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|
#!/usr/bin/perl
#
# take a multiple fasta amino acid sequence file
# and output a file that is non-redundant:
#
# the percent identity value between each pair of different sequence is below a threshold
# and each sequence name occurrs only once
#
# This script is used by the braker.pl pipeline.
# Please be extremely careful when changing this script because the braker.pl
# pipeline may fail upon custom modification of this script.
# In case of doubt, contact katharina.hoff@uni-greifswald.de
#
# Mario Stanke & Katharina Hoff, last modification on May 31 2019
use strict;
use Getopt::Long;
use File::Which qw(which where);
use File::Spec::Functions qw(rel2abs);
use Cwd 'abs_path';
use File::Path qw(rmtree make_path);
use File::Basename qw(dirname basename);
use Parallel::ForkManager; # native blastp parallelization keeps most nodes idle most of the time, therefore data parallelize in perl
use Scalar::Util qw(openhandle);
use POSIX;
#
# The maximum percent identiy allowed between any two sequences.
# Here defined as (number of identical aa)/(length of shorter sequence)
#
my $max_percent_id = 0.8;
my $BLAST_PATH;
my $DIAMOND_PATH;
my $blast_path;
my $diamond_path;
my $CPU = 1;
my $diamond;
my $v = 0;
my $help;
my $cmdString;
my $usage = "aa2nonred.pl -- make a protein file non-redundant\n";
$usage .= "Usage: aa2nonred.pl input.fa output.fa\n";
$usage .= "In output.fa the percent identity value between each pair of \n";
$usage .= "When removing redundant sequences, priority is given to the sequence occuring last.\n";
$usage .= "Options:\n";
$usage .= "--maxid=f maximum percent identity between to sequences\n";
$usage .= " (#identical aa) / (length of shorter sequence) default: 0.8\n";
$usage .= "--BLAST_PATH=s path to blast (only implemented for NCBI BLAST)\n";
$usage .= "--DIAMOND_PATH=s path to diamond\n";
$usage .= "--cores=n number of cores to be used by NCBI BLAST or DIAMOND\n";
$usage .= "--diamond use DIAMOND istead of NCBI BLAST\n";
$usage .= "--verbosity=n verbosity level for information printed to stdout\n";
$usage .= "--help print this help message\n";
GetOptions(
'maxid:f' => \$max_percent_id,
'BLAST_PATH=s' => \$blast_path,
'DIAMOND_PATH=s' => \$diamond_path,
'cores=i' => \$CPU,
'diamond!' => \$diamond,
'verbosity=i' => \$v,
'help!' => \$help
);
if ($help) {
print $usage;
exit(1);
}
if (scalar(@ARGV) < 2){
print "Too few input arguments!\n";
print $usage;
exit(1);
}
if($diamond){
set_DIAMOND_PATH();
}else{
set_BLAST_PATH();
}
my $inputfilename = $ARGV[0];
my $outputfilename = $ARGV[1];
###########################################################################################
#
# split input file
#
###########################################################################################
my $splitDir; # for parallelization
my @splitFiles;
my $SPLITF;
if ( $CPU > 1 && not($diamond)) {
my $nFastaEntries = 0;
# counter number of fasta entries
open( INPUT, "<$inputfilename" ) or die("ERROR in file " . __FILE__ ." at line ". __LINE__ ."\nCould not open $inputfilename!\n");
while (<INPUT>) {
if ($_ =~ m/^>/) {
$nFastaEntries++;
}
}
close ( INPUT ) or die("ERROR in file " . __FILE__ ." at line ". __LINE__ ."\nCould not close $inputfilename!\n");
$splitDir = dirname( abs_path($inputfilename) ) . "/split_blast";
make_path ($splitDir) or die ("ERROR in file " . __FILE__ ." at line ". __LINE__ ."\nFailed to create directory $splitDir!\n");
my $maxEntries = ceil($nFastaEntries/$CPU);
my $fileNr = 1;
my $nEntries = 0;
my $filename;
open( INPUT, "<$inputfilename" ) or die("ERROR in file " . __FILE__ ." at line ". __LINE__ ."\nCould not open $inputfilename!\n");
while (<INPUT>) {
if ($_ =~ m/^>(\S+)/) {
if ( defined ( openhandle($SPLITF)) && $nEntries == $maxEntries) {
close ($SPLITF) or die ("ERROR in file " . __FILE__ ." at line ". __LINE__ ."\nCould not close file $filename!\n");
$fileNr++;
$nEntries = 0;
}
if($nEntries==0){
$filename = "$splitDir/split_$fileNr.fa";
push @splitFiles, $filename;
open ( $SPLITF, ">", $filename) or die ("ERROR in file " . __FILE__ ." at line ". __LINE__ ."\nCould not open file $filename!\n");
}
$nEntries++;
print $SPLITF $_;
}else{
if ( defined ( openhandle($SPLITF) ) ) {
print $SPLITF $_;
}
}
}
if ( defined ( openhandle($SPLITF)) ) {
close ($SPLITF) or die ("ERROR in file " . __FILE__ ." at line ". __LINE__ ."\nCould not close file $filename!\n");
}
close ( INPUT ) or die("ERROR in file " . __FILE__ ." at line ". __LINE__ ."\nCould not close $inputfilename!\n");
foreach(@splitFiles){
print "$_\n";
}
}
###########################################################################################
#
# make a file with unique sequence names
#
###########################################################################################
open( INPUT, "<$inputfilename" ) or die("ERROR in file " . __FILE__ ." at line ". __LINE__ ."\nCould not open $inputfilename!\n");
my $tempdbname = "$inputfilename.nonreddb";
open( TEMP, ">$tempdbname " ) or die("ERROR in file " . __FILE__ ." at line ". __LINE__ ."\nCould not open $tempdbname!\n");
my %seqnames = ();
my $seqname;
$/ = "\n\>";
while (<INPUT>) {
$_ =~ s/\>//g;
$_ =~ m/^(\S+)/;
$seqname = $1;
if ( !exists( $seqnames{$seqname} ) && length($seqname) > 1) {
$seqnames{$seqname} = $'; #'
print TEMP ">$seqname" . $seqnames{$seqname} . "";
}
}
close (INPUT) or die("ERROR in file " . __FILE__ ." at line ". __LINE__ ."\nCould not close $inputfilename!\n");
close (TEMP) or die("ERROR in file " . __FILE__ ." at line ". __LINE__ ."\nCould not close $tempdbname!\n");
###########################################################################################
#
# blast that file against itself
#
###########################################################################################
my $tempoutfile = "$inputfilename.blastout";
if($diamond){
if($CPU > 1){
$cmdString = "$DIAMOND_PATH/diamond makedb --in $tempdbname -d $tempdbname --threads $CPU";
system($cmdString) == 0 or die("ERROR in file " . __FILE__ . " at line " . __LINE__ ."\nFailed to execute: $cmdString!\n");
}else{
$cmdString = "$DIAMOND_PATH/diamond makedb --in $tempdbname -d $tempdbname --threads 1";
system($cmdString) == 0 or die("ERROR in file " . __FILE__ . " at line " . __LINE__ ."\nFailed to execute: $cmdString!\n");
}
}else{
## NCBI blast
$cmdString = "$BLAST_PATH/makeblastdb -in $tempdbname -dbtype prot -parse_seqids -out $tempdbname";
system($cmdString) == 0 or die("ERROR in file " . __FILE__ . " at line " . __LINE__ ."\nFailed to execute: $cmdString!\n");
}
if ( $CPU == 1 ) {
if($diamond){
$cmdString = "$DIAMOND_PATH/diamond blastp --db $tempdbname --outfmt 0 --query $tempdbname --out $tempoutfile";
system($cmdString) == 0 or die("ERROR in file " . __FILE__ . " at line " . __LINE__ ."\nFailed to execute: $cmdString!\n");
}else{
$cmdString = "$BLAST_PATH/blastp -query $tempdbname -db $tempdbname > $tempoutfile";
system($cmdString) == 0 or die("ERROR in file " . __FILE__ . " at line " . __LINE__ ."\nFailed to execute: $cmdString!\n");
}
}else{
if($diamond){
$cmdString = "$DIAMOND_PATH/diamond blastp --db $tempdbname --outfmt 0 --query $tempdbname --threads $CPU --out $tempoutfile";
system($cmdString) == 0 or die("ERROR in file " . __FILE__ . " at line " . __LINE__ ."\nFailed to execute: $cmdString!\n");
}else{
my $pm = new Parallel::ForkManager($CPU);
foreach ( @splitFiles ) {
my $pid = $pm->start and next;
$cmdString = "$BLAST_PATH/blastp -query $_ -db $tempdbname > $_.blastout";
system($cmdString) == 0 or die("ERROR in file " . __FILE__ . " at line " . __LINE__ ."\nFailed to execute: $cmdString!\n");
$pm->finish;
}
$pm->wait_all_children;
foreach ( @splitFiles ) {
$cmdString = "cat $_.blastout >> $tempoutfile";
system($cmdString) == 0 or die("ERROR in file " . __FILE__ . " at line " . __LINE__ ."\nFailed to execute: $cmdString!\n");
}
}
}
###########################################################################################
#
# parse the blast/diamond output
#
###########################################################################################
open( BLASTOUT, "<$tempoutfile" ) or die("ERROR in file " . __FILE__ ." at line ". __LINE__ ."\nCould not open $tempoutfile!\n");
$/ = "\nQuery= ";
my ( $query, $target, $qlen, $tlen, $numid, $minlen );
while (<BLASTOUT>) {
if(not($diamond)){
next unless / producing /;
}
$_ =~ m/(\S+)\n+Length=(\d+)/;
$query = $1;
$qlen = $2;
print STDOUT "query=$query, qlen=$qlen\n" if ($v>0);
while ( $_ =~ m/>(\S+).*\nLength=(\d+)\n.*\n.*\n Identities = (\d+)/g) {
$target = $1;
$tlen = $2;
$numid = $3;
print STDOUT "target=$target, tlen=$tlen, numid=$numid\n" if ($v>0);
$minlen = ( $qlen < $tlen ) ? $qlen : $tlen;
if ( $minlen == 0 ) { $minlen = 0.0000001; }
if ( $numid / $minlen > $max_percent_id ) { # conflict: too similar
if ( $query ne $target ) {
print STDOUT "$query and $target are similar\n" if ($v>0);
if ( exists( $seqnames{$query} )
&& exists( $seqnames{$target} ) )
{
delete( $seqnames{$query} );
print STDOUT "delete $query\n" if ($v>0);
}
}
}
}
}
close (BLASTOUT) or die("ERROR in file " . __FILE__ ." at line ". __LINE__ ."\nCould not close $tempoutfile!\n");
###########################################################################################
#
# output the nonredundant file
#
###########################################################################################
open( OUT, ">", $outputfilename ) or die("ERROR in file " . __FILE__ ." at line ". __LINE__ ."\nCould not open $outputfilename!\n");
foreach $seqname ( keys %seqnames ) {
print OUT ">$seqname" . $seqnames{$seqname} ;
}
close( OUT ) or die("ERROR in file " . __FILE__ ." at line ". __LINE__ ."\nCould not close $outputfilename!\n");
###########################################################################################
#
# Clean up
#
###########################################################################################
unlink ( rel2abs($tempdbname) );
if(not($diamond)){
unlink ( rel2abs($tempdbname).".phr" );
unlink ( rel2abs($tempdbname).".pin" );
unlink ( rel2abs($tempdbname).".pog" );
unlink ( rel2abs($tempdbname).".psd" );
unlink ( rel2abs($tempdbname).".psi" );
unlink ( rel2abs($tempdbname).".psq" );
}else{
unlink (rel2abs($tempdbname).".dmnd" );
}
unlink ( rel2abs($tempoutfile) );
if ($CPU > 1 && not($diamond)) {
rmtree( ["$splitDir"] );
}
###########################################################################################
#
# finding blastp and makeblastdb executables
#
###########################################################################################
sub set_BLAST_PATH {
my $prtStr;
# try to get path from ENV
if ( defined( $ENV{'BLAST_PATH'} ) ) {
if ( -e $ENV{'BLAST_PATH'} ) {
$prtStr
= "\# "
. (localtime)
. ": Found environment variable \$BLAST_PATH.\n";
print STDOUT $prtStr;
$BLAST_PATH = $ENV{'BLAST_PATH'};
}
}
else {
$prtStr
= "\# "
. (localtime)
. ": Did not find environment variable \$BLAST_PATH\n";
print STDOUT $prtStr;
}
# try to get path from command line
if ( defined($blast_path) ) {
my $last_char = substr( $blast_path, -1 );
if ( $last_char eq "\/" ) {
chop($blast_path);
}
if ( -d $blast_path ) {
$prtStr
= "\# "
. (localtime)
. ": Setting \$BLAST_PATH to command line argument ";
$prtStr .= "--BLAST_PATH value $blast_path.\n";
print STDOUT $prtStr;
$BLAST_PATH = $blast_path;
}
else {
$prtStr
= "\# "
. (localtime)
. ": WARNING: Command line argument --BLAST_PATH was ";
$prtStr
.= "supplied but value $blast_path is not a directory. Will not set ";
$prtStr .= "\$BLAST_PATH to $blast_path!\n";
print STDOUT $prtStr;
}
}
# try to guess
if ( not( defined($BLAST_PATH) )
|| length($BLAST_PATH) == 0 )
{
$prtStr
= "\# "
. (localtime)
. ": Trying to guess \$BLAST_PATH from location of blastp";
$prtStr .= " executable that is available in your \$PATH.\n";
print STDOUT $prtStr;
my $epath = which 'blastp';
if ( -d dirname($epath) ) {
$prtStr
= "\# "
. (localtime)
. ": Setting \$BLAST_PATH to "
. dirname($epath) . "\n";
print STDOUT $prtStr;
$BLAST_PATH = dirname($epath);
}
else {
$prtStr
= "\# "
. (localtime)
. ": WARNING: Guessing the location of \$BLAST_PATH ";
$prtStr
.= "failed. " . dirname($epath) . " is not a directory!\n";
print STDOUT $prtStr;
}
}
if ( not( defined($BLAST_PATH) ) ) {
my $blast_err;
$blast_err .= "There are 3 alternative ways to set this variable for "
. " aa2nonred.pl:\n"
. " a) provide command-line argument "
. "--BLAST_PATH=/your/path\n"
. " b) use an existing environment variable "
. "\$BLAST_PATH\n"
. " for setting the environment variable, run\n"
. " export BLAST_PATH=/your/path\n"
. " in your shell. You may append this to your "
. ".bashrc or .profile file in\n"
. " order to make the variable available to all your "
. "bash sessions.\n"
. " c) aa2nonred.pl can try guessing the location of "
. "\$BLAST_PATH from the\n"
. " location of a blastp executable that is "
. "available in your \$PATH variable.\n"
. " If you try to rely on this option, you can check "
. "by typing\n"
. " which blastp\n"
. " in your shell, whether there is a blastp "
. "executable in your \$PATH\n";
$prtStr = "\# " . (localtime) . " ERROR in file " . __FILE__ ." at line ". __LINE__ ."\n\$BLAST_PATH not set!\n";
print STDERR $prtStr;
print STDERR $blast_err;
exit(1);
}
if ( not ( -x "$BLAST_PATH/blastp" ) ) {
print STDERR "\# " . (localtime) . " ERROR in file " . __FILE__ ." at line ". __LINE__ ."\n$BLAST_PATH/blastp is not an executable file!\n";
exit(1);
}elsif( not ( -x "$BLAST_PATH/makeblastdb" ) ){
print STDERR "\# " . (localtime) . " ERROR in file " . __FILE__ ." at line ". __LINE__ ."\n$BLAST_PATH/makeblastdb is not an executable file!\n";
exit(1);
}
}
###########################################################################################
#
# finding diamond executable
#
###########################################################################################
sub set_DIAMOND_PATH {
my $prtStr;
# try to get path from ENV
if ( defined( $ENV{'DIAMOND_PATH'} ) ) {
if ( -e $ENV{'DIAMOND_PATH'} ) {
$prtStr
= "\# "
. (localtime)
. ": Found environment variable \$DIAMOND_PATH.\n";
print STDOUT $prtStr;
$DIAMOND_PATH = $ENV{'DIAMOND_PATH'};
}
}
else {
$prtStr
= "\# "
. (localtime)
. ": Did not find environment variable \$DIAMOND_PATH\n";
print STDOUT $prtStr;
}
# try to get path from command line
if ( defined($diamond_path) ) {
my $last_char = substr( $diamond_path, -1 );
if ( $last_char eq "\/" ) {
chop($diamond_path);
}
if ( -d $diamond_path ) {
$prtStr
= "\# "
. (localtime)
. ": Setting \$DIAMOND_PATH to command line argument ";
$prtStr .= "--DIAMOND_PATH value $diamond_path.\n";
print STDOUT $prtStr;
$DIAMOND_PATH = $diamond_path;
}
else {
$prtStr
= "\# "
. (localtime)
. ": WARNING: Command line argument --DIAMOND_PATH was ";
$prtStr
.= "supplied but value $diamond_path is not a directory. Will not set ";
$prtStr .= "\$DIAMOND_PATH to $diamond_path!\n";
print STDOUT $prtStr;
}
}
# try to guess
if ( not( defined($DIAMOND_PATH) )
|| length($DIAMOND_PATH) == 0 )
{
$prtStr
= "\# "
. (localtime)
. ": Trying to guess \$DIAMOND_PATH from location of diamond";
$prtStr .= " executable that is available in your \$PATH.\n";
print STDOUT $prtStr;
my $epath = which 'diamond';
if ( -d dirname($epath) ) {
$prtStr
= "\# "
. (localtime)
. ": Setting \$DIAMOND_PATH to "
. dirname($epath) . "\n";
print STDOUT $prtStr;
$DIAMOND_PATH = dirname($epath);
}
else {
$prtStr
= "\# "
. (localtime)
. ": WARNING: Guessing the location of \$DIAMOND_PATH ";
$prtStr
.= "failed. " . dirname($epath) . " is not a directory!\n";
print STDOUT $prtStr;
}
}
if ( not( defined($DIAMOND_PATH) ) ) {
my $diamond_err;
$diamond_err .= "There are 3 alternative ways to set this variable for "
. " aa2nonred.pl:\n"
. " a) provide command-line argument "
. "--DIAMOND_PATH=/your/path\n"
. " b) use an existing environment variable "
. "\$DIAMOND_PATH\n"
. " for setting the environment variable, run\n"
. " export DIAMOND_PATH=/your/path\n"
. " in your shell. You may append this to your "
. ".bashrc or .profile file in\n"
. " order to make the variable available to all your "
. "bash sessions.\n"
. " c) aa2nonred.pl can try guessing the location of "
. "\$DIAMOND_PATH from the\n"
. " location of a diamond executable that is "
. "available in your \$PATH variable.\n"
. " If you try to rely on this option, you can check "
. "by typing\n"
. " which diamond\n"
. " in your shell, whether there is a diamond "
. "executable in your \$PATH\n";
$prtStr = "\# " . (localtime) . " ERROR in file " . __FILE__ ." at line ". __LINE__ ."\n\$DIAMOND_PATH not set!\n";
print STDERR $prtStr;
print STDERR $diamond_err;
exit(1);
}
if ( not ( -x "$DIAMOND_PATH/diamond" ) ) {
print STDERR "\# " . (localtime) . " ERROR in file " . __FILE__ ." at line ". __LINE__ ."\n$DIAMOND_PATH/diamond is not an executable file!\n";
exit(1);
}
}
|
