEVALUEBLASTSEARCH)\n";
print "-D require equal Pfam domain composition ".
"(best with -m cluster or -n threads)\n";
print " when defining similarity-based orthology\n";
print "-S min \%sequence identity in BLAST query/subj pairs ".
"(range [1-100],default: -S $PERCENT_IDENTITY_CUTOFF_EST_DEFAULT [BDBH|OMCL])\n";
print "-b compile core-transcriptome with minimum BLAST searches ".
"(ignores -c [BDBH])\n";
print "\nOptions that control clustering:\n";
print "-t report sequence clusters including at least t taxa ".
"(default: t=numberOfTaxa,\n".
" ".
" t=0 reports all clusters [OMCL])\n";
print "-L add redundant isoforms to clusters ".
"(optional, requires -i)\n";
print "-r reference transcriptome .fna file ".
"(by default takes file with\n".
" ".
" least sequences; with BDBH sets\n".
" ".
" first taxa to start adding genes)\n";
print "-e exclude clusters with inparalogues ".
"(by default inparalogues are\n".
" ".
" included)\n";
print "-F orthoMCL inflation value ".
"(range [1-5], default: -F $MCL_INFLATION_DEFAULT [OMCL])\n";
print "-A calculate average identity of clustered sequences, ".
"(optional, creates tab-separated matrix,\n";
print " uses blastn results ".
" [OMCL])\n";
print "-P calculate percentage of conserved sequences (POCS), ".
"(optional, creates tab-separated matrix,\n";
print " uses blastn results, best with CDS ".
" [OMCL])\n";
print "-z add soft-core to genome composition analysis ".
"(optional, requires -c [OMCL])\n";
print "\n".
"This program uses BLASTN/HMMER to define clusters of 'orthologous' transcripts\n".
"and pan/core-trancriptome sets. Different algorithm choices are available\n".
"and search parameters are customizable. It is designed to process (in a HPC computer\n".
"cluster) files contained in a directory (-d), so that new .fna/.faa files can be added\n".
"while conserving previous BLASTN/HMMER results. In general the program will try to re-use\n".
"previous results when run with the same input directory.\n";
exit;
}
# read version number from CHANGES.txt
open(CHANGES,"$Bin/CHANGES.txt");
while(<CHANGES>)
{
if(eof && /^(\d+):/){ $VERSION = $1 }
}
close(CHANGES);
if(defined($opts{'v'}))
{
print "\n$0 version $VERSION\n";
print "\nProgram written by Bruno Contreras-Moreira (1) and Pablo Vinuesa (2).\n";
print "\n 1: http://www.eead.csic.es/compbio (Estacion Experimental Aula Dei/CSIC/Fundacion ARAID, Spain)\n";
print " 2: http://www.ccg.unam.mx/~vinuesa (Center for Genomic Sciences, UNAM, Mexico)\n\n";
print "\nPrimary citation:\n\n";
print " Contreras-Moreira B, Cantalapiedra CP, Garcia Pereira MJ, Gordon S, Vogel JP, Igartua E, Casas AM,\n".
" Vinuesa P (2017) Analysis of plant pan-genomes and transcriptomes with GET_HOMOLOGUES-EST, a clustering\n".
" solution for sequences of the same species. Front. Plant Sci. 10.3389/fpls.2017.00184\n";
print "\nThis software employs code, binaries and data from different authors, please cite them accordingly:\n";
print " OrthoMCL v1.4 (www.orthomcl.org , PubMed:12952885)\n";
print " NCBI Blast-2.2 (blast.ncbi.nlm.nih.gov , PubMed=9254694,20003500)\n";
print " Bioperl v1.5.2 (www.bioperl.org , PubMed=12368254)\n";
print " HMMER 3.1b2 (hmmer.org)\n";
print " Pfam (http://pfam.xfam.org , PubMed=26673716)\n";
# check all binaries and data needed by this program and print diagnostic info
print check_installed_features(@FEATURES2CHECK);
exit(0);
}
if(defined($opts{'d'}))
{
$inputDIR = $opts{'d'};
die "# EXIT : need a valid input directory\n" if(!-e $inputDIR);
if(basename($inputDIR) =~ /([\+])/)
{
die "# EXIT : need a valid input directory name, offending char: '$1'\n";
}
if(substr($inputDIR,length($inputDIR)-1,1) eq '/'){ chop $inputDIR }
$newDIR = $pwd.basename($inputDIR)."_est_homologues";
}
else{ die "# EXIT : need a -d directory with .fasta/.fna files as input\n"; }
if(defined($opts{'m'}))
{
$runmode = $opts{'m'};
if($runmode eq 'pbs'){ $runmode = 'cluster'; } # legacy
elsif($runmode ne 'local' && $runmode ne 'cluster' && $runmode ne 'dryrun'){ $runmode = 'cluster'; }
}
else{ $runmode = 'local'; }
if($runmode eq 'local' && defined($opts{'n'}) && $opts{'n'} > 0)
{
$n_of_cpus = $opts{'n'};
$BLAST_NOCPU = $n_of_cpus;
}
if($runmode eq 'cluster')
{
# check whether file 'cluster.conf' exists & parse it
read_cluster_config( "$Bin/HPC.conf" );
if(!cluster_is_available())
{
print "# EXIT : cluster is not available, please create/edit file cluster.conf\n";
print_cluster_config();
die "# EXIT : or choose runmode -m local\n";
}
}
if(defined($opts{'o'})){ $onlyblast = 1; }
else
{
if(defined($opts{'i'}))
{
if($opts{'i'} >= $isoform_overlap)
{
$isoform_overlap = $opts{'i'};
$output_mask .= "isoover$isoform_overlap\_";
$pancore_mask = "_isoover$isoform_overlap";
}
else
{
$isoform_overlap = 0;
}
}
if(defined($opts{'L'}))
{
$add_rd_isoforms = $opts{'L'};
$output_mask .= "rediso_";
}
else{ $add_rd_isoforms = 0; } #$output_mask .= "rdiso0_"; }
#if(defined($opts{'H'})){
# $isoform_best_hit = 1;
# $output_mask .= "isohit_";
# $pancore_mask = "_isohit";
#}
}
if($opts{'r'}){ $reference_proteome_string = $opts{'r'}; }
else{ $reference_proteome_string = 0; }
if(defined($opts{'t'}) && $opts{'t'} >= 0)
{
if(!defined($opts{'M'}))
{
die "\n# WARNING: use of the default BDBH algorithm with options -c -t is not supported ".
"(please check the manual)\n\n";
}
$min_cluster_size = $opts{'t'};
$output_mask .= $min_cluster_size."taxa_";
$pancore_mask .= '_'.$min_cluster_size."taxa";
}
else{ $min_cluster_size = 'all'; $output_mask .= "alltaxa_"; }
if($opts{'I'} && $inputDIR)
{
$include_file = $opts{'I'};
$output_mask .= basename($include_file)."_";
$pancore_mask = "_".basename($include_file);
}
check_installed_features(@FEATURES2CHECK);
if(defined($opts{'A'})){ $do_ANIb_matrix = 1 }
if(defined($opts{'P'})){ $do_POCP_matrix = 1 }
if(defined($opts{'M'}))
{
if(feature_is_installed('OMCL'))
{
$doMCL = 1;
$output_mask .= "algOMCL_";
$pancore_mask .= "_algOMCL";
}
else{ warn_missing_soft('OMCL') }
}
else
{
if(defined($opts{'b'}))
{
$do_minimal_BDBHs = 1;
$output_mask .= "algBDBHmin_";
$pancore_mask .= "_algBDBHmin";
}
else
{
$output_mask .= "algBDBH_";
$pancore_mask .= "_algBDBH";
}
if($min_cluster_size eq '0')
{
die "\n# WARNING: use of the default BDBH algorithm with option -t 0 is not supported ".
"(please check the manual)\n\n";
}
elsif($do_ANIb_matrix)
{
die "\n# WARNING: use of the default BDBH algorithm with option -A is not supported ".
"(please check the manual)\n\n";
}
elsif($do_POCP_matrix)
{
die "\n# WARNING: use of the default BDBH algorithm with option -P is not supported ".
"(please check the manual)\n\n";
}
}
if(defined($opts{'D'}))
{
if(feature_is_installed('PFAM'))
{
$do_PFAM = 1;
$output_mask .= "Pfam\_";
$pancore_mask .= "_Pfam";
# in local mode increase batch size before calling _split_hmmscan.pl
if($runmode eq 'local'){ $MAXPFAMSEQS = 5000 } # should fit most bacterial proteomes
}
else{ warn_missing_soft('PFAM') }
}
if(defined($opts{'s'}) && eval{ require DB_File } ){ $saveRAM = 1; }
else{ $saveRAM = 0; }
if(defined($opts{'c'}) && !$do_minimal_BDBHs)
{
$do_genome_composition = 1;
if(defined($opts{'z'}) && $doMCL)
{
$do_soft = 1;
}
if($opts{'R'})
{
$random_number_generator_seed = $opts{'R'};
}
srand($random_number_generator_seed);
}
else{ $do_genome_composition = 0; }
if(defined($opts{'e'}))
{
$exclude_inparalogues = 1;
$output_mask .= "e1_";
}
else{ $output_mask .= "e0_"; }
if(defined($opts{'E'}))
{
$evalue_cutoff = $opts{'E'};
if($evalue_cutoff > $MAXEVALUEBLASTSEARCH){ $evalue_cutoff = $MAXEVALUEBLASTSEARCH }
$output_mask .= "E$evalue_cutoff\_"; $pancore_mask .= "_E$evalue_cutoff";
}
if(defined($opts{'C'}))
{
$pmatch_cutoff = $opts{'C'}; # BDBH|OMCL
if($pmatch_cutoff < 1){ $pmatch_cutoff = 1 }
elsif($pmatch_cutoff > 100){ $pmatch_cutoff = 100 }
$output_mask .= "C$pmatch_cutoff\_"; $pancore_mask .= "_C$pmatch_cutoff";
}
if(defined($opts{'S'}))
{
$pi_cutoff = $opts{'S'};
if($pi_cutoff < 1){ $pi_cutoff = 1 }
elsif($pi_cutoff > 100){ $pi_cutoff = 100 }
$output_mask .= "S$pi_cutoff\_"; $pancore_mask .= "_S$pi_cutoff";
}
if(defined($opts{'F'}))
{
$MCLinflation = $opts{'F'};
if($MCLinflation < 1){ $MCLinflation = 1 }
elsif($MCLinflation > 5){ $MCLinflation = 5 }
$output_mask .= "F$MCLinflation\_"; $pancore_mask .= "_F$MCLinflation";
}
print "# $0 -d $inputDIR -o $onlyblast -i $isoform_overlap -e $exclude_inparalogues -r $reference_proteome_string ".
"-t $min_cluster_size -c $do_genome_composition -z $do_soft -I $include_file -m $runmode -n $n_of_cpus -M $doMCL ".
"-C $pmatch_cutoff -S $pi_cutoff -E $evalue_cutoff -F $MCLinflation -b $do_minimal_BDBHs ".
"-s $saveRAM -D $do_PFAM -R $random_number_generator_seed -L $add_rd_isoforms -A $do_ANIb_matrix -P $do_POCP_matrix\n\n";
if($runmode eq 'cluster')
{
print "# computer cluster settings\n";
print_cluster_config();
}
###############################################################
## 0) declare most important vars
my ($infile,$new_infile,$prot_new_infile,$p2oinfile,$seq,$seqL,$comma_input_files,@newfiles,%ressize,%orth_taxa);
my ($label,%orthologues,$gene,$orth,$orth2,$para,%inparalogues,%paralogues,$FASTAresultsDIR,$order,$minlog);
my ($n_of_similar_length_orthologues,$clusterfile,$prot_clusterfile,$previous_files,$current_files,$inpara);
my ($min_proteome_size,$reference_proteome,$smallest_proteome,$proteome_size,%seq_length,$cluster,$annot);
my ($smallest_proteome_name,$reference_proteome_name,%psize,$taxon,$taxon2,$n_of_clusters,$n_of_taxa,$n_of_residues);
my ($pname,$n_of_sequences,$refOK,$genbankOK,$cluster_size,$prot_cluster_size,$prot_cluster);
my (%orth_registry,%LSE_registry,$LSE_reference,$LSE_t,$redo_inp,$redo_orth,%idclusters,%names,$generef);
my ($reparse_all,$n_of_similar_length_paralogues,$pfam_annot,$protOK,$n_of_taxa_cluster) = (0);
my ($BDBHdone,$PARANOIDdone,$orthoMCLdone,$n_of_parsed_lines,$n_of_pfam_parsed_lines) = (0,0,0,0,0);
my ($diff_BDBH_params,$diff_INP_params,$diff_HOM_params,$diff_OMCL_params,$lockcapableFS,$total_dry) = (0,0,0,0,0,0);
my ($diff_ISO_params,$redo_iso,$partial_sequences,$isof,%full_length_file,%redundant_isoforms,%total_redundant) = (0);
my ($total_clustersOK,$clgene,$clorth,%ANIb_matrix,%POCP_matrix,%GIclusters,$clustersOK,%cluster_ids) = (0);
constructDirectory($newDIR) || die "# EXIT : cannot create directory $newDIR , check permissions\n";
# 0.1) try to make sure there is only 1 instance writing to $newDIR
# http://www.perlmonks.org/?node_id=590619, flock might fail with NFS filehandles
# http://stackoverflow.com/questions/4502721/in-perl-how-can-i-check-if-a-file-is-locked
# http://perldoc.perl.org/File/Temp.html
my ($fhtest,$testlockfilename) = tempfile( DIR => $newDIR );
if(flock($fhtest,LOCK_EX|LOCK_NB))
{
$lockcapableFS = 1;
}
else
{
print "# WARNING : cannot lock files in $newDIR ,\n".
"# please ensure that no other instance of the program is running at this location\n\n";
}
unlink($testlockfilename);
open(my $fhlock,">$marfil_homology::lockfile") ||
die "# EXIT : cannot create lockfile $marfil_homology::lockfile\n";
if($lockcapableFS)
{
flock($fhlock, LOCK_EX|LOCK_NB) ||
die "# EXIT : cannot run another instance of the program with same input data while previous is running\n";
}
# 0.2) prepare to tie hash/array to db if required : allows massives,slow data structs in disk
my (@sequence_data,@sequence_prot,@sequence_dna);
my $sequence_data_filename = $newDIR."/sequence_data.db";
my $sequence_prot_filename = $newDIR."/sequence_prot.db";
my $sequence_dna_filename = $newDIR."/sequence_dna.db";
my $pfam_data_filename = $newDIR."/pfam.db";# %pfam_hash is global, imported from marfil_homology
unlink($sequence_data_filename,$sequence_prot_filename,$sequence_dna_filename,$pfam_data_filename);
my $input_order_file = $newDIR."/input_order.txt";
my $dryrun_file = $newDIR."/dryrun.txt";
print "# version $VERSION\n";
print "# results_directory=$newDIR\n";
print "# parameters: MAXEVALUEBLASTSEARCH=$MAXEVALUEBLASTSEARCH MAXPFAMSEQS=$MAXPFAMSEQS BATCHSIZE=$BATCHSIZE MINSEQLENGTH=$MINSEQLENGTH MAXSEQLENGTH=$MAXSEQLENGTH\n";
# 0.3) prepare dryrun file if required
if($runmode eq 'dryrun')
{
open(DRYRUNLOG,">",$dryrun_file);
}
################################################################
## 1) read all input files, identify format and generate input FASTA files in temporary directory
print "\n# checking input files...\n";
$min_proteome_size = -1;
$reference_proteome = $refOK = $n_of_sequences = $n_of_taxa = $n_of_residues = 0;
$previous_files = $current_files = '';
# 1.0) instantiate BerkeleyDB if required
if($saveRAM)
{
eval
{
import DB_File;
tie(@sequence_data,'DB_File',$sequence_data_filename,1,0666,$DB_File::DB_RECNO)
|| die "# EXIT : cannot create $sequence_data_filename: $! (BerkeleyDB::Error)\n";
tie(@sequence_prot,'DB_File',$sequence_prot_filename,1,0666,$DB_File::DB_RECNO)
|| die "# EXIT : cannot create $sequence_prot_filename: $! (BerkeleyDB::Error)\n";
tie(@sequence_dna,'DB_File',$sequence_dna_filename,1,0666,$DB_File::DB_RECNO)
|| die "# EXIT : cannot create $sequence_dna_filename: $! (BerkeleyDB::Error)\n";
if($do_PFAM && !$onlyblast)
{
tie(%pfam_hash,'DB_File',$pfam_data_filename,1,0666,$DB_File::DB_HASH)
|| die "# EXIT : cannot create $pfam_data_filename: $! (BerkeleyDB::Error)\n";
}
}
}
# 1.1) directory with input files
if($inputDIR)
{
# 1.1.1) open and read directory, only master .fasta/.fna files are considered
opendir(DIR,$inputDIR) || die "# EXIT : cannot list $inputDIR\n";
my @inputfiles = sort grep {
/\.fna$/i || /\.fna\.gz$/i || /\.fna.bz2$/i ||
/\.fa$/i || /\.fa\.gz$/i || /\.fa.bz2$/i ||
/\.fasta$/i || /\.fasta\.gz$/i || /\.fasta.bz2$/i ||
/_$/ || /\.clusters$/ # pre-clustered sequences
} readdir(DIR);
closedir(DIR);
# 1.1.2) sort input files and put new files towards the end of @inputfiles: LILO
if(-s $input_order_file)
{
my (@new_order_input_files,%previous_input_file,$n_of_new_infiles);
open(ORDER,$input_order_file) || die "# EXIT : cannot read $input_order_file\n";
while(<ORDER>)
{
chomp;
($order,$infile) = split(/\t/);
if(!-s $inputDIR."/".$infile)
{ die "# EXIT : cannot find previous input file $infile, please re-run everything\n"; }
$previous_input_file{$infile} = 1;
$new_order_input_files[$order] = $infile;
}
close(ORDER);
$n_of_new_infiles=0;
foreach $infile (@inputfiles)
{
next if($previous_input_file{$infile});
$new_order_input_files[++$order] = $infile;
print "# order of new input file $infile = $order\n";
$n_of_new_infiles++;
}
if($n_of_new_infiles){ print "# updating $input_order_file with $n_of_new_infiles new input files\n"; }
open(ORDER,">$input_order_file") || die "# EXIT : cannot write $input_order_file\n";
$order=0;
foreach $infile (@new_order_input_files)
{
print ORDER "$order\t$infile\n";
$order++;
}
close(ORDER);
@inputfiles = @new_order_input_files;
}
else
{
$order=0;
open(ORDER,">$input_order_file") || die "# EXIT : cannot write $input_order_file\n";
foreach $infile (@inputfiles)
{
print ORDER "$order\t$infile\n";
$order++;
}
close(ORDER);
}
# 1.1.4) open CSV protein to organism file, id to name (required by check_BDBHs.pl)
open(COGCSV,">$p2ofilename") || die "# EXIT : cannot write $p2ofilename\n";
# 1.1.5) iteratively parse input FASTA files
foreach $infile (@inputfiles)
{
my ($file_ressize,$fasta_ref,$prot_fasta_ref,@fasta_length) = (0);
($genbankOK,$protOK,$clustersOK,$proteome_size) = (0,0,0,-1);
++$n_of_taxa;
if(($infile =~ /\.clusters$/ || $infile =~ /_$/) && -d $inputDIR.'/'.$infile) # subdirectory with cluster files in FASTA format
{
opendir(CLDIR,$inputDIR.'/'.$infile) || die "# EXIT : cannot list $inputDIR/$infile\n";
my @clusterfiles = sort grep {/\.fna$/} readdir(CLDIR);
closedir(CLDIR);
my ($cluster_id,$cldnaOK) = (0);
my $clseq = $n_of_sequences + 1; # make sure sequences in clusters are correctly numbered
my (@clusters_fasta,@clusters_prot_fasta);
foreach my $clusterfile (@clusterfiles)
{
$cldnaOK = 0;
my $dnafile = $inputDIR.'/'.$infile.'/'.$clusterfile;
$fasta_ref = read_FASTA_file_array($dnafile);
# check whether an identical .fna file exists (expects sequences in same order)
my $protfile = $dnafile;
$protfile =~ s/\.fna$/\.faa/;
if($protfile ne $dnafile && -s $protfile )
{
$prot_fasta_ref = read_FASTA_file_array($protfile);
if($#{$fasta_ref} == $#{$prot_fasta_ref}){ $cldnaOK = 1 }
else
{
print "WARNING: cannot use protein sequences in file $protfile\n".
"# as they do not match those in file $dnafile\n";
}
}
# arbitrarily choose first cluster sequence as representative
$cluster_id = $clseq;
# add sequentially members of pre-processed cluster
foreach $seq ( 0 .. $#{$fasta_ref} )
{
# remember members of this cluster
push(@{$cluster_ids{$cluster_id}},$clseq) if($clseq != $cluster_id);
$clusters_fasta[$clseq] = $fasta_ref->[$seq]; #print "$clseq\n";
if($cldnaOK)
{
$clusters_prot_fasta[$clseq] = $prot_fasta_ref->[$seq];
}
$clseq++;
}
}
$fasta_ref = \@clusters_fasta;
$prot_fasta_ref = \@clusters_prot_fasta;
if($#{$fasta_ref} == $#{$prot_fasta_ref}){ $protOK = 1 }
$clustersOK = scalar(@clusterfiles);
$total_clustersOK++;
if($do_genome_composition)
{
die "\n# WARNING: -c genome composition analyses cannot be done with pre-clustered input sequences ".
"(please check the manual)\n\n";
}
}
else # fasta files
{
my $dnafile = $inputDIR."/".$infile;
$fasta_ref = read_FASTA_file_array($dnafile);
# check whether an identical .faa file exists (expects sequences in same order)
my $protfile = $dnafile;
$protfile =~ s/\.fna$/\.faa/;
$protfile =~ s/\.fna.gz$/\.faa.gz/;
$protfile =~ s/\.fna.bz2$/\.faa.bz2/; #print "$dnafile\n$protfile\n";
if($dnafile ne $protfile && -s $protfile )
{
$prot_fasta_ref = read_FASTA_file_array($protfile);
if($#{$fasta_ref} == $#{$prot_fasta_ref}){ $protOK = 1 }
else
{
print "WARNING: cannot use protein sequences in file $protfile\n".
"# as they do not match those in file $dnafile\n";
}
}
}
if(!defined($fasta_ref)){ die "# ERROR: could not extract nucleotide sequences from file $inputDIR/$infile\n"; }
$proteome_size = scalar(@$fasta_ref);
$psize{$infile} = $proteome_size;
# print files that will be parsed
if($clustersOK)
{
if($protOK){ print "# $infile $proteome_size ($clustersOK clusters, found twin .faa file) "; }
else{ print "# $infile $proteome_size ($clustersOK clusters) "; }
}
elsif($protOK){ print "# $infile $proteome_size (found twin .faa file) "; }
else
{
print "# $infile $proteome_size ";
if($do_PFAM)
{
die "\n# WARNING: -D Pfam domain scans cannot be performed without input protein sequences ".
"(please check the manual)\n\n";
}
}
$new_infile = basename($infile) . ".nucl.$FASTAEXTENSION";
$p2oinfile = basename($infile);
if($infile =~ /$FULLENGTHFLAG/)
{
print "[full length sequences]";
$full_length_file{basename($infile).'.nucl'} = 1;
}
# calculate median sequence length and warn if too long sequence are found
my ($total_long_seqs,$len) = (0,0);
foreach $seq ( 0 .. $#{$fasta_ref} )
{
$len = length($fasta_ref->[$seq][SEQ]);
push(@fasta_length,$len);
if($len > $MAXSEQLENGTH){ $total_long_seqs++ }
}
@fasta_length = sort {$a<=>$b} @fasta_length;
printf(" median length = %d",$fasta_length[int($#fasta_length/2)]);
if($total_long_seqs > 0)
{
printf(" WARNING: %d seqs > $MAXSEQLENGTH b",$total_long_seqs)
}
print "\n";
if($protOK)
{
$prot_new_infile = $new_infile;
$prot_new_infile =~ s/nucl/amino/;
open(INPUTFAA,">$newDIR/$prot_new_infile") || die "# EXIT : cannot create $newDIR/$prot_new_infile\n";
}
open(INPUT,">$newDIR/$new_infile") || die "# EXIT : cannot create $newDIR/$new_infile\n";
# sort sequences by nucleotide length (longest to shortest) to support isoform clustering
my @length_sorted_seqs =
map { $_->[0],$_->[1] }
sort { $b->[1]<=>$a->[1] }
map { [ $_ , length($fasta_ref->[$_][SEQ]) ] } (0 .. $#{$fasta_ref});
while( ($seq,$seqL) = splice(@length_sorted_seqs,0,2) ) #foreach $seq ( 0 .. $#{$fasta_ref})
{
if(!$fasta_ref->[$seq][SEQ] || $fasta_ref->[$seq][SEQ] eq '')
{
chomp $fasta_ref->[$seq][NAME];
print "# skipped void sequence ($fasta_ref->[$seq][NAME])\n" if($fasta_ref->[$seq][NAME]);
next;
}
if($seqL < $MINSEQLENGTH)
{
print "# skipped short sequence ($fasta_ref->[$seq][NAME] , $seqL)\n";
next;
}
elsif($seqL > $MAXSEQLENGTH)
{
print "# skipped long sequence ($fasta_ref->[$seq][NAME] , $seqL)\n";
next;
}
# skip amino sequences in supposed-to-be DNA files
if($fasta_ref->[$seq][SEQ] =~ /^[^ACGTXN\-\s]+$/i)
{
print "# skipped protein sequence ($fasta_ref->[$seq][NAME])\n";
next;
}
# @sequence_... global arrays take large amounts of RAM
# first sequence is [1]
++$n_of_sequences;
$file_ressize += $seqL; #$file_ressize += length($fasta_ref->[$seq][SEQ]);
if(!$onlyblast)
{
# save simplified FASTA header
#$sequence_data[$n_of_sequences] = "$fasta_ref->[$seq][NAME] |[$p2oinfile]";
$sequence_data[$n_of_sequences] = (split(/\s+/,$fasta_ref->[$seq][NAME]))[0] ." [$p2oinfile]";
$sequence_dna[$n_of_sequences] = $fasta_ref->[$seq][SEQ];
if($fasta_ref->[$seq][SEQ] eq '')
{
printf("# cannot parse nucleotide sequence of %s... (%s)\n",
substr($fasta_ref->[$seq][NAME],0,15),$infile);
}
if($protOK)
{
$sequence_prot[$n_of_sequences] = $prot_fasta_ref->[$seq][SEQ];
if($prot_fasta_ref->[$seq][SEQ] eq '')
{
printf("# cannot parse protein sequence of %s... (%s)\n",
substr($fasta_ref->[$seq][NAME],0,15),$infile);
}
}
}
# do not print redundant pre-clustered sequences, take only 1/cluster
next if($clustersOK && !$cluster_ids{$seq});
print INPUT ">$n_of_sequences\n$fasta_ref->[$seq][SEQ]\n";
if($protOK){ print INPUTFAA ">$n_of_sequences\n$prot_fasta_ref->[$seq][SEQ]\n"; }
$pname = $fasta_ref->[$seq][NAME];
print COGCSV "$n_of_sequences,$p2oinfile,$pname\n";
}
close(INPUT);
close(INPUTFAA);
$comma_input_files .= "$new_infile,";
push(@newfiles,$new_infile);
$n_of_residues += $file_ressize;
$ressize{$infile} = $ressize{$new_infile} = $file_ressize;
}
close(COGCSV);
if($min_cluster_size eq 'all'){ $min_cluster_size = $n_of_taxa; } # size of clusters in section 4
}
# 1.2.5) read taxa labels and concatenate FASTA files ($all_fa_file is put in $newDIR/tmp, global @taxa is filled in here)
chop($comma_input_files);
if($inputDIR){ %seq_length = %{ constructAllFasta($comma_input_files,$n_of_sequences) };}
else{ %seq_length = %{ constructAllFasta($comma_input_files,$n_of_sequences,$all_fa_file) }; }
# 1.4) correct list of included files, must be done after reading them all for correct numbering
if($include_file)
{
my ($included,$includedfull,$n_of_matched_included,@Itaxa);
open(INCL,$include_file) || die "# EXIT : cannot read $include_file\n";
while(<INCL>)
{
next if(/^#/ || /^$/);
$included_input_files{(split)[0]} = $.;
}
close(INCL);
print "# included input files (".scalar(keys(%included_input_files))."):\n";
$refOK = $n_of_sequences = $n_of_residues = $n_of_matched_included = 0;
TAXON: foreach $included (sort {$included_input_files{$a}<=>$included_input_files{$b}}
keys(%included_input_files))
{
$includedfull = "$included.nucl";
foreach $taxon (@taxa)
{
if($taxon =~ /^$includedfull$/) #if($includedfull =~ /$taxon/)
{
push(@Itaxa,$taxon);
$n_of_sequences += $psize{$included};
$n_of_residues += $ressize{$included};
print ": $taxon $included $psize{$included}\n";
$n_of_matched_included++;
next TAXON;
}
}
}
print "\n";
if($n_of_matched_included < scalar(keys(%included_input_files)))
{
die "# EXIT : failed to match taxa included in $include_file ($n_of_matched_included), ".
"please make sure their names match those of input files\n";
}
# update @taxa, $min_cluster_size
@taxa = @Itaxa;
$n_of_taxa = scalar(@Itaxa);
if($min_cluster_size eq 'all'){ $min_cluster_size = $n_of_taxa; }
elsif($min_cluster_size > $n_of_taxa){ $min_cluster_size = $n_of_taxa; }
}
printf("# taxa considered = %d sequences = %d residues = %d\n\n",$n_of_taxa,$n_of_sequences,$n_of_residues);
if($n_of_taxa<2){ die "# EXIT: need at least two taxa to make clusters\n" }
#my $RAM = estimate_RAM($n_of_sequences); # not benchmarked for ESTs
#printf("# estimated memory requirements: %s\n\n",$RAM) if(!$onlyblast && $RAM);
# 1.5) set reference proteome index and mask (by default, select proteome with smallest number of sequences)
# it is necessary for BDBH in particular
for($taxon=0;$taxon<scalar(@taxa);$taxon++)
{
$proteome_size = $psize{$taxa[$taxon]};
# update minimal proteome size
if((defined($min_proteome_size) && $min_proteome_size== -1) ||
(defined($min_proteome_size) && defined($proteome_size) && $proteome_size < $min_proteome_size))
{
$min_proteome_size = $proteome_size;
$smallest_proteome_name = $taxa[$taxon];
$smallest_proteome = $taxon;
}
# check user-defined reference proteome
if($reference_proteome_string ne '0' && !$refOK && $taxa[$taxon] =~ /$reference_proteome_string/)
{
$reference_proteome = $taxon;
$reference_proteome_name = $taxa[$taxon];
$refOK=1;
}
}
if(!$refOK && $reference_proteome_string ne '0')
{
print "# WARNING: cannot find reference proteome ($reference_proteome_string), taking default\n";
}
if($reference_proteome_string eq '' || !$refOK)
{
$reference_proteome_name = $smallest_proteome_name;
$reference_proteome = $smallest_proteome;
}
$reference_proteome_name = (split(/\./,$reference_proteome_name))[0];
$reference_proteome_name =~ s/[\s+|_]//g;
$output_mask = $reference_proteome_name."\_" . $output_mask;
print "# mask=$output_mask ($pancore_mask)\n" if(!$onlyblast);
# 1.6) check previously processed input files to decide whether blast parsing
# is needed again and update $selected_genomes_file
if($do_minimal_BDBHs){ $minlog = "minimal_BDBHs$reference_proteome" } # reference is crucial for -b runs
else{ $minlog = 'minimal_BDBHsNO' }
$current_files = join('',sort(@taxa,$minlog));
if(-s $selected_genomes_file)
{
$previous_files = get_string_with_previous_genomes($selected_genomes_file);
if($current_files eq $previous_files)
{
print "\n# skipped transcriptome parsing (".short_path($selected_genomes_file,$pwd).")\n\n";
} #else{ print "$current_files ne $previous_files\n" }
}
open(SEL,">$selected_genomes_file") || die "# cannot create $selected_genomes_file\n";
foreach $taxon (@taxa){ print SEL "$taxon\n"; }
print SEL "$minlog\n"; #
close(SEL);
# 1.7) run Pfam searches if requested (recommended only in cluster mode or with -n otherwise it is painfully sluggish)
if($do_PFAM)
{
my ($pfamout,$hmmerout,$total_seqs_group,$group,$group_fasta_file,$group_clusterfile,$command,@group_pfamfiles);
my ($group_pfamfile,$totalseqs,%cluster_PIDs,%pfamgroups);
print "\n# submitting Pfam HMMER jobs ... \n"; #(MAXPFAMSEQS=$MAXPFAMSEQS) ...\n";
foreach $new_infile (@newfiles)
{
$prot_new_infile = $new_infile;
$prot_new_infile =~ s/nucl/amino/;
next if(-s $newDIR ."/_". $prot_new_infile .".pfam"); # comprueba que no exista ya
$infile = (split(/\.amino/,$prot_new_infile))[0];
next if($include_file && !$included_input_files{$infile});
# split Pfam searches in groups of $MAXPFAMSEQS searches
my $fasta_ref = read_FASTA_file_array($newDIR ."/". $prot_new_infile); # minimize RAM usage, file by file
$totalseqs = scalar(@$fasta_ref);
$total_seqs_group = $group = 0;
$group_fasta_file = $newDIR . "/_". $prot_new_infile . $group;
$group_pfamfile = $newDIR . "/_". $prot_new_infile . $group . '.pfam';
$group_clusterfile = $newDIR . "/_". $prot_new_infile . $group . '.queue';
push(@group_pfamfiles,$group_fasta_file,$group_pfamfile,$group_clusterfile);
open(FGR,">$group_fasta_file") || die "# EXIT : cannot create fasta group file $group_fasta_file : $!\n";
foreach $seq (0 .. $#{$fasta_ref})
{
$total_seqs_group++;
print FGR ">$fasta_ref->[$seq][NAME]\n$fasta_ref->[$seq][SEQ]\n";
if($seq+1 == $totalseqs || ($MAXPFAMSEQS && $total_seqs_group == $MAXPFAMSEQS))
{
close(FGR);
if(!-s $group_pfamfile) # scan Pfam only if necessary
{
$command = format_HMMPFAM_command()." $group_fasta_file > $group_pfamfile";
if($runmode eq 'cluster')
{
submit_cluster_job($prot_new_infile.$group,$command,$group_clusterfile,$newDIR,\%cluster_PIDs);
}
elsif($runmode eq 'dryrun')
{
print DRYRUNLOG "$command\n";
$total_dry++;
}
else # 'local' runmode
{
$command = format_SPLITHMMPFAM_command()."$BATCHSIZE $command ";
system("$command");
if($? != 0)
{
die "# EXIT: failed while running local Pfam search ($command)\n";
}
}
}
$pfamgroups{$prot_new_infile}{$group} = $group_pfamfile;
if($seq < $totalseqs) # take care of remaining sequences/groups
{