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get_homologues.pl
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get_homologues.pl
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#!/usr/bin/env perl
# 2013-2024 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 BLAST (and DIAMOND/HMMER/Pfam) to define clusters of 'orthologous' ORF/intergenic
# sequences and pan/core-genome gene sets. Several algorithms are available and search parameters
# are customizable. It is designed to process (in a HPC computer cluster) files contained in a
# directory (-d), so that new .faa/.gbk files can be added while conserving previous BLAST 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 = 100;
## global variables that control some algorithmic choices
my $NOFSAMPLESREPORT = 10; # 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 $MININTERGENESIZE = 200; # minimum length (nts) required for intergenic segments to be considered
my $MAXINTERGENESIZE = 700;
my $INTERGENEFLANKORF = 180; # length in nts of intergene flanks borrowed from neighbor ORFs (/3 for amino acids)
my $PRINTCLUSTERSSCREEN = 0; # whether cluster names should be printed to screen
my $KEEPSCNDHSPS = 1; # by default only the first BLAST hsp is kept for further calculations
my $COMPRESSBLAST = 1; # set to 0 if gzip is not available
my $DISSABLEPRND = 1; # dissable paranoid (PRND, -P) options due to no redistribution license;
## list of features/binaries required by this program (do not edit)
my @FEATURES2CHECK = ('EXE_BLASTP','EXE_BLASTN','EXE_FORMATDB','EXE_MCL','EXE_MAKEHASH',
'EXE_READBLAST','EXE_COGLSE','EXE_COGTRI','EXE_HMMPFAM','EXE_DMNDP','EXE_DMNFT',
'EXE_INPARA','EXE_ORTHO','EXE_HOMOL','EXE_SPLITBLAST','EXE_SPLITHMMPFAM');
if(!$DISSABLEPRND){ push(@FEATURES2CHECK,'EXE_MULTIPARANOID'); }
################################################################
my ($newDIR,$output_mask,$pancore_mask,$include_file,$onlyblast,%included_input_files,%opts) = ('','','',0,0);
my ($exclude_inparalogues,$doMCL,$doPARANOID,$doCOG,$do_PFAM,$reference_proteome_string) = (0,0,0,0,0,0);
my ($inputDIR,$input_FASTA_file,$filter_by_length,$cluster_list_file,$do_intergenic,$do_features) = (0,0);
my ($n_of_cpus,$COGmulticluster,$do_minimal_BDBHs,$do_soft) = ($BLAST_NOCPU,0,0,0);
my ($do_ANIb_matrix,$do_POCP_matrix,$do_diamond) = (0,0,0);
my ($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, # see marfil_homology.pm
$PERCENT_IDENTITY_CUTOFF_DEFAULT,$PERCENT_MATCH_CUTOFF_DEFAULT);
my ($MCLinflation,$neighbor_corr_cutoff) = ($MCL_INFLATION_DEFAULT,$MIN_NEIGHBOR_CORR_DEFAULT);
my ($bitscore_cutoff,$segment_cover_cutoff) = ($MIN_BITSCORE_DEFAULT,$MIN_SEGMENT_COVER_DEFAULT);
my $random_number_generator_seed = 0;
my $pwd = getcwd(); $pwd .= '/';
my $start_time = new Benchmark();
getopts('hvbcesgPADGMpXoxza:f:n:m:d:r:t:i:I:E:S:C:F:N:B:O:R:', \%opts);
if(($opts{'h'})||(scalar(keys(%opts))==0))
{
print "\nusage: $0 [options]\n\n";
print "-h this message\n";
print "-v print version, credits and checks installation\n";
print "-d directory with input FASTA files ( .faa / .fna ), (overrides -i,\n";
print " GenBank files ( .gbk ), 1 per genome, or a subdirectory use of pre-clustered sequences\n".
" ( subdir.clusters / subdir_ ) with pre-clustered sequences ignores -c, -g)\n".
" ( .faa / .fna ); allows for new files to be added later; \n".
" creates output folder named 'directory_homologues'\n";
print "-i input amino acid FASTA file with [taxon names] in headers, ".
"(required unless -d is set)\n".
" creates output folder named 'file_homologues'\n";
print "\nOptional parameters:\n";
print "-o only run BLAST/Pfam searches and exit ".
"(useful to pre-compute searches)\n";
print "-c report genome composition analysis ".
"(follows order in -I file if enforced,\n".
" ".
" ignores -r,-t,-e)\n";
print "-R set random seed for genome composition analysis ".
"(optional, requires -c, example -R 1234,\n".
" ".
" required for mixing -c with -c -a runs)\n";
if(eval{ require DB_File } ) # show only if DB_File is available (it should as it is core)
{
print "-s save memory by using BerkeleyDB; default parsing stores\n".
" sequence hits in RAM\n";
}
print "-m runmode [local|cluster|dryrun] ".
"(default local)\n";
print "-n nb of threads for BLAST/HMMER/MCL in 'local' runmode (default=$n_of_cpus)\n";
print "-I file with .faa/.gbk files in -d to be included ".
"(takes all by default, requires -d)\n";
print "\nAlgorithms instead of default bidirectional best-hits (BDBH):\n";
print "-G use COGtriangle algorithm (COGS, PubMed=20439257) ".
"(requires 3+ genomes|taxa)\n";
print "-M use orthoMCL algorithm (OMCL, PubMed=12952885)\n";
print "-p use PARANOID algorithm (PRND, PubMed=16873526)\n" if(!$DISSABLEPRND);
print "\nOptions that control sequence similarity searches:\n";
print "-X use diamond instead of blastp (optional, set threads with -n)\n";
print "-C min \%coverage in BLAST pairwise alignments ";
print "(range [1-100],default=$PERCENT_MATCH_CUTOFF_DEFAULT)\n";
print "-E max E-value ".
"(default=$BLAST_PVALUE_CUTOFF_DEFAULT,max=$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=$PERCENT_IDENTITY_CUTOFF_DEFAULT [BDBH|OMCL])\n";
print "-N min BLAST neighborhood correlation PubMed=18475320 ".
"(range [0,1],default=$MIN_NEIGHBOR_CORR_DEFAULT [BDBH|OMCL])\n";
print "-b compile core-genome 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|COGS])\n";
print "-a report clusters of sequence features in GenBank files ".
"(requires -d and .gbk files,\n";
print " instead of default 'CDS' GenBank features ".
" example -a 'tRNA,rRNA',\n".
" ".
" NOTE: uses blastn instead of blastp,\n".
" ".
" ignores -g,-D)\n";
print "-g report clusters of intergenic sequences flanked by ORFs ".
"(requires -d and .gbk files)\n";
print " in addition to default 'CDS' clusters\n";
print "-f filter by \%length difference within clusters ".
"(range [1-100], by default sequence\n".
" ".
" length is not checked)\n";
print "-r reference proteome .faa/.gbk 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 "-x allow sequences in multiple COG clusters ".
"(by default sequences are allocated\n".
" ".
" to single clusters [COGS])\n";
print "-F orthoMCL inflation value ".
"(range [1-5], default=$MCL_INFLATION_DEFAULT [OMCL])\n";
print "-B min bit-score (in bits) ".
"(default=$MIN_BITSCORE_DEFAULT [PRND])\n" if(!$DISSABLEPRND);
print "-A calculate average identity of clustered sequences, ".
"(optional, creates tab-separated matrix,\n";
print " by default uses blastp results but can use blastn with -a ".
" recommended with -t 0 [OMCL|COGS])\n";
print "-P calculate percentage of conserved proteins (POCP), ".
"(optional, creates tab-separated matrix,\n";
print " by default uses blastp results but can use blastn with -a ".
" recommended with -t 0 [OMCL|COGS])\n";
print "-z add soft-core to genome composition analysis ".
"(optional, requires -c [OMCL|COGS])\n";
print "\n".
" This program uses BLAST (and optionally HMMER/Pfam) to define clusters of 'orthologous'\n".
" genomic sequences and pan/core-genome gene sets. Several algorithms are available\n".
" and search parameters are customizable. It is designed to process (in a HPC computer\n".
" cluster) files contained in a directory (-d), so that new .faa/.gbk files can be added\n".
" while conserving previous BLAST results. In general the program will try to re-use\n".
" previous results when run with the same input directory.\n\n";
exit;
}
# read version bumber from CHANGES.txt
open(CHANGES,"$Bin/CHANGES.txt");
while(<CHANGES>)
{
if(eof && /^(\d+):/){ $VERSION = $1 }
}
close(CHANGES);
if(defined($opts{'v'}))
{
print "\n$0 version $VERSION\n";
print "\nProgram written by Bruno Contreras-Moreira (1) and Pablo Vinuesa (2).\n";
print "\n 1: http://www.eead.csic.es/compbio (Estacion Experimental Aula Dei/CSIC/Fundacion ARAID, Spain)\n";
print " 2: http://www.ccg.unam.mx/~vinuesa (Center for Genomic Sciences, UNAM, Mexico)\n";
print "\nPrimary citations:\n\n";
print " Contreras-Moreira B, Vinuesa P (2013) GET_HOMOLOGUES, a versatile software package for scalable and\n".
" robust microbial pangenome analysis. Appl Environ Microbiol 79(24):7696-701. (PubMed:24096415)\n\n";
print " Vinuesa P and Contreras-Moreira B (2015) Robust Identification of Orthologues and Paralogues for \n".
" Microbial Pan-Genomics Using GET_HOMOLOGUES: A Case Study of pIncA/C Plasmids. In Bacterial Pangenomics,\n".
" Methods in Molecular Biology Volume 1231, 203-232, edited by A Mengoni, M Galardini and M Fondi.\n";
print "\nThis software employs code, binaries and data from different authors, please cite them accordingly:\n";
print " OrthoMCL v1.4 (www.orthomcl.org , PubMed:12952885)\n";
print " INPARANOID v3.0 (inparanoid.sbc.su.se , PubMed:16873526)\n" if(!$DISSABLEPRND);
print " COGtriangles v2.1 (sourceforge.net/projects/cogtriangles , PubMed=20439257)\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";
print " Diamond v0.8.25 (https://github.com/bbuchfink/diamond, PubMed=25402007)\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)."_homologues";
}
elsif(defined($opts{'i'}))
{
$input_FASTA_file = $opts{'i'};
die "# EXIT : need a valid input FASTA file\n" if(!-e $input_FASTA_file);
my $root = basename($input_FASTA_file);
if($root =~ /([\+])/)
{
die "# EXIT : need a valid input FASTA file, offending char: '$1'\n";
}
while($root =~ /\./){ $root = substr($root,0,-1) }
$newDIR = $pwd.$root."_homologues";
}
else{ die "# EXIT : need a -d directory with .faa/.gbk files or a -i .faa file as input\n"; }
#if($input_FASTA_file){ $runmode = 'local'; }
if(defined($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'})){ $onlyblast = 1; }
if($opts{'r'}){ $reference_proteome_string = $opts{'r'}; }
else{ $reference_proteome_string = 0; }
check_installed_features(@FEATURES2CHECK);
if(defined($opts{'X'}))
{
if(feature_is_installed('DIAMOND'))
{
$do_diamond = 1;
$output_mask .= "dmd_";
$pancore_mask .= "_dmd";
}
else{ warn_missing_soft('DIAMOND') }
}
if(defined($opts{'f'}) && $opts{'f'} > 0 && $opts{'f'} <= 100)
{
$filter_by_length = $opts{'f'};
$output_mask .= "f$filter_by_length\_";
}
else{ $filter_by_length = 0; $output_mask .= "f0_"; }
if(defined($opts{'t'}) && $opts{'t'} >= 0)
{
$min_cluster_size = $opts{'t'};
$output_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);
}
if($opts{'a'} && defined($opts{'d'}))
{
if($do_diamond)
{
die "# EXIT : cannot run diamond (-X) with DNA features (-a)\n";
}
$do_features = $opts{'a'};
$do_features =~ s/\s+//g;
my $features = $do_features;
$features =~ s/,//g;
$output_mask .= "$features\_";
$pancore_mask .= "_$features";
rename_blast_outfiles('features');
}
else{ $do_features = 0 }
if(defined($opts{'A'})){ $do_ANIb_matrix = 1 }
if(defined($opts{'P'})){ $do_POCP_matrix = 1 }
if(defined($opts{'g'}) && defined($opts{'d'}) && !$do_features)
{
if($min_cluster_size ne 'all')
{
die "\n# WARNING: intergenic sequences are extracted between syntenic core clusters, ".
"please remove option -t $min_cluster_size\n\n";
}
$do_intergenic = 1;
}
else{ $do_intergenic = 0 }
if(defined($opts{'M'}))
{
if(feature_is_installed('OMCL'))
{
$doMCL = 1;
$output_mask .= "algOMCL_";
$pancore_mask .= "_algOMCL";
}
else{ warn_missing_soft('OMCL') }
}
elsif(defined($opts{'p'}) && !$DISSABLEPRND)
{
if(feature_is_installed('PRND'))
{
$doPARANOID = 1;
$output_mask .= "algPRND_";
$pancore_mask .= "_algPRND";
}
else{ warn_missing_soft('PRND') }
}
elsif(defined($opts{'G'})) # && $inputDIR ne '')
{
if(feature_is_installed('COGS'))
{
$doCOG = 1;
if(defined($opts{'x'}))
{
$COGmulticluster = 1;
$output_mask .= "algCOGx_";
$pancore_mask .= "_algCOGx";
}
else
{
$COGmulticluster = 0;
$output_mask .= "algCOG_";
$pancore_mask .= "_algCOG";
}
}
else{ warn_missing_soft('COGS') }
}
else
{
if(defined($opts{'b'}))
{
if($do_diamond)
{
die "# EXIT : cannot run diamond (-X) with -b option\n";
}
$do_minimal_BDBHs = 1;
$output_mask .= "algBDBHmin_";
$pancore_mask .= "_algBDBHmin";
}
else
{
$output_mask .= "algBDBH_";
$pancore_mask .= "_algBDBH";
}
#if(defined($opts{'D'}) && !$do_features){}
if($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($opts{'D'}) && !$do_features)
{
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 || $doCOG))
{
$do_soft = 1;
}
if($opts{'R'})
{
$random_number_generator_seed = $opts{'R'};
}
elsif($do_features)
{
die "\n# WARNING: use of -c and -a options usually requires setting -R ".
"(please check the manual)\n\n";
}
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 }
$segment_cover_cutoff = $pmatch_cutoff; # COGS|PRND
$output_mask .= "C$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";
}
if(defined($opts{'N'}))
{
$neighbor_corr_cutoff = $opts{'N'};
if($neighbor_corr_cutoff < 0){ $neighbor_corr_cutoff = 0 }
elsif($neighbor_corr_cutoff > 1){ $neighbor_corr_cutoff = 1 }
$output_mask .= "N$neighbor_corr_cutoff\_"; $pancore_mask .= "_N$neighbor_corr_cutoff";
}
if(defined($opts{'B'}) && !$DISSABLEPRND)
{
$bitscore_cutoff = $opts{'B'};
if($bitscore_cutoff < 0){ $bitscore_cutoff = 0 }
$output_mask .= "B$bitscore_cutoff\_"; $pancore_mask .= "_B$bitscore_cutoff";
}
print "# $0 -i $input_FASTA_file -d $inputDIR -o $onlyblast -X $do_diamond -e $exclude_inparalogues -f $filter_by_length -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 -G $doCOG -p $doPARANOID ".
"-C $pmatch_cutoff -S $pi_cutoff -E $evalue_cutoff -F $MCLinflation -N $neighbor_corr_cutoff -B $bitscore_cutoff -b $do_minimal_BDBHs ".
"-s $saveRAM -D $do_PFAM -g $do_intergenic -a '$do_features' -x $COGmulticluster -R $random_number_generator_seed -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,$total,$total_blast,$p2oinfile,$seq,$comma_input_files,$n_of_residues);
my ($label,%orthologues,$gene,$orth,$orth2,$para,%inparalogues,%paralogues,$FASTAresultsDIR,$order,$minlog);
my ($n_of_similar_length_orthologues,$clusterfile,$dna_clusterfile,$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);
my ($pname,$n_of_sequences,$refOK,$genbankOK,$cluster_size,$dna_cluster_size,$dna_cluster,$prot_cluster);
my (%orth_registry,%LSE_registry,$LSE_reference,$LSE_t,$redo_inp,$redo_orth,%idclusters,%names,$generef);
my ($reparse_all,$total_genbankOK,$n_of_similar_length_paralogues,$pfam_annot,$dnaOK,$n_of_taxa_cluster) = (0,0);
my ($BDBHdone,$PARANOIDdone,$orthoMCLdone,$COGdone,$n_of_parsed_lines,$n_of_pfam_parsed_lines) = (0,0,0,0,0,0);
my ($diff_BDBH_params,$diff_INP_params,$diff_HOM_params,$diff_OMCL_params,$lockcapableFS,$total_dry) = (0,0,0,0,0,0);
my ($total_clustersOK,$clgene,$clorth,%ANIb_matrix,%POCP_matrix,%GIclusters,$clustersOK,%cluster_ids) = (0);
my (@sorted_clusters,%taxa_clusters,%orth_taxa,@newfiles,%ressize,%intergenic_FNA_files);
constructDirectory($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 $diamond_file = $newDIR."/diamond.txt";
my $dryrun_file = $newDIR."/dryrun.txt";
my $paranoidDIR = $newDIR."/paranoid_sql_C$pmatch_cutoff\_B$bitscore_cutoff\_O$segment_cover_cutoff/";
print "# version $VERSION\n";
print "# results_directory=$newDIR\n";
print "# parameters: MAXEVALUEBLASTSEARCH=$MAXEVALUEBLASTSEARCH MAXPFAMSEQS=$MAXPFAMSEQS ".
"BATCHSIZE=$BATCHSIZE KEEPSCNDHSPS=$KEEPSCNDHSPS\n";
# 0.3) make sure that DIAMOND jobs are not mixed with BLASTP jobs
if(-s $diamond_file)
{
open(DMDFILE,$diamond_file) || die "# EXIT : cannot read $diamond_file\n";
while(<DMDFILE>)
{
if(/^diamond job:(\d)/)
{
if($1 != $do_diamond)
{
$do_diamond = $1;
print "# diamond job:$do_diamond (forced to match previous jobs)\n";
}
else{ print "# diamond job:$do_diamond\n" }
}
}
close(DMDFILE);
}
else
{
if(-s $input_order_file)
{
# assume this was a BLASTP job to be sure
if($do_diamond == 1)
{
$do_diamond = 0;
print "# diamond job:$do_diamond (forced to match previous jobs)\n";
}
else{ print "# diamond job:$do_diamond\n" }
}
else{ print "# diamond job:$do_diamond\n" }
open(DMDFILE,'>',$diamond_file) || die "# EXIT : cannot create $diamond_file\n";
print DMDFILE "diamond job:$do_diamond\n";
close(DMDFILE);
}
# 0.4) 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 .fa/.gb files are considered
opendir(DIR,$inputDIR) || die "# EXIT : cannot list $inputDIR\n";
my @inputfiles = sort grep {/\.fa[a-z]*\S*$/i||/\.gb\S*$/||/_$/||/\.clusters$/} 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";
$order++;
}
close(ORDER);
}
# 1.1.3) check if all input files have GenBank format (-a/-g options)
if($do_intergenic || $do_features)
{
foreach $infile (@inputfiles)
{
if($infile !~ /\.gb\S*$/i)
{
die "\n# EXIT : cannot analyze feature/intergenic sequences when input file is not in GenBank format\n\n";
}
}
}
# 1.1.4) open CSV protein to organism file, id to name (required by COGtriangles)
open(COGCSV,">$p2ofilename") || die "# EXIT : cannot write $p2ofilename\n";
# 1.1.5) iteratively parse input files
foreach $infile (@inputfiles)
{
my ($file_ressize,$intergenic_size,$fasta_ref,$dna_fasta_ref) = (0,0);
($genbankOK,$dnaOK,$clustersOK,$proteome_size) = (0,0,0,-1);
++$n_of_taxa;
# 1.1.5.1) genbank files
if($infile =~ /\.gb\S*$/i)
{
my $prot_tmp_file = $newDIR ."/" .basename($infile) . ".faa";
my $dna_tmp_file = $newDIR ."/" .basename($infile) . ".fna";
my $feature_dna_tmp_file = $newDIR ."/" .basename($infile) . "_features.fna";
my $interg_tmp_file = $newDIR ."/" .basename($infile) .
"_intergenic$MININTERGENESIZE\_$MAXINTERGENESIZE\_$INTERGENEFLANKORF.fna";
if($do_features)
{
if(-s $feature_dna_tmp_file ||
extract_features_from_genbank($inputDIR."/".$infile,
$feature_dna_tmp_file,$do_features) > 0)
{
$fasta_ref = read_FASTA_file_array($feature_dna_tmp_file);
if(!@$fasta_ref)
{
die "# ERROR: could not extract feature sequences from file ".
"$inputDIR/$infile\n";
}
}
else{ die "# ERROR: could not extract feature sequences from file $inputDIR/$infile\n"; }
}
else
{
if(-s $prot_tmp_file ||
extract_CDSs_from_genbank($inputDIR."/".$infile,$prot_tmp_file,$dna_tmp_file) > 0)
{
$fasta_ref = read_FASTA_file_array($prot_tmp_file);
if(-s $dna_tmp_file && @$fasta_ref)
{
$dna_fasta_ref = read_FASTA_file_array($dna_tmp_file);
$genbankOK = 1;
$total_genbankOK++;
}
else
{
print "# WARNING: could not extract nucleotide sequences from file $inputDIR/$infile\n";
}
}
elsif(extract_genes_from_genbank($inputDIR."/".$infile,$prot_tmp_file,$dna_tmp_file) > 0)
{
print "# WARNING: can only extract genes (not CDSs) from file $inputDIR/$infile\n";
# both dna & prot sequences are always generated with extract_genes
$fasta_ref = read_FASTA_file_array($prot_tmp_file);
$dna_fasta_ref = read_FASTA_file_array($dna_tmp_file);
$genbankOK = 1;
$total_genbankOK++;
}
else{ die "# ERROR: could not extract sequences from file $inputDIR/$infile\n"; }
if($do_intergenic)
{
if(!-s $interg_tmp_file)
{
$intergenic_size =
extract_intergenic_from_genbank($inputDIR."/".$infile,
$interg_tmp_file,$MININTERGENESIZE,
$MAXINTERGENESIZE,$INTERGENEFLANKORF);
}
else
{
my $intergfna_ref = read_FASTA_file_array($interg_tmp_file);
$intergenic_size = scalar(@$intergfna_ref);
}
if(!$intergenic_size)
{
print "# WARNING: could not extract intergenic sequences from file $inputDIR/$infile\n";
}
else{ $intergenic_FNA_files{$infile} = $interg_tmp_file }
}
}
}
elsif(($infile =~ /\.clusters$/ || $infile =~ /_$/) && -d $inputDIR.'/'.$infile) # 1.1.5.2) subdirectory with cluster files in FASTA format
{
if($total_clustersOK++)
{
die "\n# WARNING: can only take one folder of pre-clustered sequences\n\n";
}
opendir(CLDIR,$inputDIR.'/'.$infile) || die "# EXIT : cannot list $inputDIR/$infile\n";
my @clusterfiles = sort grep {/\.fa[a-z]*$/i} readdir(CLDIR);
closedir(CLDIR);
my ($cluster_id,$cldnaOK) = (0);
my $clseq = $n_of_sequences + 1; # make sure sequences in clusters are correctly numbered
my (@clusters_fasta,@clusters_dna_fasta);
foreach my $clusterfile (@clusterfiles)
{
$cldnaOK = 0;
my $protfile = $inputDIR.'/'.$infile.'/'.$clusterfile;
$fasta_ref = read_FASTA_file_array($protfile);
# check whether an identical .fna file exists (expects sequences in same order)
my $dnafile = $protfile;
$dnafile =~ s/\.fa+.*?$/\.fna/;
if($dnafile ne $protfile && -s $dnafile )
{
$dna_fasta_ref = read_FASTA_file_array($dnafile);
if($#{$fasta_ref} == $#{$dna_fasta_ref}){ $cldnaOK = 1 }
else
{
print "WARNING: cannot use nucleotide sequences in file $dnafile\n".
"# as they do not match those in file $protfile\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)
{
$clusters_dna_fasta[$clseq] = $dna_fasta_ref->[$seq];
}
$clseq++;
}
}
$fasta_ref = \@clusters_fasta;
$dna_fasta_ref = \@clusters_dna_fasta;
if($#{$fasta_ref} == $#{$dna_fasta_ref}){ $dnaOK = 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 # 1.1.5.3) fasta files
{
my $protfile = $inputDIR."/".$infile;
$fasta_ref = read_FASTA_file_array($protfile);
# check whether an identical .fna file exists (expects sequences in same order)
my $dnafile = $protfile;
$dnafile =~ s/\.fa+.*?$/\.fna/;
if($dnafile ne $protfile && -s $dnafile )
{
$dna_fasta_ref = read_FASTA_file_array($dnafile);
if($#{$fasta_ref} == $#{$dna_fasta_ref}){ $dnaOK = 1 }
else
{
print "WARNING: cannot use nucleotide sequences in file $dnafile\n".
"# as they do not match those in file $protfile\n";
}
}
}
if(!defined($fasta_ref)){ die "# ERROR: could not extract sequences from $inputDIR/$infile\n"; }
$proteome_size = scalar(@$fasta_ref);
$psize{$infile} = $proteome_size;
if($do_intergenic){ print "# $infile $proteome_size (intergenes=$intergenic_size)\n"; }
elsif($clustersOK)
{
if($dnaOK){ print "# $infile $proteome_size ($clustersOK clusters, found twin .fna file)\n"; }
else{ print "# $infile $proteome_size ($clustersOK clusters)\n"; }
}
elsif($dnaOK){ print "# $infile $proteome_size (found twin .fna file)\n"; }
else{ print "# $infile $proteome_size\n"; }
$new_infile = basename($infile) . ".$FASTAEXTENSION";
$p2oinfile = basename($infile);
if($do_features)
{
$new_infile = basename($infile) . "_features.$FASTAEXTENSION";
$p2oinfile = basename($infile) . '_features';
}
open(INPUT,">$newDIR/$new_infile") || die "# EXIT : cannot create $newDIR/$new_infile\n";
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;
}
# skip DNA sequences in supposed-to-be amino acid files
if(!$do_features && $fasta_ref->[$seq][SEQ] =~ /^[ACGTUXN\-\s]+$/i)
{
print "# skipped nucleotide sequence ($fasta_ref->[$seq][NAME])\n";
next;
}
# @sequence_... global arrays take large amounts of RAM
# first sequence is [1]
++$n_of_sequences;
$file_ressize += length($fasta_ref->[$seq][SEQ]);
if(!$onlyblast)
{
$sequence_data[$n_of_sequences] = $fasta_ref->[$seq][NAME];
$sequence_prot[$n_of_sequences] = $fasta_ref->[$seq][SEQ]; # with -a actually stores dna
if($fasta_ref->[$seq][SEQ] eq '')
{
printf("# cannot parse protein sequence of %s... (%s)\n",
substr($fasta_ref->[$seq][NAME],0,15),$infile);
}
if($genbankOK || $dnaOK)
{
$sequence_dna[$n_of_sequences] = $dna_fasta_ref->[$seq][SEQ];
if(!$dna_fasta_ref->[$seq][SEQ] || $dna_fasta_ref->[$seq][SEQ] eq '')
{
printf("# cannot parse DNA 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";
$pname = $fasta_ref->[$seq][NAME];$pname =~ s/#/hash/g; # COG does not like hashes in names
print COGCSV "$n_of_sequences,$p2oinfile,$pname\n";
}
close(INPUT);
$comma_input_files .= "$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
}
else # 1.2) input single FASTA file with [taxon names] in headers, order not checked
{
print "# $input_FASTA_file\n";
# 1.2.0) open CSV protein to organism file required by COGtriangles
open(COGCSV,">$p2ofilename") || die "# EXIT : cannot write $p2ofilename\n";
my $fasta_ref = read_FASTA_file_array($input_FASTA_file);
# allow taxon/genome name to be a single word (1)
my %taxa_in_headers = find_taxa_FASTA_array_headers($fasta_ref,1);
# 1.2.1) print sequences of the same taxon in separate files
foreach $taxon (sort (keys(%taxa_in_headers)))
{
my $taxon_ressize = 0;