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OPERA-MS.pl
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OPERA-MS.pl
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#use strict;
use warnings;
use Cwd;
use Switch;
use File::Which;
use File::Spec;
use File::Spec::Functions qw(rel2abs);
use File::Basename;
#
use Statistics::Basic;
use Statistics::R;
#
use Getopt::Std;
use Getopt::Long;
use Getopt::Long qw(GetOptionsFromArray);
#use Getopt::long::Parser;
my %opera_ms_option = ();
my $opera_ms_full_path = dirname(rel2abs($0)) . "/";
$opera_ms_option{"OPERA_MS_DIR"} = $opera_ms_full_path;
$opera_ms_option{"VERSION"} = "v0.9.0";
my %opera_ms_dependency = ();
require "$opera_ms_full_path/bin/test_time.pl";
#Error message
my $help_line = "To configure OPERA-MS, please look at the example config file inside the OPERA-MS folder.\nUsage: \n\npath/OPERA-MS/OPERA-MS.pl <config_file>\n\n";
my $incorrect_arguments="Please input the correct arguments. Refer to the documentation for more information or use:\n\n path/OPERA-MS/OPERA-MS.pl -h. \n\n";
if ( @ARGV == 0 ){
print "\n" . print_help();exit(0);
}
#To setup and check if the depndency are functionning
check_dependency(\%opera_ms_option, \%opera_ms_dependency, "CHECK_DEPENDENCY") if($ARGV[0] eq "check-dependency");
#To setup the database
setup_opera_ms_database(\%opera_ms_option) if($ARGV[0] eq "install-db");
#read from config file
set_default_value(\%opera_ms_option);
if ( $ARGV[0] ne "--help" && @ARGV <= 2){
read_config_file(\%opera_ms_option, @ARGV);
}
else{
read_argument(\%opera_ms_option,\@ARGV);#Write config file
#read_config_file(\%opera_ms_option, $opera_ms_option{"CONFIG_PATH"});
#die $incorrect_arguments;
}
update_option(\%opera_ms_option);
#Make paths absolute if they are not already.
setup_directory_path(\%opera_ms_option);
#Write the config file used for OPERA-MS-UTILS
write_utils_config_file(\%opera_ms_option);
#Check if dependencies are found. Otherwise, die.
check_dependency(\%opera_ms_option, \%opera_ms_dependency, $opera_ms_option{"STAGE"});
#Check data integrity
check_data(\%opera_ms_option);
#Check if the database contains all the information
if(($opera_ms_option{"REF_CLUSTERING"}) && !check_database(\%opera_ms_option)){
die "OPERA-MS default database not found. You can download the default OPERA-MS-DB using the following command: perl OPERA-MS.pl install-db";
}
#Starting OPERA-MS pipeline
my ($start_time, $end_time);
print STDERR "version\t" . $opera_ms_option{"VERSION"} . "\n";
short_read_assembly(\%opera_ms_option, \%opera_ms_dependency);
assembly_graph_creation(\%opera_ms_option, \%opera_ms_dependency);
if($opera_ms_option{"SINGLE_GENOME"}){#Single genome pipeline
single_genome_assembly(\%opera_ms_option, \%opera_ms_dependency);
}
else{#Normal metagenome pipeleine
hierarchical_clustering(\%opera_ms_option, \%opera_ms_dependency);
reference_clustering(\%opera_ms_option, \%opera_ms_dependency);#Add check for reference clustering
strain_clustering_and_assembly(\%opera_ms_option, \%opera_ms_dependency);
}
gap_filling(\%opera_ms_option, \%opera_ms_dependency);
#
generate_assembly_stats(\%opera_ms_option, \%opera_ms_dependency);
write_final_assembly(\%opera_ms_option, \%opera_ms_dependency);
#
#
polishing(\%opera_ms_option, \%opera_ms_dependency);
$time = localtime;
print "\n[$time]\tOPERA-MS completed\n";# if($STAGE_TO_RUN eq "ALL");
sub generate_sigma_config_file{
my ($dir_cov_estimate, $dir_mapping, $dir_sigma) = @_;
#
#long read sigma config
open(OUT, ">$dir_sigma/sigma.config");
open(FILE, "$dir_cov_estimate/sigma.config");
my $max_edge_set = 5;
my $str_edge = "";
while(<FILE>){
@line = split(/\=/, $_);
#no mapping file
next if($line[0] eq "mapping_files");
#Change the output
if($line[0] eq "output_dir"){
print OUT "output_dir=$dir_sigma\n";
next;
}
#Get the edge file name
if($line[0] eq "edges_files"){
for(my $i = 0; $i <= $max_edge_set; $i++){
$str_edge .= "$dir_sigma/pairedEdges_i$i/pairedEdges_i$i,";
#Creat the directory
run_exe("mkdir $dir_sigma/pairedEdges_i$i");
#Link to the correct file
run_exe("ln -s $dir_mapping/pairedEdges_i$i $dir_sigma/pairedEdges_i$i/pairedEdges_i$i");
#create the lib file
open(LIB, ">$dir_sigma/pairedEdges_i$i/lib.txt");
@val_mean_std = `grep -A2 pairedEdges_i$i $dir_mapping/config | cut -d "=" -f 2`;
#
print LIB "For the mapping file: \n";
print LIB "Mean length of the library is: $val_mean_std[1]";
print LIB "Standard deviation of the library is: $val_mean_std[2]";
print LIB "Orientation of paired-reads is: forward ->...-> 1st read...2nd read\n";
print LIB "Number of used paired reads for estimating the mean and the standard deviation of the library is: NA\n";
print LIB "Percentage of outliers is: NA%";
}
chop $str_edge;
print OUT "edges_files=$str_edge\n";
next
}
print OUT $_;
}
#print OUT "samtools_dir=$samtools_dir\n";
close(OUT);
close(FILE);
}
sub generate_opera_config_file{
my ($dir_mapping, $dir_ref_clustering, $dir_opera_lr, $contig_file) = @_;
#
#long read sigma config
open(FILE, "$dir_opera_lr/../lib_1_bundles/lib_1.ebconfig");
open(OUT, ">$dir_opera_lr/opera.config");
my $max_edge_set = 5;
while(<FILE>){
if($_ eq "[LIB]\n"){
for(my $i = 0; $i <= $max_edge_set; $i++){
#create the lib file
@val_mean_std = `grep -A2 pairedEdges_i$i $dir_mapping/config | cut -d "=" -f 2`;
print STDERR " *** $dir_mapping/config\n";
print OUT "[LIB]\n";
print OUT "map_type=opera\n";
print OUT "map_file=$dir_ref_clustering/NO_REPEAT/filtered_pairedEdges_i$i\n";
print OUT "lib_mean=$val_mean_std[1]\n";
print OUT "lib_std=$val_mean_std[2]\n";
}
next;
}
@line = split(/\=/, $_);
#no mapping file
next if($line[0] eq "mapping_files" || $line[0] eq "map_file" || $line[0] eq "filter_repeat");
if($line[0] eq "output_folder"){
print OUT "output_folder=$dir_opera_lr\n";
next;
}
if($line[0] eq "contig_file"){
print OUT "contig_file=$contig_file\n";
next;
}
if($line[0] eq "cluster_threshold"){
print OUT "cluster_threshold=1\n";
next;
}
print OUT $_;
}
print OUT "keep_repeat=yes\n";
#print OUT "samtools_dir=$samtools_dir\n";
close(FILE);
close(OUT);
}
sub compute_Nx{
my ($x, $length, $ptr_tab) = @_;
my $nb_x_base = $length * ($x / 100);
my $total = 0;
my $i = 0;
for($i = 0; $total < $nb_x_base; $i++){
$total += $ptr_tab->[$i];
}
return $i -1;
}
sub setup_directory_path{
my ($opera_ms_option) = @_;
#Grab the executing directory; turn all paths into absolute paths.
my $main_dir = getcwd;
$main_dir .= "\/";
$opera_ms_option->{"MAIN_DIR"} = $main_dir;
my $output_dir = $opera_ms_option{"OUTPUT_DIR"};
my $inter = "intermediate_files";
#
$output_dir = $main_dir . $output_dir."/" if (substr($output_dir, 0, 1) ne "/");
run_exe("mkdir -p $output_dir/$inter") if(! -d "$output_dir/$inter");
$opera_ms_option->{"OUTPUT_DIR"} = $output_dir;
$opera_ms_option->{"INTER_DIR"} = "$output_dir/$inter";
#Make paths absolute if they are not already.
$opera_ms_option->{"LONG_READ"} = $main_dir . $opera_ms_option->{"LONG_READ"} if(substr($opera_ms_option->{"LONG_READ"}, 0, 1) ne "/");
$opera_ms_option->{"ILLUMINA_READ_1"} = $main_dir . $opera_ms_option->{"ILLUMINA_READ_1"} if(substr($opera_ms_option->{"ILLUMINA_READ_1"}, 0, 1) ne "/");
$opera_ms_option->{"ILLUMINA_READ_2"} = $main_dir . $opera_ms_option->{"ILLUMINA_READ_2"} if(substr($opera_ms_option->{"ILLUMINA_READ_2"}, 0, 1) ne "/");
if(defined $opera_ms_option->{"CONTIGS_FILE"}){
$opera_ms_option->{"CONTIGS_FILE"} = $main_dir . $opera_ms_option->{"CONTIGS_FILE"} if(substr($opera_ms_option->{"CONTIGS_FILE"}, 0, 1) ne "/");
}
}
sub check_data{
my ($opera_ms_option) = @_;
data_exists($opera_ms_option->{"ILLUMINA_READ_1"});
data_exists($opera_ms_option->{"ILLUMINA_READ_2"});
data_exists($opera_ms_option->{"LONG_READ"});
#
if ($opera_ms_option->{"LONG_READ"} =~ /\.gz$/){
die $opera_ms_option->{"LONG_READ"} . " appears to be in gzipped format. The current version of OPERA-MS does not accept compressed long read file, please unzip.\n";
}
}
sub data_exists{
my ($data_file) = @_;
if(! -e $data_file){
die "\nError : $data_file does not exist\n";
}
}
sub check_dependency{
my ($opera_ms_option, $opera_ms_dependency, $stage) = @_;
#
my $opera_ms_dir = $opera_ms_option->{"OPERA_MS_DIR"};
my $utils_dir = "$opera_ms_dir/tools_opera_ms/";
$opera_ms_dependency->{"OPERA_MS_TOOLS"} = $utils_dir;
#
#bundler
#check_software("$opera_ms_dir/bin", "bundler", $sofware_name, $test_command, $opera_ms_dependency) = @_;
#For the perl verion
if($stage eq "CHECK_DEPENDENCY"){
my $perl_path = "$utils_dir/perl";
`rm $perl_path` if(-e "$perl_path");
`ln -s $^X $perl_path`;
}
$opera_ms_dependency->{"perl"} = $utils_dir;
#OPERA-LG
check_software("$opera_ms_dir/OPERA-LG/bin/", "OPERA-LG", "OPERA-LG", $opera_ms_dependency, $stage);
#Check for sigma
check_software("$opera_ms_dir/bin/", "sigma", "sigma help", $opera_ms_dependency, $stage);
#samtools
check_software($utils_dir, "samtools", "samtools 2>&1 | grep Version", $opera_ms_dependency, $stage);
#blasr
check_software($utils_dir, "blasr", "blasr -h", $opera_ms_dependency, $stage);
#bwa
check_software($utils_dir, "bwa", "bwa 2>&1 | grep Version", $opera_ms_dependency, $stage);
#minimap2
check_software($utils_dir, "minimap2", "minimap2 --version", $opera_ms_dependency, $stage);
#mash
check_software($utils_dir, "mash", "mash --version", $opera_ms_dependency, $stage);
#mummer
check_software("$utils_dir/MUMmer3.23/", "mummer", "nucmer --version", $opera_ms_dependency, $stage);
#megahit
check_software("$utils_dir/megahit_v1.0.4-beta_LINUX_CPUONLY_x86_64-bin/", "megahit", "megahit --version", $opera_ms_dependency, $stage);
#spades
check_software("$utils_dir/SPAdes-3.13.0-Linux/bin/", "spades", "spades.py --version", $opera_ms_dependency, $stage);
#Check for racon.
check_software($utils_dir, "racon", "racon 2>&1 | grep options", $opera_ms_dependency, $stage);
#Check for pilon.
check_software($utils_dir, "pilon", "java -jar $utils_dir/pilon.jar --version", $opera_ms_dependency, $stage);
#
#
$opera_ms_dependency->{"kraken"} = "NULL";
if($stage eq "CHECK_DEPENDENCY"){
print STDOUT "\n *** All compiled software are functional.\n";
print STDOUT " *** Please try to run OPERA-MS on the test dataset.\n\n";
exit(0);
}
}
sub check_software{
my ($software_dir, $software_name, $test_command, $opera_ms_dependency, $stage) = @_;
if($stage eq "CHECK_DEPENDENCY"){
$exe_check = "${software_dir}$test_command";
$exe_check = "$test_command" if(index($test_command, "java") != -1);
#print STDOUT "\n" . $exe_check . "\n";
`$exe_check 2> /dev/null > /dev/null`;
if($?){
print STDERR "\n$software_name found in $software_dir is not functional. Checking path for $software_name.\n";
my $valid_path = which("$software_name");
die "$software_name not found in path. Exiting.\n" if (!$valid_path);
print STDERR "\n$software_name found in path.\n";
$valid_path = `which $software_name`;chop $valid_path;
run_exe("mv ${software_dir}/$software_name ${software_dir}/$software_name.bk");
run_exe("ln -s $valid_path $software_dir");
}
print STDOUT " *** $software_name functional\n" if($stage eq "CHECK_DEPENDENCY");
}
$opera_ms_dependency->{$software_name} = $software_dir;
}
sub check_database{
my ($opera_ms_option) = @_;
#
my $opera_ms_db_dir = $opera_ms_option->{"OPERA_MS_DB"};
#print STDERR "$opera_ms_db_dir/genomes_list.txt" . "\n";
return (-e "$opera_ms_db_dir/genomes.msh");
}
sub setup_opera_ms_database{
my ($opera_ms_option) = @_;
my $opera_ms_db_dir = $opera_ms_option->{"OPERA_MS_DIR"}."/OPERA-MS-DB";
$opera_ms_option->{"OPERA_MS_DB_DIR"} = $opera_ms_db_dir;
my $tracking_file = "$opera_ms_db_dir/database_download_completed";
my %downloaded_file = ();
if (-e $tracking_file){
open(FILE, $tracking_file);
while(<FILE>){
chop $_;
$downloaded_file{$_} = 1;
}
close(FILE);
}
my %figshare = (
"genome_1.tar.gz", "https://ndownloader.figshare.com/files/22364238",
"genome_2.tar.gz", "https://ndownloader.figshare.com/files/22378161",
"genome_3.tar.gz", "https://ndownloader.figshare.com/files/22385391",
"genome_4.tar.gz", "https://ndownloader.figshare.com/files/22392252",
"genome_5.tar.gz", "https://ndownloader.figshare.com/files/22405236",
"genome_6.tar.gz", "https://ndownloader.figshare.com/files/22409529",
"genome_7.tar.gz", "https://ndownloader.figshare.com/files/22420533",
"genomes.msh.tar.gz", "https://ndownloader.figshare.com/files/22423779",
"genomes_lists.txt.tar.gz", "https://ndownloader.figshare.com/files/22423785",
"genomes_length.txt.tar.gz", "https://ndownloader.figshare.com/files/22423782"
);
my %dropbox = (
"genomes.msh.tar.gz", "https://www.dropbox.com/s/0zt2h1zp4p53qgh/genomes.msh.tar.gz?dl=0",
"genomes_list.txt.tar.gz", "https://www.dropbox.com/s/e8ozv99fe60w9ny/genomes_list.txt.tar.gz?dl=0",
"genomes_length.txt.tar.gz", "https://www.dropbox.com/s/7xr6w3g4pl9u0vn/genomes_length.txt.tar.gz?dl=0",
"genome_1.tar.gz", "https://www.dropbox.com/s/91dfti1xzdk3760/genomes_1.tar.gz?dl=0",
"genome_2.tar.gz", "https://www.dropbox.com/s/jklrpkvcjm066e6/genomes_2.tar.gz?dl=0",
"genome_3.tar.gz", "https://www.dropbox.com/s/2vmaa6ine92w4fh/genomes_3.tar.gz?dl=0",
"genome_4.tar.gz", "https://www.dropbox.com/s/jvd9uspg5luh7xi/genomes_4.tar.gz?dl=0",
"genome_5.tar.gz", "https://www.dropbox.com/s/tffy3bynnin6rkr/genomes_5.tar.gz?dl=0",
"genome_6.tar.gz", "https://www.dropbox.com/s/71x0myfp7asembr/genomes_6.tar.gz?dl=0",
"genome_7.tar.gz", "https://www.dropbox.com/s/ja4ju0t6x3pxjtf/genomes_7.tar.gz?dl=0"
);
if(! download_db(\%dropbox, $opera_ms_option, \%downloaded_file, $tracking_file)){
print STDERR "\n *** Automatated database download from dropbox failed. \n *** Using figshare, may take > 15h\n";
if(!download_db(\%figshare, $opera_ms_option, \%downloaded_file, $tracking_file)){
die"\nAutomated download of the genome data base failed.\n"
}
}
print STDERR " *** Database download completed\n";
exit(1)
}
sub download_db{
my($db_url, $opera_ms_option, $downloaded_file, $tracking_file) = @_;
my $opera_ms_dir = $opera_ms_option->{"OPERA_MS_DIR"};
my $opera_ms_db_dir = $opera_ms_option->{"OPERA_MS_DB_DIR"};
my $cmp = 1;
my $nb_step = keys(%{$db_url}) * 2;
#Download the genome reference data base for the first utilasation
print STDOUT " *** Set-up of reference genome databases [35Gb disk space required]. Please wait ...\n";
#
foreach $file (keys %{$db_url}){
$url = $db_url->{$file};
$file_path = "$opera_ms_dir/$file";
$skip_flag = "";
if(exists $downloaded_file->{$file}){
print STDERR " *** ($cmp/$nb_step) Download $file [File previously downloaded skip]\n";
$cmp++;
print STDOUT " *** ($cmp/$nb_step) Extraction of $file [File extracted skip]\n";
$cmp++;
next;
}
print STDERR " *** ($cmp/$nb_step) Download $file $skip_flag\n";
run_exe("wget --no-check-certificate -O $file_path $url");
$cmp++;
print STDOUT " *** ($cmp/$nb_step) Extraction of $file\n";
print STDERR " ***************************** $file_path\n";
if(system ("tar -xvzf $file_path --directory $opera_ms_dir")){
return 0;
}
else{
`rm $file_path`;
`echo -e \"$file\" >> $tracking_file`;
$cmp++;
$downloaded_file->{$file} = 1;
}
}
#
return 1;
}
#### OPERA-MS pipeline stage
sub short_read_assembly{
my ($opera_ms_option, $opera_ms_dependency) = @_;
#
#
if($opera_ms_option->{"STAGE"} eq "ALL" || $opera_ms_option->{"STAGE"} eq "SHORT_READ_ASSEMBLY"){
$opera_ms_option->{"STAGE"} = "ALL" if($opera_ms_option->{"STAGE_FOLLOW"} == 1);
#Check if the contig file is given in the config file
if(defined $opera_ms_option->{"CONTIGS_FILE"}){
$opera_ms_option->{"ASSEMBLED_CONTIG_FILE"} = 0;
$time = localtime;
print STDOUT "\n[$time]\tShort read assembly [1/" . $opera_ms_option->{"NB_TOTAL_STAGE"} ."]\n";
print STDOUT "[$time]\tSkip [contig file provided as input]\n";
process_user_assembly($opera_ms_option->{"CONTIGS_FILE"}, $opera_ms_option);
}
else{
$opera_ms_option->{"ASSEMBLED_CONTIG_FILE"} = 1;
$start_time = time();
if($opera_ms_option->{"SHORT_READ_ASSEMBLER"} eq "megahit"){
run_megahit($opera_ms_option, $opera_ms_dependency);
}
if($opera_ms_option->{"SHORT_READ_ASSEMBLER"} eq "spades"){
run_spades($opera_ms_option, $opera_ms_dependency);
}
$end_time = time();
write_time($opera_ms_option->{"INTER_DIR"}, "short_read_assembly", ($end_time - $start_time));
}
}
}
sub process_user_assembly{
my ($user_contig_file, $opera_ms_option) = @_;
my $inter_dir = $opera_ms_option->{"INTER_DIR"};
my $assembly_out_dir = "$inter_dir/user_assembly";
init_dir($assembly_out_dir);
$opera_ms_option->{"CONTIGS_FILE"} = "$assembly_out_dir/contigs.fasta";
multi_to_linear_fa($user_contig_file, $opera_ms_option->{"CONTIGS_FILE"});
}
sub run_megahit{
my ($opera_ms_option, $opera_ms_dependency) = @_;
my $inter_dir = $opera_ms_option->{"INTER_DIR"};
my $megahit_out_dir = "$inter_dir/megahit_assembly";
$opera_ms_option->{"CONTIGS_FILE"} = "$megahit_out_dir/final.contigs.fa";
#Stage 0: short assembly using megahit if the contig file is not provided in the config file
if(! check_completed($opera_ms_option->{"CONTIGS_FILE"}, "Short read assembly [MEGAHIT]", 1)){
run_exe("rm -r $megahit_out_dir") if(-d $megahit_out_dir);
run_exe($opera_ms_dependency->{"megahit"}."/megahit " .
"--num-cpu-threads " . $opera_ms_option->{"NUM_PROCESSOR"} . " " .
"-1 " . $opera_ms_option->{"ILLUMINA_READ_1"} . " " .
"-2 " . $opera_ms_option->{"ILLUMINA_READ_2"} . " " .
"-o " . $megahit_out_dir . "> $inter_dir/megahit.out 2> $inter_dir/megahit.err");
if($?){
die "Error during MEGAHIT assembly. Please see $inter_dir/megahit.out and $inter_dir/megahit.err for details.\n";
}
#Delete all the meggahit intermidate files
run_exe("rm -r $megahit_out_dir/intermediate_contigs");
}
}
sub run_spades{
my ($opera_ms_option, $opera_ms_dependency) = @_;
my $inter_dir = $opera_ms_option->{"INTER_DIR"};
my $assembly_out_dir = "$inter_dir/spades_assembly";
$opera_ms_option->{"CONTIGS_FILE"} = "$assembly_out_dir/contigs.fasta";
#Stage 0: short assembly using megahit if the contig file is not provided in the config file
if(! check_completed($opera_ms_option->{"CONTIGS_FILE"}, "Short read assembly [SPAdes]", 1)){
run_exe("rm -r $assembly_out_dir") if(-d $assembly_out_dir);
run_exe($opera_ms_dependency->{"spades"}."/spades.py " .
"--meta " .
"--threads " . $opera_ms_option->{"NUM_PROCESSOR"} . " " .
"-1 " . $opera_ms_option->{"ILLUMINA_READ_1"} . " " .
"-2 " . $opera_ms_option->{"ILLUMINA_READ_2"} . " " .
"-k 21,33,55,81 " .
"-o " . $assembly_out_dir . "> $inter_dir/spades.out 2> $inter_dir/spades.err");
if($?){
die "Error during SPAdes assembly. Please see $inter_dir/spades.out and $inter_dir/spades.err for details.\n";
}
#Delete all the meggahit intermidate files
run_exe("rm -r $assembly_out_dir/K* $assembly_out_dir/corrected* $assembly_out_dir/misc");
my $contig_file = $opera_ms_option->{"CONTIGS_FILE"};
multi_to_linear_fa($contig_file, "$contig_file.single");
run_exe("mv $contig_file.single $contig_file");
}
}
sub multi_to_linear_fa{
my ($multi_file, $single_file) = @_;
open(FILE, $multi_file);
open(OUT, ">$single_file");
my $first = 1;
while (<FILE>)
{
$line = $_;
chomp $line;
if ($line =~ />(.*)/) {
print OUT "\n" if(! $first);
print OUT $line . "\n";
$first = 0
}
else { print OUT $line; }
}
close(OUT);
close(FILE);
}
sub assembly_graph_creation{
my ($opera_ms_option, $opera_ms_dependency) = @_;
#
#
my $inter_dir = $opera_ms_option->{"INTER_DIR"};
my $opera_ms_dir = $opera_ms_option->{"OPERA_MS_DIR"};
my $cov_dir = "$inter_dir/coverage_estimation";
$opera_ms_option->{"COV_DIR"} = $cov_dir;
my $mapping_dir = "$inter_dir/read_mapping";
$opera_ms_option->{"MAPPING_DIR"} = $mapping_dir;
#my $DIR_COV = "$output_dir/$inter/coverage_estimation/";
#my $DIR_MAPPING = "$output_dir/$inter/long-read-mapping";
if($opera_ms_option->{"STAGE"} eq "ALL" || $opera_ms_option->{"STAGE"} eq "ASSEMBLY_GRAPH"){
$opera_ms_option->{"STAGE"} = "ALL" if($opera_ms_option->{"STAGE_FOLLOW"} == 1);
$start_time = time;
#Stage 1: Construction of scaffold graph and contig coverage estimation
#
#Short and long read mapping stage
if(! check_completed("$mapping_dir/config", "Long-read mapping and assembly graph generation", 2)){
init_dir($mapping_dir);
#print STDERR "\n--- STARTING OPERA-long-read.pl ----\n";
$start_time_sub = time;
$long_read_mapper_option = " --minimap2 " . $opera_ms_dependency->{"minimap2"} if($opera_ms_option->{"LONG_READ_MAPPER"} eq "minimap2");
$long_read_mapper_option = " --blasr " . $opera_ms_dependency->{"blasr"} if($opera_ms_option->{"LONG_READ_MAPPER"} eq "blasr");
run_exe($opera_ms_dependency->{"perl"}."perl " .
$opera_ms_dependency->{"OPERA-LG"}."/OPERA-long-read.pl" .
" --contig-file " . $opera_ms_option->{"CONTIGS_FILE"} .
" --kmer " . $opera_ms_option->{"KMER_SIZE"} .
" --long-read-file " . $opera_ms_option->{"LONG_READ"} .
" --output-prefix " . "opera" .
" --output-directory " . $mapping_dir .
" --num-of-processors " . $opera_ms_option->{"NUM_PROCESSOR"} .
" --opera " . $opera_ms_dependency->{"OPERA-LG"} .
" --illumina-read1 " . $opera_ms_option->{"ILLUMINA_READ_1"} .
" --illumina-read2 " . $opera_ms_option->{"ILLUMINA_READ_2"} .
" --samtools-dir " . $opera_ms_dependency->{"samtools"} .
" --perl-dir " . $opera_ms_dependency->{"perl"} .
$long_read_mapper_option .
" --short-read-tooldir " . $opera_ms_dependency->{"bwa"} .
" --skip-opera " . "1 " .
" --skip-short-read-mapping " . "1 " .
"> $mapping_dir/OPERA-long-read.out 2> $mapping_dir/OPERA-long-read.err"
);
#
#Error in STAGE 1
if($?){
die "Error in the long read processing. Please see log for details $mapping_dir/OPERA-long-read.out $mapping_dir/OPERA-long-read.err.\n";
}
$end_time_sub = time;
write_time($inter_dir, "graph_construction", ($end_time_sub - $start_time_sub));
}
### run cov_estimate
if(! check_completed($cov_dir."/".$opera_ms_option{"CONTIGS_COV_FILE"}, "Short-read mapping and coverage estimation", 3)){
run_exe("mkdir $cov_dir") if(! -d $cov_dir);
#Generate the config file
my $cov_estimate_config = "$cov_dir/cov_estimate.config";
run_exe("cp " . $opera_ms_option{"CONFIG_PATH"} . " " . $cov_estimate_config);
open(CONF, '>>', $cov_estimate_config);
if($opera_ms_option->{"ASSEMBLED_CONTIG_FILE"}){#Add the contig file name to the config file
print CONF "\nCONTIGS_FILE " . $opera_ms_option->{"CONTIGS_FILE"} ."\n";
}
if(index($opera_ms_option->{"ILLUMINA_READ_1"}, ",") == -1){#single sample assembly
print CONF "\nLIB $mapping_dir/opera.bam\n";
print CONF "\nMAPPING_FILES $mapping_dir/opera.bam\n";
}
else{
@illum_read1_tab = split(/,/, $opera_ms_option->{"ILLUM_READ1"});
my $str_bam = "";
for(my $i = 0; $i < @illum_read1_tab; $i++){
$str_bam .= "$mapping_dir/opera_$i.bam,";
}
chop $str_bam;
#
print CONF "\nLIB $mapping_dir/opera_1.bam\n";#NEED TO MERGE THE BAM
#
#
print CONF "\nMAPPING_FILES $str_bam\n";
}
#
print CONF "SAMTOOLS_DIR " . $opera_ms_dependency->{"samtools"} . "\n";
print CONF "TOOLS_DIR " . $opera_ms_dependency->{"OPERA_MS_TOOLS"} . "\n";
close(CONF);
##
#SIGMA on short reads used to estimate the contig read coverage
#bundling of short reads (can be use if fix the problem with short reads)
$start_time_sub = time;
run_exe($opera_ms_dependency->{"perl"}."perl ${opera_ms_dir}/bin/cov_estimate.pl $cov_estimate_config > $cov_dir/cov_estimate.out 2> $cov_dir/cov_estimate.err");
if($?){
die "Error in during cov_estimate.pl. Please see $cov_dir/cov_estimate.out and $cov_dir/cov_estimate.err for details.\n";
}
$end_time_sub = time;
write_time($inter_dir, "cov_estimation", ($end_time_sub - $start_time_sub));
#Remove edge with an outlier edge support/contig coverage ratio => good to remove error due to missmapping
#This step must be skipped in case of multi sample assembly
#
if(! $opera_ms_option->{"MULTI_SAMPLE"}){
$start_time_sub = time;
run_exe($opera_ms_dependency->{"perl"}."perl ${opera_ms_dir}bin/filter_edge_coverage_ratio.pl $mapping_dir " . "$cov_dir/".$opera_ms_option{"CONTIGS_COV_FILE"} . " 2> $mapping_dir/filter_edge_coverage_ratio.err");
write_time($inter_dir, "cov_filtering", ($end_time_sub - $start_time_sub));
$end_time_sub = time;
if($?){
die "Error in during filter_edge_coverage_ratio.pl. Please see $mapping_dir/filter_edge_coverage_ratio.err for details.\n";
}
}
else{
#create soft links
}
}
}
}
sub hierarchical_clustering{
my ($opera_ms_option, $opera_ms_dependency) = @_;
#
#
my $inter_dir = $opera_ms_option->{"INTER_DIR"};
my $opera_ms_dir = $opera_ms_option->{"OPERA_MS_DIR"};
my $cov_dir = $opera_ms_option->{"COV_DIR"};
my $sigma_dir = "$inter_dir/hierarchical_clustering";
$opera_ms_option->{"SIGMA_DIR"} = $sigma_dir;
if($opera_ms_option->{"STAGE"} eq "ALL" || $opera_ms_option->{"STAGE"} eq "CLUSTERING"){
$opera_ms_option->{"STAGE"} = "ALL" if($opera_ms_option->{"STAGE_FOLLOW"} == 1);
if(! check_completed("$sigma_dir/r_value_evaluation_summary.dat", "Hierarchical clustering", 4)){
init_dir($sigma_dir);
$start_time = time;
### Run sigma for long read (filtered edge obtain using filter_edge_coverage_ratio.pl###
#
generate_sigma_config_file($cov_dir, $opera_ms_option->{"MAPPING_DIR"}, $sigma_dir);
#Run sigma
run_exe("${opera_ms_dir}bin/sigma $sigma_dir/sigma.config > $sigma_dir/sigma.out 2> $sigma_dir/sigma.err");
if($?){
die "Error during ${opera_ms_dir}bin/sigma execution. Please see $sigma_dir/sigma.out and $sigma_dir/sigma.err for details.\n";
}
#Refine the sigma R parameter
run_exe($opera_ms_dependency->{"perl"}."perl ${opera_ms_dir}bin/refine_r_estimate.pl " .
$cov_dir."/".$opera_ms_option->{"CONTIGS_COV_FILE"} . " " .
$sigma_dir . " " .
$opera_ms_dependency->{"perl"} . " " .
$opera_ms_option->{"NUM_PROCESSOR"} . " " .
$opera_ms_option->{"CLUSTER_LEN_THRESHOLD"} . " " .
$opera_ms_dir . " 2> $sigma_dir/refine_r.err"
);
$end_time = time;
write_time($inter_dir, "sigma", ($end_time - $start_time));
}
}
}
sub reference_clustering{
my ($opera_ms_option, $opera_ms_dependency) = @_;
#
#
my $inter_dir = $opera_ms_option->{"INTER_DIR"};
my $opera_ms_dir = $opera_ms_option->{"OPERA_MS_DIR"};
my $ref_clustering_dir = "$inter_dir/reference_clustering";
$opera_ms_option->{"REF_CLUSTERING_DIR"} = $ref_clustering_dir;
if($opera_ms_option->{"STAGE"} eq "ALL" || $opera_ms_option->{"STAGE"} eq "REF_CLUSTERING"){
$opera_ms_option->{"STAGE"} = "ALL" if($opera_ms_option->{"STAGE_FOLLOW"} == 1);
if(! check_completed("$ref_clustering_dir/cluster_species.dat", "Reference based clustering", 5, !$opera_ms_option->{"REF_CLUSTERING"})){
init_dir($ref_clustering_dir);
$start_time = time;
#Run reference mapping to merge clusters, use both long read and short read.
#Use the short read and add a link if support > 5, gap estimated < 500 and validated on a reference
#NEED TO REMOVE LINKS SUPPORTED BY BOTH SHORT AND LONG READS
run_exe($opera_ms_dependency->{"perl"}."perl ${opera_ms_dir}bin/sequence_similarity_clustering.pl " .
$inter_dir . " " .
$ref_clustering_dir . " " .
$opera_ms_option->{"MAPPING_DIR"} . " " .
$opera_ms_option->{"SIGMA_DIR"} . " " .
$opera_ms_option->{"CONTIGS_FILE"} . " " .
$opera_ms_option->{"NUM_PROCESSOR"} . " " .
$opera_ms_option->{"OPERA_MS_DB"} . " " .
$opera_ms_dir . " " .
$opera_ms_dependency->{"mash"} . " " .
$opera_ms_dependency->{"mummer"} . " " .
$opera_ms_dependency->{"kraken"} . " 2> $ref_clustering_dir/sequence_similarity_clustering.err");
if($?){
die "Error in during reference based clustering. Please see $ref_clustering_dir/sequence_similarity_clustering.err for details.\n";
}
$end_time = time;
write_time($inter_dir, "ref_clustering", ($end_time - $start_time));
}
}
}
sub strain_clustering_and_assembly{
my ($opera_ms_option, $opera_ms_dependency) = @_;
#
#
my $inter_dir = $opera_ms_option->{"INTER_DIR"};
my $opera_ms_dir = $opera_ms_option->{"OPERA_MS_DIR"};
my $sigma_dir = $opera_ms_option->{"SIGMA_DIR"};
my $ref_clustering_dir = $opera_ms_option->{"REF_CLUSTERING_DIR"};
my $strain_dir = "$inter_dir/strain_analysis";
$opera_ms_option->{"STRAIN_DIR"} = $strain_dir;
#
my $opera_lr_dir = "$inter_dir/opera_long_read";
$opera_ms_option->{"OPERA_LR_DIR"} = $opera_lr_dir;
if(($opera_ms_option->{"STAGE"} eq "ALL" || $opera_ms_option->{"STAGE"} eq "STRAIN_CLUSTERING")){
$opera_ms_option->{"STAGE"} = "ALL" if($opera_ms_option->{"STAGE_FOLLOW"} == 1);
if( ! check_completed("$strain_dir/done", "Strain clustering and assembly", 6, !($opera_ms_option->{"REF_CLUSTERING"} && $opera_ms_option->{"STRAIN_CLUSTERING"}))){#Add check for reference clustering
init_dir($strain_dir);
$start_time = time;
#Plug-in the strain level assembly
#->if a species contains the strain each strain will be assembled independently by opera-lg
#->if a species does not contain the strain they are all pulled together and assembled in a single run by opera-lg
run_exe($opera_ms_dependency->{"perl"}."perl ${opera_ms_dir}bin/coverage_clustering.pl " .
$opera_ms_option->{"INTER_DIR"} . " " .
$strain_dir . " " .
$opera_ms_option->{"CONTIGS_FILE"} . " " .
$ref_clustering_dir . " " .
$opera_ms_option->{"OPERA_MS_DB"} . " " .
$opera_ms_dir . " " .
$opera_ms_dependency->{"perl"} . " " .
"1 " . "2> $strain_dir/coverage_clustering.err");
if($?){
die "Error in during strain clustering. Please see $strain_dir/coverage_clustering.err for details.\n";
}
#Assembly of species with multiple strains
my $global_r_value = `tail -n1 $sigma_dir/r_value_evaluation_summary.dat | cut -f1`;chop $global_r_value;
#
my $r_value_interval = `head -n1 $sigma_dir/r_estimate_value.dat`;chop $r_value_interval;
my @r_value_interval_tab = split(/\s+/, $r_value_interval);
my $r_value_step = ($r_value_interval_tab[1] - $r_value_interval_tab[0]) / 10;
#
open(FILE, "$strain_dir/reference_length.dat");
my $cmd_strain = "$strain_dir/assembly.cmd";
open(CMD_STRAIN, ">$cmd_strain");
my ($species, $species_length);
while(<FILE>){
@line = split(/\t/, $_);
$species = $line[0];
$species_length = $line[1];
print CMD_STRAIN $opera_ms_dependency->{"perl"}."perl ${opera_ms_dir}bin/cluster_strain.pl $strain_dir/$species $species_length $global_r_value $r_value_step $opera_ms_dir 2> $strain_dir/$species.log" . "\n";
}
close(FILE);
close(CMD_STRAIN);
my $num_processor = $opera_ms_option->{"NUM_PROCESSOR"};
run_exe("cat $cmd_strain | xargs -L 1 -P $num_processor -I COMMAND sh -c \"COMMAND\"");
if($?){
die "Error in during strain clustering assembly. Please see $strain_dir/*.log for details.\n";
}
$end_time = time;
run_exe("touch $strain_dir/done");
write_time($inter_dir, "strain_clustering", ($end_time - $start_time));
}
########## SMALL SPECIES CLUSTER AND NO SPECIES CLUSTER ASSEMBLY
if(! check_completed("$opera_lr_dir/statistics", "Assembly of other clusters",7)){
init_dir($opera_lr_dir);
$start_time = time;
#Add the contigs in cluster strain 0 back to the opera assembly
if($opera_ms_option->{"REF_CLUSTERING"} && $opera_ms_option->{"STRAIN_CLUSTERING"}){
run_exe("rm $opera_lr_dir/*single*") if(-e "$opera_lr_dir/contigs_single");#Deletion required to avoid re-using previous assemblies for the gapfilling
run_exe("cat $ref_clustering_dir/single_strain_species.fa $strain_dir/*/excluded_contigs.fa > $ref_clustering_dir/single_strain_species_with_excluded.fa");
}
#Assembly of all the other contigs were no multiple strain of the same species have inferred
#Filter that remove contigs with a coverage 1.5 times higher than the mean
#remove the contigs that belong to clusters that are associated to species with multiple strains
my $clustering_edge_dir = $opera_ms_option->{"SIGMA_DIR"};
my $reference_cluster_file = "$clustering_edge_dir/clusters";
my $contig_file = $opera_ms_option->{"CONTIGS_FILE"};
#
if($opera_ms_option->{"REF_CLUSTERING"}){
$clustering_edge_dir = $ref_clustering_dir;
$reference_cluster_file = "$ref_clustering_dir/clusters_seq_similarity";
if($opera_ms_option->{"STRAIN_CLUSTERING"}){
$contig_file = "$ref_clustering_dir/single_strain_species_with_excluded.fa";
$reference_cluster_file = "$ref_clustering_dir/clusters_single_strain"
}
}
$contig_coverage_file = $opera_ms_option->{"COV_DIR"} . "/" . $opera_ms_option->{"CONTIGS_COV_FILE"};
run_exe($opera_ms_dependency->{"perl"}."perl ${opera_ms_dir}bin/filter_cluster_coverage.pl $contig_coverage_file $reference_cluster_file $clustering_edge_dir 1.5 $clustering_edge_dir/NO_REPEAT 2> $clustering_edge_dir/filter_cluster_coverage.err");
if($?){
die "Error in during filter_cluster_coverage. Please see $clustering_edge_dir/filter_cluster_coverage.err for details.\n";
}
#Generate the OPERA-long-read config file
generate_opera_config_file($opera_ms_option->{"MAPPING_DIR"}, $clustering_edge_dir, $opera_lr_dir, $contig_file);
#finally, run opera
run_opera_lg($opera_lr_dir, "$opera_lr_dir/opera.config", $opera_ms_dependency);
$end_time = time;
write_time($inter_dir, "other_assembly", ($end_time - $start_time));
}
}
}
sub single_genome_assembly{
my ($opera_ms_option, $opera_ms_dependency) = @_;
my $inter_dir = $opera_ms_option->{"INTER_DIR"};
my $mapping_dir = $opera_ms_option->{"MAPPING_DIR"};
my $opera_lr_dir = "$inter_dir/opera_long_read";
$opera_ms_option->{"OPERA_LR_DIR"} = $opera_lr_dir;
$opera_lr_result_dir = "$opera_lr_dir/results";
if($opera_ms_option->{"STAGE"} eq "ALL" || $opera_ms_option->{"STAGE"} eq "SINGLE_GENOME"){
$opera_ms_option->{"STAGE"} = "ALL" if($opera_ms_option->{"STAGE_FOLLOW"} == 1);
if(! check_completed("$opera_lr_dir/scaffoldSeq.fasta", "Single genome assembly", 4, !$opera_ms_option->{"SINGLE_GENOME"})){
#Create a softlink of the read_mapping directory, this directory contains all the edges and the config files required to run OPERA-LG
$start_time = time;
run_exe("rm $opera_lr_dir") if(-e $opera_lr_dir);
run_exe("ln -s $mapping_dir $opera_lr_dir");
init_dir($opera_lr_result_dir);
#Create the coverage file
convert_sigma_to_opera_lg_coverage_file($opera_ms_option->{"COV_DIR"}."/".$opera_ms_option->{"CONTIGS_COV_FILE"}, "$opera_lr_result_dir/contigs");
#run OPERA-LG
run_opera_lg($opera_lr_dir, "$opera_lr_dir/config", $opera_ms_dependency);
#Move the output files
run_exe("mv $opera_lr_result_dir/scaffolds.scaf $opera_lr_result_dir/scaffoldSeq.fasta $opera_lr_dir");
$end_time = time;
write_time($inter_dir, "assembly", ($end_time - $start_time));
}
}
}
sub convert_sigma_to_opera_lg_coverage_file{
my ($sigma_coverage_file, $opera_lg_cov_file) = @_;
print STDERR " *** convert_sigma_to_opera_lg_coverage_file " . "\t" . $sigma_coverage_file . "\t" . $opera_lg_cov_file . "\n";
$READ_SIZE_FACTOR = 101;
open(OUT, ">$opera_lg_cov_file");
open(FILE, $sigma_coverage_file);
<FILE>;#Skip the header
while(<FILE>){
@line = split(/\t/, $_);
$contig_ID = $line[0];
$contig_length = $line[1];
$read_count = <FILE>;
$coverage = ($READ_SIZE_FACTOR * $read_count) / $contig_length;
print OUT $contig_ID . "\t" . $contig_length . "\t" . $coverage . "\n";
<FILE>;#Skip the window coverage line
}
close(FILE);
close(OUT);
}
sub run_opera_lg{
my ($opera_lr_dir, $config_file, $opera_ms_dependency) = @_;
my $opera_lg_exe_dir = $opera_ms_dependency->{"OPERA-LG"};
run_exe("timeout 5m $opera_lg_exe_dir/OPERA-LG $config_file > $opera_lr_dir/opera_lg.out 2> $opera_lr_dir/opera_lg.err");
if(! -e "$opera_lr_dir/scaffoldSeq.fasta"){#NEED TO IN THE MAKE FILE THE COMMAND TO GET THE FASTS OPERA-MS
run_exe("$opera_lg_exe_dir/OPERA-LG-fast $config_file > $opera_lr_dir/opera_lg_fast.out 2> $opera_lr_dir/opera_lg_fast.err");
}
if($?){
die "Error in during OPERA-LG assembly. Please see $opera_lr_dir/opera_lg*.out and $opera_lr_dir/opera_lg*.err for details.\n";
}
}
sub gap_filling{
my ($opera_ms_option, $opera_ms_dependency) = @_;
#
#
my $final_output_dir = $opera_ms_option->{"OUTPUT_DIR"};
my $inter_dir = $opera_ms_option->{"INTER_DIR"};
my $opera_ms_dir = $opera_ms_option->{"OPERA_MS_DIR"};
my $opera_lg_dir = $opera_ms_dependency->{"OPERA-LG"};
my $strain_dir = $opera_ms_option->{"STRAIN_DIR"};
my $opera_lr_dir = $opera_ms_option->{"OPERA_LR_DIR"};
my $gap_filling_dir = "$opera_lr_dir/GAPFILLING";
$opera_ms_option->{"GAPFILLING_DIR"} = $gap_filling_dir;
if($opera_ms_option->{"STAGE"} eq "ALL" || $opera_ms_option->{"STAGE"} eq "GAP_FILLING"){
$opera_ms_option->{"STAGE"} = "ALL" if($opera_ms_option->{"STAGE_FOLLOW"} == 1);
if(! check_completed("$opera_lr_dir/scaffoldSeq.fasta.filled", "Gap filling", 8)){
init_dir($gap_filling_dir);
$start_time = time;
$start_time_sub = time;
#Add the scaffold obtain during the strain level assembly back in
my $opera_scaff_file = "$opera_lr_dir/scaffolds.scaf";
if($opera_ms_option->{"REF_CLUSTERING"} && $opera_ms_option->{"STRAIN_CLUSTERING"}){
my $opera_scaff_file_single = "$opera_lr_dir/scaffolds_single.scaf";
run_exe("mv $opera_scaff_file $opera_scaff_file_single") if(! -e $opera_scaff_file_single);
run_exe("cp $opera_scaff_file_single $opera_scaff_file");
}
my $opera_scaff_seq_file = "$opera_lr_dir/scaffoldSeq.fasta";
if($opera_ms_option->{"REF_CLUSTERING"} && $opera_ms_option->{"STRAIN_CLUSTERING"}){
my $opera_scaff_seq_file_single = "$opera_lr_dir/scaffoldSeq_single.fasta";
run_exe("mv $opera_scaff_seq_file $opera_scaff_seq_file_single") if(! -e $opera_scaff_seq_file_single);
run_exe("cp $opera_scaff_seq_file_single $opera_scaff_seq_file");
}