/
TRAVIS_Core.pl
1376 lines (1248 loc) · 51.9 KB
/
TRAVIS_Core.pl
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#!/usr/bin/env perl
use warnings;
use strict;
use File::Path qw(make_path);
use File::Basename;
use File::Spec;
use Data::Dumper;
use Parallel::Loops;
no warnings 'experimental::smartmatch';
unless (@ARGV){
die "usage: perl TRAVIS_Core.pl <TRAVIS_control_center.csv> <optional: sample>\n";
}
my $TCC_file = shift @ARGV;
print '#' x 70,"\n";
print '#' x 70,"\n";
print "\t\t\tThis is TRAVIS Core v20230214\n";
print '#' x 70,"\n";
print '#' x 70,"\n";
my $subset;
if (@ARGV){
$subset = shift @ARGV;
if ($subset eq 'preparation'){
print "will prepare all databases and perform no searches\n";
} else {
print "will only search in samples that match $subset\n";
}
} else {
print "will search in all samples\n";
$subset = 'all';
}
###reading TRAVIS Control Center
my %TCC; #contains configuration parameters from $TCC_file
&read_TCC(
\$TCC_file, #file path
\%TCC #undef hash
);
my @supported_search_tools = ('blastp', 'hmmer', 'jackhmmer', 'mmseqs', 'diamond');
print "connected to TRAVIS Control Center\n";
my %status_of_ORF_fasta;
###loop through sample library and get the ORFs
print "checking ORF_dir..\n";
&check_dir(\$TCC{'ORF_dir'});
my @skipped; #collect filenames that are in the sample file but cannot be found
my $ORF_fastas_content; #collect the ORF fasta paths that can actually be searched
open (my $SAMPLE_LIBRARY, '<', $TCC{'sample_library'}) or die "could not read from $TCC{'sample_library'}: $!\n";
my $c1;
while (my $line = <$SAMPLE_LIBRARY>){
chomp $line;
next if ($line =~ m/^#/);
next if ($line =~ m/^$/);
next if ($line =~ m/^filename/);
if (($subset ne 'preparation') and ($subset ne 'all')){
next if ($line !~ m/$subset/);
}
my @cols = split (',', $line);
my $sample_file = File::Spec -> catfile ($TCC{'sample_dir'} , $cols[0]);
#~ my $crt_sample_ORF_data = File::Spec -> catfile ($TCC{'ORF_dir'} , $cols[1].'ORFs.fasta');
unless (-s $sample_file){
push (@skipped, $cols[0]);
next;
}
#~ my $min_ORF_length = 50;
#~ (my $crt_filename = basename($sample_file)) =~ s/\.[^\.]+$//;
my $sample_ORF_data = File::Spec -> catfile($TCC{'ORF_dir'} , $cols[1].'_ORF_data.csv');
unless ((-s $sample_ORF_data) or ($TCC{'purge_data'})){
print "getting ORF data from $sample_ORF_data...\n";
&get_sample_ORF_data(\$sample_file, \$sample_ORF_data, \'fasta', \$TCC{'min_ORF_length'},\$TCC{'max_ORF_length'});
$c1++;
#~ last if ($c1 >= 5 );
} else {
print "$sample_ORF_data already exists...skipping...\n";
}
my $sample_ORF_fasta = File::Spec -> catfile($TCC{'ORF_dir'} , $cols[1].'_ORFs_AA.fasta');
my $sample_ORF_fasta_basename = basename($sample_ORF_fasta);
unless (-s $sample_ORF_fasta ){
if (-s $sample_ORF_data){
print "converting $sample_ORF_data to $sample_ORF_fasta...\n";
&ORF_data_2_fasta(\$sample_ORF_data, \$sample_ORF_fasta);
$ORF_fastas_content .= $sample_ORF_fasta."\n";
} else {
print "no ORFs found that match the criteria\n";
}
} else {
print "$sample_ORF_fasta already exists...skipping...\n";
$ORF_fastas_content .= $sample_ORF_fasta_basename."\n";
#~ next;
}
$status_of_ORF_fasta{$sample_ORF_fasta_basename} = 'clean';
push (@{$status_of_ORF_fasta{'order'}}, $sample_ORF_fasta_basename);
#~ print "read $sample_file\n";
print '#' x 70,"\n";
}
print "checking result_dir..\n";
&check_dir(\$TCC{'result_dir'});
if ($subset eq 'preparation'){
$status_of_ORF_fasta{'preparation_dummy'} = 'positive';
push (@{$status_of_ORF_fasta{'order'}}, 'preparation_dummy');
} else {
my $ORF_fastas = File::Spec -> catfile ($TCC{'result_dir'}, $subset.'__ORF_fastas.txt');
&write_file(\$ORF_fastas ,\$ORF_fastas_content);
foreach my $skipped (@skipped){
print "skipped $skipped because it was empty or non existent!\n";
}
}
my $ORF_fastas_done = File::Spec -> catfile ($TCC{'result_dir'}, $subset.'__ORF_fastas_done.txt');
my $ORF_results = File::Spec -> catfile ($TCC{'result_dir'}, $subset.'__ORF_results.csv');
my %already_done;
my $already_done_ORF_results;
if ((-s $ORF_fastas_done) and ($TCC{'resume_calculation'})){
print "will try to resume calculation!\n";
open (my $ORF_FASTAS_DONE, '<', $ORF_fastas_done) or die "could not read from '$ORF_fastas_done': $!\n";
while (my $line = <$ORF_FASTAS_DONE>){
chomp $line;
print $line, "\n";
my @cols = split (',', $line);
$already_done{$cols[0]} = $cols[1];
$status_of_ORF_fasta{$cols[0]} = $cols[1];
}
close($ORF_FASTAS_DONE);
open (my $ORF_RESULTS_DONE, '<', $ORF_results) or die "could not read from '$ORF_results': $!\n";
while (my $line = <$ORF_RESULTS_DONE>){
chomp $line;
#~ print $line, "\n";
my @cols = split (',', $line);
my $crt_ORF_fasta = File::Spec -> catfile ($TCC{'ORF_dir'}, $cols[0].'_ORFs_AA.fasta');
$already_done_ORF_results .= $line."\n" if $already_done{$crt_ORF_fasta};
}
close($$ORF_RESULTS_DONE);
}
#~ print Dumper %already_done;
open (my $ORF_FASTAS_DONE, '>', $ORF_fastas_done) or die "could not write to '$ORF_fastas_done': $!\n";
open (my $ORF_RESULTS, '>', $ORF_results) or die "could not write to '$ORF_results': $!\n";
print $ORF_RESULTS "#Sample,Sequence,Reference,Reference_Set,Runtime,Search_Tool,E_Value,Score,BitScore\n";
print $ORF_RESULTS $already_done_ORF_results if ( $already_done_ORF_results);
if ($TCC{'purge_data'}){
my $collection= File::Spec -> catfile ($TCC{'reference_fastas'}, '*AA*collection*');
my @cfiles = glob $collection;
if (@cfiles){
print "removing previous collections...\n";
system(qq(rm -f $collection));
} else {
print "no previous collections found that should be deleted\n";
}
}
my %collection_contents;#dim1: all/main_positive, dim2: search tool, dim3: main/company, = array of fasta paths
my %reference_descriptions;
open (my $TTT_FH, '<', $TCC{'TTT'}) or die "could not read from '$TCC{'TTT'}': $!\n";
while (my $line = <$TTT_FH>){
chomp $line;
next if ($line =~ m/^#/);
next if ($line =~ m/^$/);
#~ print $line, "\n";
my @cols = split (',', $line);
next if ($cols[7] ne 'on');
#~ print "preparing search for sequence(s) in $cols[0]\n";
$reference_descriptions{$cols[0]} = $cols[1];
$cols[1] =~ m/^([^_]+)/;
my $main_or_company = $1;
#~ my $main_or_company = 'main';
#~ print "$main_or_company\n";
if ($subset eq 'preparation'){
$cols[5] = 'all';
if (($cols[4] > 1) and ($cols[0] ne 'company_unclustered')) {
$cols[6] = 'hmmer&blastp&mmseqs&jackhmmer&diamond';
} else {
$cols[6] = 'blastp&mmseqs&jackhmmer&diamond';
}
}
my @search_tools = split ('&', $cols[6]);
foreach my $search_tool (@search_tools){
die "$search_tool is not supported!\n" unless ($search_tool ~~ @supported_search_tools);
my $reference_file_path;
if ($search_tool eq 'hmmer'){
$reference_file_path = File::Spec -> catfile ($TCC{'reference_fastas'}, $cols[3]);
}else {
$reference_file_path = File::Spec -> catfile ($TCC{'reference_fastas'}, $cols[2]);
}
push (@{$collection_contents{$cols[5]}{$search_tool}{$main_or_company}{$cols[0]}}, $reference_file_path);
push (@{$collection_contents{'main_positive'}{$search_tool}{$main_or_company}{$cols[0]}}, $reference_file_path) if ($subset eq 'preparation');
}
}
my $c;
foreach my $crt_sample (@{$status_of_ORF_fasta{'order'}}){
print '#' x 70,"\n";
print '#' x 70,"\n";
print '#' x 70,"\n";
print "searching in $crt_sample...\n" unless ($subset eq 'preparation');
$c++;
#~ last if ($c >= 7);
if ($already_done{$crt_sample}){
print "already did that! Skipping...\n";
print $ORF_FASTAS_DONE "$crt_sample,$already_done{$crt_sample}\n";
next;
}
my $crt_sample_filepath = File::Spec -> catfile ($TCC{'ORF_dir'}, $crt_sample);
my @suspicious_sequences;
foreach my $search_tool (sort keys %{$collection_contents{'all'}}){
#~ foreach my $search_tool ('mmseqs'){
print '#' x 70,"\n";
print "\trunning $search_tool for main sequences...\n" unless ($subset eq 'preparation');;
if ($search_tool eq 'hmmer'){
foreach my $reference (keys %{$collection_contents{'all'}{$search_tool}{'main'}}){
foreach my $reference_file (@{$collection_contents{'all'}{$search_tool}{'main'}{$reference}}){
next if ($reference_file =~ m/NA$/);
print "\tvs. $reference_file\n";
#~ print "\t\t\t###running hmmer...";
&run_hmmer(
\$reference, #name of the reference file
\$reference_descriptions{$reference} , #description
\$reference_file, #reference alignment
$ORF_RESULTS, #filehandle
\%TCC, #path to config
\%status_of_ORF_fasta, #contains all ORF fastas as keys
\$crt_sample_filepath,
\@suspicious_sequences,
\'hmmer'
);
}
}
} else {
my $crt_reference_fasta = File::Spec -> catfile ($TCC{'reference_fastas'}, $search_tool.'_AA_all_main.fasta');
unless (-s $crt_reference_fasta){
print "\tjoining fastas to $crt_reference_fasta\n";
&join_fastas(\%{$collection_contents{'all'}{$search_tool}{'main'}}, \$crt_reference_fasta);
}
if ($search_tool eq 'jackhmmer'){
#~ print "\t\t\t###running jackhmmer...";
&run_jackhmmer(
\'jackhmmer_AA_all_main_collection', #name of the reference file
\'jackhmmer_AA_all_main', #description
\$crt_reference_fasta , #reference alignment
$ORF_RESULTS, #filehandle
\%TCC, #path to config
\%status_of_ORF_fasta, #contains all ORF fastas as keys
\$crt_sample_filepath,
\@suspicious_sequences,
\'jackhmmer'
);
} elsif ($search_tool eq 'blastp'){
#~ print "\t\t\t###running blastp...";
&run_blastp(
\'blastp_AA_all_main_collection', #name of the reference file
\'blastp_AA_all_main', #description
\$crt_reference_fasta , #reference alignment
$ORF_RESULTS, #filehandle
\%TCC, #path to config
\%status_of_ORF_fasta, #contains all ORF fastas as keys
\$crt_sample_filepath,
\@suspicious_sequences,
\'blastp'
);
} elsif ($search_tool eq 'mmseqs'){
#~ print "\t\t\t###running mmseqs...";
&run_mmseqs(
\'mmseqs_AA_all_main_collection', #name of the reference file
\'mmseqs_AA_all_main', #description
\$crt_reference_fasta , #reference alignment
$ORF_RESULTS, #filehandle
\%TCC, #path to config
\%status_of_ORF_fasta, #contains all ORF fastas as keys
\$crt_sample_filepath,
\@suspicious_sequences,
\'mmseqs'
);
} elsif ($search_tool eq 'diamond'){
#~ print "\t\t\t###running diamond...";
&run_diamond(
\'diamond_AA_all_main_collection', #name of the reference file
\'diamond_AA_all_main', #description
\$crt_reference_fasta , #reference alignment
$ORF_RESULTS, #filehandle
\%TCC, #path to config
\%status_of_ORF_fasta, #contains all ORF fastas as keys
\$crt_sample_filepath,
\@suspicious_sequences,
\'diamond'
);
}
}
print '#' x 70,"\n";
if ($search_tool eq 'hmmer'){
foreach my $reference (keys %{$collection_contents{'all'}{$search_tool}{'company'}}){
foreach my $reference_file (@{$collection_contents{'all'}{$search_tool}{'company'}{$reference}}){
next if ($reference_file =~ m/NA$/);
print "\tvs. $reference_file\n" unless ($subset eq 'preparation');
print "\t\t\t###running hmmer..." unless ($subset eq 'preparation');
&run_hmmer(
\$reference, #name of the reference file
\$reference_descriptions{$reference} , #description
\$reference_file, #reference alignment
$ORF_RESULTS, #filehandle
\%TCC, #path to config
\%status_of_ORF_fasta, #contains all ORF fastas as keys
\$crt_sample_filepath,
\@suspicious_sequences,
\'hmmer'
);
}
}
} else {
my $crt_reference_fasta = File::Spec -> catfile ($TCC{'reference_fastas'}, $search_tool.'_AA_all_company.fasta');
unless (-s $crt_reference_fasta){
print "\tjoining fastas to $crt_reference_fasta\n";
&join_fastas(\%{$collection_contents{'all'}{$search_tool}{'company'}}, \$crt_reference_fasta);
}
if ($search_tool eq 'jackhmmer'){
#~ print "\t\t\t###running jackhmmer...";
&run_jackhmmer(
\'jackhmmer_AA_all_company_collection', #name of the reference file
\'jackhmmer_AA_all_company', #description
\$crt_reference_fasta , #reference alignment
$ORF_RESULTS, #filehandle
\%TCC, #path to config
\%status_of_ORF_fasta, #contains all ORF fastas as keys
\$crt_sample_filepath,
\@suspicious_sequences,
\'jackhmmer'
);
} elsif ($search_tool eq 'blastp'){
#~ print "\t\t\t###running blastp...";
&run_blastp(
\'blastp_AA_all_company_collection', #name of the reference file
\'blastp_AA_all_company', #description
\$crt_reference_fasta , #reference alignment
$ORF_RESULTS, #filehandle
\%TCC, #path to config
\%status_of_ORF_fasta, #contains all ORF fastas as keys
\$crt_sample_filepath,
\@suspicious_sequences,
\'blastp'
);
} elsif ($search_tool eq 'mmseqs'){
#~ print "\t\t\t###running mmseqs...";
&run_mmseqs(
\'mmseqs_AA_all_company_collection', #name of the reference file
\'mmseqs_AA_all_company', #description
\$crt_reference_fasta , #reference alignment
$ORF_RESULTS, #filehandle
\%TCC, #path to config
\%status_of_ORF_fasta, #contains all ORF fastas as keys
\$crt_sample_filepath,
\@suspicious_sequences,
\'mmseqs'
);
} elsif ($search_tool eq 'diamond'){
#~ print "\t\t\t###running diamond...";
&run_diamond(
\'diamond_AA_all_company_collection', #name of the reference file
\'diamond_AA_all_company', #description
\$crt_reference_fasta , #reference alignment
$ORF_RESULTS, #filehandle
\%TCC, #path to config
\%status_of_ORF_fasta, #contains all ORF fastas as keys
\$crt_sample_filepath,
\@suspicious_sequences,
\'diamond'
);
}
}
}
if (($status_of_ORF_fasta{$crt_sample} eq 'clean') and ($subset ne 'preparation')){
print "no hits found for main set. Will skip company sequence search for main_positive\n" unless ($subset eq 'preparation');
} else {
foreach my $search_tool (keys %{$collection_contents{'main_positive'}}){
print '#' x 70,"\n";
print "\trunning $search_tool for company sequences in main_positive...\n" unless ($subset eq 'preparation');;
if ($search_tool eq 'hmmer'){
foreach my $reference (keys %{$collection_contents{'main_positive'}{$search_tool}{'company'}}){
foreach my $reference_file (@{$collection_contents{'main_positive'}{$search_tool}{'company'}{$reference}}){
print "\tvs. $reference_file\n";
#~ print "\t\t\t###running hmmer...";
&run_hmmer(
\$reference, #name of the reference file
\$reference_descriptions{$reference} , #description
\$reference_file, #reference alignment
$ORF_RESULTS, #filehandle
\%TCC, #path to config
\%status_of_ORF_fasta, #contains all ORF fastas as keys
\$crt_sample_filepath,
\@suspicious_sequences,
\'hmmer'
);
}
}
} else {
my $crt_reference_fasta = File::Spec -> catfile ($TCC{'reference_fastas'}, $search_tool.'_AA_main_positive_company.fasta');
unless (-s $crt_reference_fasta){
print "\tjoining fastas to $crt_reference_fasta\n";
&join_fastas(\%{$collection_contents{'main_positive'}{$search_tool}{'company'}}, \$crt_reference_fasta);
}
if ($search_tool eq 'jackhmmer'){
#~ print "\t\t\t###running jackhmmer...";
&run_jackhmmer(
\'jackhmmer_AA_main_positive_company_collection', #name of the reference file
\'jackhmmer_AA_main_positive_company', #description
\$crt_reference_fasta , #reference alignment
$ORF_RESULTS, #filehandle
\%TCC, #path to config
\%status_of_ORF_fasta, #contains all ORF fastas as keys
\$crt_sample_filepath,
\@suspicious_sequences,
\'jackhmmer'
);
} elsif ($search_tool eq 'blastp'){
#~ print "\t\t\t###running blastp...";
&run_blastp(
\'blastp_AA_main_positive_company_collection', #name of the reference file
\'blastp_AA_main_positive_company', #description
\$crt_reference_fasta , #reference alignment
$ORF_RESULTS, #filehandle
\%TCC, #path to config
\%status_of_ORF_fasta, #contains all ORF fastas as keys
\$crt_sample_filepath,
\@suspicious_sequences,
\'blastp'
);
} elsif ($search_tool eq 'mmseqs'){
#~ print "\t\t\t###running mmseqs...";
&run_mmseqs(
\'mmseqs_AA_main_positive_company_collection', #name of the reference file
\'mmseqs_AA_main_positive_company', #description
\$crt_reference_fasta , #reference alignment
$ORF_RESULTS, #filehandle
\%TCC, #path to config
\%status_of_ORF_fasta, #contains all ORF fastas as keys
\$crt_sample_filepath,
\@suspicious_sequences,
\'mmseqs'
);
} elsif ($search_tool eq 'diamond'){
#~ print "\t\t\t###running diamond...";
&run_diamond(
\'diamond_AA_main_positive_company_collection', #name of the reference file
\'diamond_AA_main_positive_company', #description
\$crt_reference_fasta , #reference alignment
$ORF_RESULTS, #filehandle
\%TCC, #path to config
\%status_of_ORF_fasta, #contains all ORF fastas as keys
\$crt_sample_filepath,
\@suspicious_sequences,
\'diamond'
);
}
}
}
}
###checking suspicious sequences vs NR via blastp
# print Dumper @suspicious_sequences;
print '#' x 70,"\n";
my $crt_basename = basename($crt_sample);
$crt_basename =~ s/\.[^\.]+$//;
(my $crt_sample_ID = $crt_basename) =~ s/_ORFs_AA//;
if (@suspicious_sequences){
print "$crt_sample_ID: ", scalar @suspicious_sequences," sequence(s)!\n";
my $crt_ORF_CSV = File::Spec -> catfile ($TCC{'ORF_dir'}, $crt_sample_ID.'_ORF_data.csv');
###loading all ORF data from the sample ORF file
print "retrieving ORF data from '$crt_ORF_CSV'...\n";
my %ORF_data;
my %ORF_data_by_sequence;
open (my $CRT_ORF_CSV, '<', $crt_ORF_CSV) or die "could not read from '$crt_ORF_CSV': $!\n";
my $crt_header;
while (my $line = <$CRT_ORF_CSV>){
chomp $line;
$line =~ m/^([^,]+),(.+)$/;
my $crt_ORF_header = $1;
my $crt_ORF_data = $2;
$ORF_data{$crt_ORF_header} = $crt_ORF_data;
if ($line =~ m/^>([^,]+)/){
$crt_header = $1;
} else {
$ORF_data_by_sequence{$crt_header}{$crt_ORF_header} = $crt_ORF_data;
}
}
###getting the suspicious sequences from that sample
my %suspicious_ORFs_AA; #collects the AA sequences of the ORFs of the sample
my $crt_sample_suspicious_NT_fasta = File::Spec -> catfile ($TCC{'result_dir'}, $crt_sample_ID.'_suspicious_NT.fasta');
open (my $CRT_SUSPICIOUS_NT, '>', $crt_sample_suspicious_NT_fasta) or die "could not write to '$crt_sample_suspicious_NT_fasta': $!\n";
my $crt_sample_suspicious_fasta = File::Spec -> catfile ($TCC{'result_dir'}, $crt_sample_ID.'_suspicious_AA.fasta');
open (my $CRT_SUSPICIOUS, '>', $crt_sample_suspicious_fasta) or die "could not write to '$crt_sample_suspicious_fasta': $!\n";
print "writing to '$crt_sample_suspicious_fasta'...\n";
foreach my $sequence_header (@suspicious_sequences){
print "\t\t$sequence_header\n";
my $crt_full_seq = $sequence_header.','.$ORF_data{'>'.$sequence_header};
print $CRT_SUSPICIOUS_NT ">$sequence_header\n$ORF_data{'>'.$sequence_header}\n";
foreach my $crt_ORF (sort keys %{$ORF_data_by_sequence{$sequence_header}}){
my @cols = split (',', $ORF_data_by_sequence{$sequence_header}{$crt_ORF});
print $CRT_SUSPICIOUS ">$crt_ORF\n$cols[7]\n";
$suspicious_ORFs_AA{$crt_ORF} = $cols[7];
}
}
close ($CRT_SUSPICIOUS);
close ($CRT_SUSPICIOUS_NT);
if ($subset eq 'preparation'){
next;
}
###checking all sample sequences vs the full database (nr) with blastp
if ($TCC{'blastp_db_full'} and ($TCC{'blastp_db_full'} !~ m/skip/)){
my $crt_sample_suspicious_blasted = File::Spec -> catfile ($TCC{'result_dir'}, $crt_sample_ID.'_suspicious_AA_vs_full_blastp.csv');
print "blasting suspicious ORFs from $crt_sample_ID vs $TCC{'blastp_db_full'}...\n";
my $t1 = time();
unless ( $TCC{'only_evaluate'}){
system(qq($TCC{'blastp'} -query $crt_sample_suspicious_fasta -out $crt_sample_suspicious_blasted $TCC{'blastp_settings'} -db $TCC{'blastp_db_full'} -num_threads $TCC{'nCPU'} -outfmt \"10 qseqid qlen qframe sframe length pident nident qcovhsp qstart qend sstart send qseq sseq evalue score bitscore sacc\")) and die "Fatal: blast failed for '$crt_sample_suspicious_fasta': $!\n";
# (system(qq($blastp -query $$sref_crt_sample -out $crt_result $blastp_settings -db $blast_db_path -num_threads $ncpu -outfmt \"10 qseqid qlen qframe sframe length stitle pident nident qcovhsp qstart qend sstart send qseq sseq evalue score bitscore sacc\")) and die "Fatal: blast failed for '$$sref_crt_sample': $!\n") unless ($href_TCC -> {'only_evaluate'});
}
my $t2 = time();
my $tdiff = $t2 - $t1;
my $hitcounter;
print "reading BLAST output '$crt_sample_suspicious_blasted'...\n";
open (my $BLAST_OUT, '<', $crt_sample_suspicious_blasted) or die "could not read from '$crt_sample_suspicious_blasted': $!\n";
while (my $line = <$BLAST_OUT>){
chomp $line;
next if ($line =~ m/^#/);
next if ($line =~ m/^$/);
my @cols = split (',', $line);
$cols[-1] =~ s/\.\d\s*$//; #get rid of version number
$hitcounter++;
print $ORF_RESULTS "$crt_sample_ID,$cols[0],$cols[-1],$TCC{'blastp_db_full'},$tdiff,blastp_vs_full,$cols[-4],$cols[-3],$cols[-2]\n";
}
if ($hitcounter){
print "\t\tfound $hitcounter hit(s)!\n";
} else {
print "\t\tno hits found\n";
print $ORF_RESULTS "$crt_sample_ID,NA,NA,$TCC{'blastp_db_full'},$tdiff,blastp_vs_full,NA,NA,NA\n";
}
}
###checking all sample sequences vs the full database (nr) with diamond
if ($TCC{'diamond_db_full'} and ($TCC{'diamond_db_full'} !~ m/skip/)){
my $crt_sample_suspicious_diamonded = File::Spec -> catfile ($TCC{'result_dir'}, $crt_sample_ID.'_suspicious_AA_vs_full_diamond.tsv');
print "diamond vs suspicious ORFs from $crt_sample_ID vs $TCC{'diamond_db_full'}...\n";
my $t1 = time();
unless ( $TCC{'only_evaluate'}){
####system(qq($TCC{'blastp'} -query $crt_sample_suspicious_fasta -out $crt_sample_suspicious_blasted $TCC{'blastp_settings'} -db $TCC{'blastp_db_full'} -num_threads $TCC{'nCPU'} -outfmt \"10 qseqid qlen qframe sframe length pident nident qcovhsp qstart qend sstart send qseq sseq evalue score bitscore sacc\")) and die "Fatal: blast failed for '$crt_sample_suspicious_fasta': $!\n";
# (system(qq($blastp -query $$sref_crt_sample -out $crt_result $blastp_settings -db $blast_db_path -num_threads $ncpu -outfmt \"10 qseqid qlen qframe sframe length stitle pident nident qcovhsp qstart qend sstart send qseq sseq evalue score bitscore sacc\")) and die "Fatal: blast failed for '$$sref_crt_sample': $!\n") unless ($href_TCC -> {'only_evaluate'});
#print "diamond call:\n\t$TCC{'diamond'} blastp -q $crt_sample_suspicious_fasta -o $crt_sample_suspicious_diamonded $TCC{'diamond_settings'} -d $TCC{'diamond_db_full'} -num_threads $TCC{'nCPU'} -f6 qseqid qlen qframe sframe length pident nident qcovhsp qstart qend sstart send qseq sseq evalue score bitscore sacc\n";
system(qq($TCC{'diamond'} blastp -q $crt_sample_suspicious_fasta -o $crt_sample_suspicious_diamonded $TCC{'diamond_settings'} --quiet -d $TCC{'diamond_db_full'} -p $TCC{'nCPU'} -f6 qseqid qlen qframe length pident nident qstart qend sstart send qseq sseq evalue score bitscore sseqid)) and die "Fatal: diamond failed for '$crt_sample_suspicious_fasta': $!\n";
}
my $t2 = time();
my $tdiff = $t2 - $t1;
my $hitcounter;
print "reading DIAMOND output '$crt_sample_suspicious_diamonded'...\n";
open (my $DIAMOND_OUT, '<', $crt_sample_suspicious_diamonded) or die "could not read from '$crt_sample_suspicious_diamonded': $!\n";
while (my $line = <$DIAMOND_OUT>){
chomp $line;
next if ($line =~ m/^#/);
next if ($line =~ m/^$/);
my @cols = split ('\t', $line);
$cols[-1] =~ s/\.\d\s*$//; #get rid of version number
$hitcounter++;
print $ORF_RESULTS "$crt_sample_ID,$cols[0],$cols[-1],$TCC{'diamond_db_full'},$tdiff,diamond_vs_full,$cols[-4],$cols[-3],$cols[-2]\n";
}
if ($hitcounter){
print "\t\tfound $hitcounter hit(s)!\n";
} else {
print "\t\tno hits found\n";
print $ORF_RESULTS "$crt_sample_ID,NA,NA,$TCC{'diamond_db_full'},$tdiff,diamond_vs_full,NA,NA,NA\n";
}
}
} else {
print "no suspicious sequences in sample!\n" unless ($subset eq 'preparation');;
# print $ORF_RESULTS "$crt_sample_ID,NA,NA,$TCC{'blastp_db_full'},NA,blastp_vs_full,NA,NA,NA\n";
}
#~ $status_of_ORF_fasta{$crt_sample}
print $ORF_FASTAS_DONE "$crt_sample,$status_of_ORF_fasta{$crt_sample}\n" unless ($subset eq 'preparation');;
}
close ($ORF_FASTAS_DONE);
close ($ORF_RESULTS);
if ($subset eq 'preparation'){
print "preparation done!\n";
system(qq(rm -f $ORF_fastas_done $ORF_results));
}
print "TRAVIS Core completed!\n";
###subroutines
sub join_fastas {
my $href_mother_hash = $_[0];
my $sref_outfile = $_[1];
my %sequences;
foreach my $reference (keys %{$href_mother_hash}){
foreach my $reference_file (@{$href_mother_hash -> {$reference}}){
print "\t\t\t$reference_file\n";
open (my $FASTA, '<', $reference_file) or print "could not read from '$reference_file': $!\n";
my $current_header;
my $skip_flag = 0;
while (my $line = <$FASTA>){
chomp $line;
if ($line =~ m/^>/){
#~ print "before: $line\n";
my @header_elements= split ('__', $line);
pop @header_elements unless ((scalar @header_elements <= 1) or ($TCC{'reference_library'} eq 'blind'));
$current_header = join ('__', @header_elements);
#~ print "after: $current_header\n";
if ($sequences{$current_header}) {
$skip_flag = 1;
} else {
$skip_flag = 0;
}
#~ print "header: $line\n";
} else {
$sequences{$current_header} .= $line unless ($skip_flag);
}
}
}
}
open (my $FH, '>',$$sref_outfile ) or die "could not write to '$$sref_outfile : $!\n";
foreach my $seq (keys %sequences){
print $FH "$seq\n$sequences{$seq}\n";
}
close ($FH);
}
sub write_file {
#takes a file path and a string to create a file with the string as content
my $sref_file = $_[0]; #file path
my $sref_content = $_[1]; #content to be written into the file
open (my $FH, '>',$$sref_file) or die "could not write to '$$sref_file': $!\n";
print $FH $$sref_content;
close $FH;
#~ print "$$sref_file written\n";
}
sub slurp_fasta {
my $sref_fasta_file = $_[0];
my $sref_seq_container = $_[1];
open (my $FASTA, '<', $$sref_fasta_file) or print "could not read from '$$sref_fasta_file': $!\n";
my $current_header;
while (my $line = <$FASTA>){
$$sref_seq_container .= $line;
}
}
sub fasta_2_hash {
my $sref_fasta_file = $_[0];
my $href_seq_container = $_[1];
open (my $FASTA, '<', $$sref_fasta_file) or print "could not read from '$$sref_fasta_file': $!\n";
my $current_header;
while (my $line = <$FASTA>){
chomp $line;
if ($line =~ m/^>/){
$current_header = $line;
#~ print "header: $line\n";
} else {
$href_seq_container -> {$current_header} .= uc $line;
}
}
print "hashed $$sref_fasta_file...\n";
}
sub run_mmseqs{
#~ return;
my $sref_mmseqs_db_name = $_[0];
my $sref_description = $_[1];
my $sref_fasta = $_[2]; #file path
my $sFHref_ORF_RESULTS = $_[3];
my $href_TCC = $_[4];
my $href_status_of_ORF_fasta = $_[5];
my $sref_crt_sample = $_[6];
my $aref_suspicious_sequences = $_[7];
my $sref_rundesc = $_[8];
unless (-s $$sref_fasta ){
print "$$sref_fasta does not exist or is empty...no need to run a search\n";
return;
}
my $mmseqs_db_path = File::Spec -> catfile ($href_TCC -> {'reference_fastas'}, $$sref_mmseqs_db_name.'.mmseqsdb');
my $mmseqs_results = File::Spec -> catdir ($href_TCC -> {'result_dir'}, 'mmseqs_results');
&check_dir(\$mmseqs_results);
my $mmseqs = $href_TCC -> {'mmseqs'};
my $mmseqs_search_settings = $href_TCC -> {'mmseqs_search_settings'};
unless (-s $mmseqs_db_path){
print "\tbuilding mmseqs database '$mmseqs_db_path'...\n";
(system(qq($mmseqs createdb $$sref_fasta $mmseqs_db_path)) and die "Fatal: could not build mmseqs database from '$$sref_fasta'\: $!\n") unless ($href_TCC -> {'only_evaluate'});
} else {
print "\t'$mmseqs_db_path' already exists...\n";
}
return if ($subset eq 'preparation');
#~ my $blastp = $href_TCC -> {'blastp'};
my $ncpu = $href_TCC -> {'nCPU'};
#~ my $blastp_settings = $href_TCC -> {'blastp_settings'};
my $hitcounter;
(my $mmseqs_query_db_path = $$sref_crt_sample) =~ s/fasta$/mmseqsdb/;
unless (-s $mmseqs_query_db_path){
print "\tbuilding mmseqs database '$mmseqs_query_db_path'...\n";
my $mmseqs = $href_TCC -> {'mmseqs'};
(system(qq($mmseqs createdb $$sref_crt_sample $mmseqs_query_db_path)) and die ("Fatal: could not build mmseqs database from '$$sref_crt_sample'\: $!\n")) unless ($href_TCC -> {'only_evaluate'});
} else {
print "\t'$mmseqs_query_db_path' already exists...\n";
}
my $crt_basename = basename($$sref_crt_sample);
$crt_basename =~ s/\.[^\.]+$//;
(my $crt_sample_ID = $crt_basename) =~ s/_ORFs_AA//;
my $crt_tmp_results = File::Spec -> catdir ($mmseqs_results, $$sref_mmseqs_db_name.'_vs_'.$crt_basename.'_tmp');
&check_dir(\$crt_tmp_results);
my $mmseqs_result_db_path = File::Spec -> catdir ($mmseqs_results, $$sref_mmseqs_db_name.'_vs_'.$crt_basename.'.mmseqsdb');
if (-s $mmseqs_result_db_path){
(system(qq(rm -rf $mmseqs_result_db_path))) unless ($href_TCC -> {'only_evaluate'});
}
#~ #print "$crt_basename\n";
my $crt_result = File::Spec -> catfile ($mmseqs_results, $$sref_mmseqs_db_name.'_vs_'.$crt_basename.'.tsv');
#~ # print "$crt_result\n";
my $t1 = time();
unless ( $href_TCC -> {'only_evaluate'}){
system(qq(rm -fr $crt_tmp_results/* $mmseqs_result_db_path*));
system(qq($mmseqs search $mmseqs_query_db_path $mmseqs_db_path $mmseqs_result_db_path $crt_tmp_results --threads $ncpu $mmseqs_search_settings)) and die "Fatal: mmseqs failed for '$$sref_crt_sample': $!\n";
print "search done...converting DBs...\n";
system(qq($mmseqs convertalis $mmseqs_query_db_path $mmseqs_db_path $mmseqs_result_db_path $crt_result --threads $ncpu)) and die "Fatal: mmseqs conversion failed for '$$sref_crt_sample': $!\n";
}
my $t2 = time();
my $tdiff = $t2 - $t1;
system(qq(rm -rf $crt_tmp_results));
open (my $CRT_RESULT, '<', $crt_result) or die "could not read from '$crt_result': $!\n";
while (my $line = <$CRT_RESULT>){
chomp $line;
next if ($line =~ m/^#/);
next if ($line =~ m/^$/);
my @cols = split (/\t/, $line);
$hitcounter++;
print $sFHref_ORF_RESULTS "$crt_sample_ID,$cols[0],$cols[1],$$sref_mmseqs_db_name,$tdiff,$$sref_rundesc,$cols[-2],$cols[-1],NA\n";
$cols[0] =~ m/(.+)_(ORF_\d+)/;
my $seq_pt1 = $1;
push(@$aref_suspicious_sequences, $seq_pt1) unless ($seq_pt1 ~~ @$aref_suspicious_sequences);
}
if ($hitcounter){
print "\t\tfound $hitcounter hit(s)!\n";
$href_status_of_ORF_fasta -> {basename($$sref_crt_sample)} = 'positive';
} else {
print "\t\tno hits found\n";
print $sFHref_ORF_RESULTS "$crt_sample_ID,NA,NA,$$sref_mmseqs_db_name,$tdiff,$$sref_rundesc,NA,NA,NA\n";
}
}
sub run_diamond{
#~ return;
my $sref_diamond_db_name = $_[0];
my $sref_description = $_[1];
my $sref_fasta = $_[2]; #file path
my $sFHref_ORF_RESULTS = $_[3];
my $href_TCC = $_[4];
my $href_status_of_ORF_fasta = $_[5];
my $sref_crt_sample = $_[6];
my $aref_suspicious_sequences = $_[7];
my $sref_rundesc = $_[8];
unless (-s $$sref_fasta ){
print "$$sref_fasta does not exist or is empty...no need to run a search\n";
return;
}
my $diamond_db_path = File::Spec -> catfile ($href_TCC -> {'reference_fastas'}, $$sref_diamond_db_name.'.diamond');
my $diamond_results = File::Spec -> catdir ($href_TCC -> {'result_dir'}, 'diamond_results');
my $diamond = $href_TCC -> {'diamond'};
&check_dir(\$diamond_results);
unless (-s $diamond_db_path.'.dmnd'){
print "\tbuilding diamond database '$diamond_db_path'...\n";
# my $makeblastdb = $href_TCC -> {'makeblastdb'};
(system(qq($diamond makedb --in $$sref_fasta --db $diamond_db_path)) and die "Fatal: could not build blast database from '$$sref_fasta '\: $!\n") unless ($href_TCC -> {'only_evaluate'});
# (system(qq($makeblastdb --in $$sref_fasta -parse_seqids -dbtype prot -out $blast_db_path -title $blast_db_path )) and die "Fatal: could not build blast database from '$$sref_fasta '\: $!\n") unless ($href_TCC -> {'only_evaluate'});
} else {
print "\t'$diamond_db_path' already exists...\n";
}
return if ($subset eq 'preparation');
my $ncpu = $href_TCC -> {'nCPU'};
my $diamond_settings = '--fast';
$diamond_settings = $href_TCC -> {'diamond_settings'} if ($href_TCC -> {'diamond_settings'});
my $hitcounter;
my $crt_basename = basename($$sref_crt_sample);
$crt_basename =~ s/\.[^\.]+$//;
(my $crt_sample_ID = $crt_basename) =~ s/_ORFs_AA//;
#~ #print "$crt_basename\n";
my $crt_result = File::Spec -> catfile ($diamond_results, $$sref_diamond_db_name.'_vs_'.$crt_basename.'.tsv');
# print "$crt_result\n";
my $t1 = time();
unless ( $href_TCC -> {'only_evaluate'}){
#fromblast: (system(qq($diamond -query $$sref_crt_sample -out $crt_result $blastp_settings -db $blast_db_path -num_threads $ncpu -outfmt \"10 qseqid qlen qframe sframe length stitle pident nident qcovhsp qstart qend sstart send qseq sseq evalue score bitscore sacc\")) and die "Fatal: blast failed for '$$sref_crt_sample': $!\n") unless ($href_TCC -> {'only_evaluate'});
(system(qq($diamond blastp -q $$sref_crt_sample -o $crt_result $diamond_settings -d $diamond_db_path -p $ncpu --quiet -f6 qseqid qlen qframe length pident nident qstart qend sstart send qseq sseq evalue score bitscore sseqid)) and die "Fatal: diamond failed for '$$sref_crt_sample': $!\n") unless ($href_TCC -> {'only_evaluate'});
}
my $t2 = time();
my $tdiff = $t2 - $t1;
open (my $CRT_RESULT, '<', $crt_result) or die "could not read from '$crt_result': $!\n";
while (my $line = <$CRT_RESULT>){
chomp $line;
next if ($line =~ m/^#/);
next if ($line =~ m/^$/);
my @cols = split ('\t', $line);
$cols[5] =~ s/\s+$//;
$hitcounter++;
#if ($TCC{'reference_library'} eq 'blind'){
# print $sFHref_ORF_RESULTS "$crt_sample_ID,$cols[0],$cols[15],$$sref_diamond_db_name,$tdiff,$$sref_rundesc,$cols[-4],$cols[-3],$cols[-2]\n";
#} else {
print $sFHref_ORF_RESULTS "$crt_sample_ID,$cols[0],$cols[15],$$sref_diamond_db_name,$tdiff,$$sref_rundesc,$cols[-4],$cols[-3],$cols[-2]\n";
#}
$cols[0] =~ m/(.+)_(ORF_\d+)/;
my $seq_pt1 = $1;
push(@$aref_suspicious_sequences, $seq_pt1) unless ($seq_pt1 ~~ @$aref_suspicious_sequences);
}
if ($hitcounter){
print "\t\tfound $hitcounter hit(s)!\n";
$href_status_of_ORF_fasta -> {basename($$sref_crt_sample)} = 'positive';
} else {
print "\t\tno hits found\n";
print $sFHref_ORF_RESULTS "$crt_sample_ID,NA,NA,$$sref_diamond_db_name,$tdiff,$$sref_rundesc,NA,NA,NA\n";
}
}
sub run_blastp{
#~ return;
my $sref_blast_db_name = $_[0];
my $sref_description = $_[1];
my $sref_fasta = $_[2]; #file path
my $sFHref_ORF_RESULTS = $_[3];
my $href_TCC = $_[4];
my $href_status_of_ORF_fasta = $_[5];
my $sref_crt_sample = $_[6];
my $aref_suspicious_sequences = $_[7];
my $sref_rundesc = $_[8];
unless (-s $$sref_fasta ){
print "$$sref_fasta does not exist or is empty...no need to run a search\n";
return;
}
my $blast_db_path = File::Spec -> catfile ($href_TCC -> {'reference_fastas'}, $$sref_blast_db_name.'.blastdb');
my $blast_results = File::Spec -> catdir ($href_TCC -> {'result_dir'}, 'blast_results');
&check_dir(\$blast_results);
unless (-s $blast_db_path.'.phr'){
print "\tbuilding blast database '$blast_db_path'...\n";
my $makeblastdb = $href_TCC -> {'makeblastdb'};
#(system(qq($makeblastdb -in $$sref_fasta -parse_seqids -dbtype prot -out $blast_db_path -title $blast_db_path )) and die "Fatal: could not build blast database from '$$sref_fasta '\: $!\n") unless ($href_TCC -> {'only_evaluate'});
(system(qq($makeblastdb -in $$sref_fasta -dbtype prot -out $blast_db_path -title $blast_db_path )) and die "Fatal: could not build blast database from '$$sref_fasta '\: $!\n") unless ($href_TCC -> {'only_evaluate'});
} else {
print "\t'$blast_db_path' already exists...\n";
}
return if ($subset eq 'preparation');
my $blastp = $href_TCC -> {'blastp'};
my $ncpu = $href_TCC -> {'nCPU'};
my $blastp_settings = '-evalue 10';
$blastp_settings = $href_TCC -> {'blastp_settings'} if ($href_TCC -> {'blastp_settings'});
my $hitcounter;
my $crt_basename = basename($$sref_crt_sample);
$crt_basename =~ s/\.[^\.]+$//;
(my $crt_sample_ID = $crt_basename) =~ s/_ORFs_AA//;
#~ #print "$crt_basename\n";
my $crt_result = File::Spec -> catfile ($blast_results, $$sref_blast_db_name.'_vs_'.$crt_basename.'.csv');
# print "$crt_result\n";
my $t1 = time();
unless ( $href_TCC -> {'only_evaluate'}){
(system(qq($blastp -query $$sref_crt_sample -out $crt_result $blastp_settings -db $blast_db_path -num_threads $ncpu -outfmt \"10 qseqid qlen qframe sframe length stitle pident nident qcovhsp qstart qend sstart send qseq sseq evalue score bitscore sacc\")) and die "Fatal: blast failed for '$$sref_crt_sample': $!\n") unless ($href_TCC -> {'only_evaluate'});
}
my $t2 = time();
my $tdiff = $t2 - $t1;
open (my $CRT_RESULT, '<', $crt_result) or die "could not read from '$crt_result': $!\n";
while (my $line = <$CRT_RESULT>){
chomp $line;
next if ($line =~ m/^#/);
next if ($line =~ m/^$/);
my @cols = split (',', $line);
$cols[5] =~ s/\s+$//;
$hitcounter++;
if ($TCC{'reference_library'} eq 'blind'){
print $sFHref_ORF_RESULTS "$crt_sample_ID,$cols[0],$cols[-1],$$sref_blast_db_name,$tdiff,$$sref_rundesc,$cols[-4],$cols[-3],$cols[-2]\n";
} else {
print $sFHref_ORF_RESULTS "$crt_sample_ID,$cols[0],$cols[5],$$sref_blast_db_name,$tdiff,$$sref_rundesc,$cols[-4],$cols[-3],$cols[-2]\n";
}
$cols[0] =~ m/(.+)_(ORF_\d+)/;
my $seq_pt1 = $1;
push(@$aref_suspicious_sequences, $seq_pt1) unless ($seq_pt1 ~~ @$aref_suspicious_sequences);
}
if ($hitcounter){
print "\t\tfound $hitcounter hit(s)!\n";
$href_status_of_ORF_fasta -> {basename($$sref_crt_sample)} = 'positive';
} else {
print "\t\tno hits found\n";
print $sFHref_ORF_RESULTS "$crt_sample_ID,NA,NA,$$sref_blast_db_name,$tdiff,$$sref_rundesc,NA,NA,NA\n";
}
}
sub run_jackhmmer{
#~ return;
my $sref_reference_name = $_[0];
my $sref_description = $_[1];
my $sref_fasta = $_[2]; #file path
my $FH_ORF_RESULTS = $_[3];
my $href_TCC = $_[4];
my $href_status_of_ORF_fasta = $_[5];
my $sref_crt_sample = $_[6];
my $aref_suspicious_sequences = $_[7];
my $sref_rundesc = $_[8];
unless (-s $$sref_fasta ){
print "$$sref_fasta does not exist or is empty...no need to run a search\n";
return;
}
return if ($subset eq 'preparation');
my $jackhmmer_results = File::Spec -> catdir ($href_TCC -> {'result_dir'}, 'jackhmmer_results');
&check_dir(\$jackhmmer_results);
my $jackhmmer = $href_TCC -> {'jackhmmer'};
my $ncpu = $href_TCC -> {'nCPU'};
my $jackhmmer_settings = '-E 10';
$jackhmmer_settings = $href_TCC -> {'jackhmmer_settings'} if ($href_TCC -> {'jackhmmer_settings'});
my $hitcounter;
my $crt_basename = basename($$sref_crt_sample);
$crt_basename =~ s/\.[^\.]+$//;
(my $crt_sample_ID = $crt_basename) =~ s/_ORFs_AA//;
my $crt_result = File::Spec -> catfile ($jackhmmer_results, $$sref_reference_name.'_vs_'.$crt_basename);
my $t1 = time();
unless ( $href_TCC -> {'only_evaluate'}){
(system(qq($jackhmmer $jackhmmer_settings --cpu $ncpu -o /dev/null --tblout $crt_result $$sref_fasta $$sref_crt_sample)) and die "Fatal: jackhmmerfailed for '$$sref_crt_sample': $!\n") unless ($href_TCC -> {'only_evaluate'}) ;
}
my $t2 = time();
my $tdiff = $t2 - $t1;
open (my $CRT_RESULT, '<', $crt_result) or die "could not read from '$crt_result': $!\n";
while (my $line = <$CRT_RESULT>){
chomp $line;
next if ($line =~ m/^#/);
$hitcounter++;
$line =~ s/\h+/\t/g;