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hamstr.v12.pl
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hamstr.v12.pl
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#!/usr/bin/perl
use strict;
use Getopt::Long;
use lib '/Users/ingo/src/hamstr/hamstr.v12/bin/lib';
use lib '/Users/ingo/src/hamstr/hamstr.v12/bin/lib/Bio';
use Bio::SearchIO;
use Bio::Search::Hit::BlastHit;
use run_genewise_hamstr;
# PROGRAMNAME: hamstrsearch_local.pl
# Copyright (C) 2009 INGO EBERSBERGER, [email protected]
# This program is free software; you can redistribute it and/or modify it
# under the terms of the GNU General Public License as published
# by the Free Software Foundation; either version 3 of the License
# or any later version.
# This program is distributed in the hope that it will be useful
# but WITHOUT ANY WARRANTY; without even the implied warranty of
# MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU
# General Public License for more details.
# You should have received a copy of the GNU General Public License
# along with this program; If not, see http://www.gnu.org/licenses
# PROGRAM DESCRIPTION:
# DATE: Wed Dec 19 10:41:09 CEST 2007
# Date last modified:
##23. 07. 2010: found a bug in the extraction of the
## hmm hit sequence from the sequnence_file. A end-of-line char was missing.
##09.08.2010: added the option to choose the new blastp program from ncbi. Just comment
##out line 45 in the script and uncomment line 46. Note, in order to make this work I have
##to slightly modify the blast output since otherwise it will not be parsed by the Bioperl
##Blast parser. Currently this is a pretty dirty $sedprog hack. It will also take care of removin
##the string lcl| that is added in some instances to the hit id in the blast output.
## I added the option that one can now provide a comma-separated string of phmm names as an
## argument for the option -hmm
## 08.03.2011:
## 1) BUG-FIX: Hamstr will now remove automatically newlines from the input sequence file
## 2) BUG-FIX: The sequence header remains now the same whether or not the flag -representative
## has been chosen.
## 10.04.2011
## 1) added some information to the log file.
## 20.05.2011
## 1) BUG-FIX: The grep for the EST sequence in the sub-routine predictORF received also a hit when
## the search pattern was only a substring of the EST sequence identifier. In some cases the wrong EST
## was then used to predict the ORF. This has been fixed.
## 30.05.2011
## 1) Extension: a command line option -longhead has been added. The user can now specify that the
## full sequence id including whitespaces will considered throughout the hamstr search. Note, the
## whitespaces will be replaced by the string specified in the variabel $idsep.
## 2) Modification from the bug fix from 20.05.2011. In the grep for the original EST it is no longer
## necessary that the search string and the EST id are identical over their entire length. Instead the
## search string may be a prefix of the EST id ending with a whitespace.
## 27.06.2011
## 1) Extension: I added the option to run a true reciprocal best hit search. Only the best hit from the
## hmmer search is used to check for reciprocity.
## 06.12.2011
## 1) Extension: I added the option -hit_limit to set the number of hmmsearch hits that HaMStR uses for
## the re-blast.
## 10.02.2012
## 1) Extension: I added checks for the appropriate hmmsearch version (HMMER 3) and for genewise and
## its environmental variable WISECONFIGDIR.
## 2) Bug fix in the -rbh option.
## 11.09.2012
## 1) Bug fix: -hitlimit, even if not set explicitely has been invoked resulting in a more stringent
## behaviour of HaMStR. This has been fixed resulting in longer run-times.
## 18.12.2012
## 1) Bug fix: There was a bug in the CDS extraction for reverse complemented
## sequences. A new line was moved to the beginning of the sequence
## leading to index errors.
## 18.12.2013
## 1) Bug fix: I have now adapted the script such that it no longer requires the default directory structure
######################## start main #############################
my $version = "hamstrsearch_server-hmmer3.v12.pl\n";
print "$version\n";
### EDIT THE FOLLOWING LINES TO CUSTOMIZE YOUR SCRIPT
my $prog = 'hmmsearch'; #program for the hmm search
my $path = '/Users/ingo/src/hamstr/hamstr.v12';
my $sedprog = 'sed';
my $grepprog = 'grep';
my $alignmentprog = 'clustalw2';
########## EDIT THE FOLLOWING TWO LINES TO CHOOSE YOUR BLAST PROGRAM ##########
my $blast_prog = 'blastall';
#my $blast_prog = 'blastp';
###############################################################################
my $hmmpath = "$path/core_orthologs"; #path where the hmms are located
my $blastpath = "$path/blast_dir"; #path to the blast-dbs
my $tmpdir = 'tmp';
my $eval = 1; # eval cutoff for the hmm search
my $idsep = '__'; #character used to replace whitespaces in the sequence header with (flag -longhead)
my $hmm_dir = 'hmm_dir';
my $fa_dir = 'fa_dir';
##############################
my $pid = $$;
my $help;
my $seq2store_file='';
my $cds2store_file='';
my $hmm;
my @hmms;
my $fa;
my $fafile;
my @seqs2store;
my @cds2store;
my $dbpath = '.';
my $ep2eg;
my $estfile;
my $aln;
my $idfile;
my $taxon_check = 0;
my $hmmset;
my $show_coreortholog_sets;
my $hmmsearch_dir;
my $dbfile = ''; # the file hmmsearch is run against
my $dbfile_short;
my $taxon_file;
my $refspec_string;
my @refspec = qw();
my @primer_taxa;
my $refspec_name = '';
my $taxon_global;
my $fileobj;
my $fa_dir_neu = '';
my $gwrefprot;
my $seqtype;
my $align;
my $rep;
my $estflag;
my $proteinflag;
my $refseq;
my $strict;
my $relaxed;
my $refspec_final = '';
my $concat;
my $seqs2store_file;
my $append;
my $longhead;
my $check = 1;
my @log = qw();
my $filter = 'T';
my $bhh;
my $hitlimit;
#####determine the hostname#######
push @log, "VERSION\n\t$version\n";
my $hostname = `hostname`;
chomp $hostname;
print "hostname is $hostname\n";
push @log, "HOSTNAME\n\t$hostname\n";
#################################
if (@ARGV==0) {
$help = 1;
}
## help message
my $helpmessage = "
This program is freely distributed under a GPL. See -version for more info
Copyright (c) GRL limited: portions of the code are from separate copyrights
\nUSAGE: hamstrsearch_local.pl -sequence_file=<> -hmmset=<> -taxon=<> -refspec=<> [-est|-protein] [-hmm=<>] [-representative] [-h]
OPTIONS:
REQUIRED
-sequence_file
path and name of the file containing the sequences hmmer is run against.
-est
set this flag if you are searching in ESTs. Note, if neither the -est nor the -protein flag is set, HaMStR will
guess the sequence type.
-protein
set this flag if you are searching in protein sequences. Note, if neither the -est nor the -protein flag is set, HaMStR will
guess the sequence type.
-hmmset
specifies the name of the core-ortholog set.
The program will look for the files in the default directory 'core-orthologs' unless you specify
a different path via the option -hmmpath.
-taxon
You need to specify a default taxon name from which your ESTs or protein sequences are derived.
-refspec
sets the reference species. Note, it has to be a species that contributed sequences
to the hmms you are using. NO DEFAULT IS SET! For a list of possible reference
taxa you can have a look at the speclist.txt file in the default core-ortholog sets
that come with this distribution. Please use the abreviations in this list. If you choose
to use core-orthologs where not every taxon is represented in all core-orthologs, you
can provide a comma-separated list with the preferred refspec first. The lower-ranking
reference species will only be used if a certain gene is not present in the preferred
refspecies due to alternative paths in the transitive closure to define the core-orthologs.
CURRENTLY NO CHECK IS IMPLEMENTED!
NOTE: A BLAST-DB FOR THE REFERENCE SPECIES IS REQUIRED!
USING NON-DEFAULT PATHS
-blastpath
Lets you specify the absolute or relative path to the blast databases. DEFAULT: $blastpath
-hmmpath
Lets you specify the absolute or relative path to the core ortholog set. DEFAULT: $hmmpath
ADDITIONAL OPTIONS
-append
set this flag if the output should be appended to the files *.out and *_cds.out. This becomes relevant when running
hamstrsearch with individual hmms and you want to combine the results.
-eval_limit=<>
This options allows to set the e-value cut-off for the HMM search.
DEFAULT: 1
-filter=<T|F>
set this flag to F if the re-blast should be performed without low-complexity filtering. Default is T.
-hit_limit=<>
By default, HaMStR will re-blast all hmmsearch hits against the reference proteome. Reduce the number
of hits for reblast with this option.
-hmm
option to provide only a single hmm to be used for the search.
Note, this file has to end with .hmm
-show_hmmsets
setting this flag will list all available core ortholog sets in the specified path. Can be combined with -hmmpath.
-rbh
set this flag if you want to use a reciprocal best hit criterion. Only the highest scoring
hit from the hmmer search will be used for re-blast.
-relaxed
set this flag if the reciprocity criterion is fulfilled when the re-blast against
any of the primer taxa was successfull. Note that setting this flag will substantially decrease the
stringency of the ortholog assignment with the consequence of an increased number of false positives.
-representative
From all sequences that fulfill the reciprocity criterion the one showing the highest similarity to the
core ortholog sequence in the reference species is identified and selected as representative.
-strict
set this flag if the reciprocity criterion is only fulfilled when the re-blast against
all primer taxa was successfull
-longhead
set this flag in the case your sequence identifier contain whitespaces and you whish to keep
the entire sequence identifier throughout your analysis. HaMStR will then replace the whitespaces with
a '__'. If this flag is not set, HaMStR will truncate the sequence
Identifier at the first whitespace, however if and only if the sequence identifier then remain unique.
\n\n";
GetOptions ("h" => \$help,
"hmm=s" => \$hmm,
"est" => \$estflag,
"protein"=> \$proteinflag,
"sequence_file=s" => \$dbfile,
"fasta_file=s" => \$fafile,
"hmmset=s" => \$hmmset,
"hmmpath=s" => \$hmmpath,
"taxon_file=s" => \$taxon_file,
"taxon=s" => \$taxon_global,
"eval_limit=s" => \$eval,
"refspec=s" => \$refspec_string,
"estfile=s" => \$estfile,
"representative" => \$rep,
"strict" => \$strict,
"relaxed" => \$relaxed,
"append" => \$append,
"filter=s" => \$filter,
"longhead" => \$longhead,
"rbh" => \$bhh,
"hit_limit=s" => \$hitlimit,
"blastpath=s" => \$blastpath,
"show_hmmsets" => \$show_coreortholog_sets);
if ($help) {
print $helpmessage;
exit;
}
## 1) check if all information is available to run HaMStR
($check, @log) = &checkInput();
if ($check == 0) {
print "$helpmessage";
print "#######################################\n";
print "There was an error running $version\n";
print join "\n", @log;
exit;
}
else {
open (OUT, ">hamstrsearch.log") or die "could not open logfile\n";
print OUT join "\n", @log;
close OUT;
}
### read in of the core-ortholog sequences
my $co_seqs = parseSeqfile("$fafile");
## 2) loop through the hmms
## process each hmm file separately
for (my $i = 0; $i < @hmms; $i++) {
$fileobj = undef;
my @seqs = qw();
my @newseqs = qw();## var to contain the sequences to be added to the orthologous cluster
my @newcds = qw();
my $hmm = $hmms[$i];
my $hmmout = $hmm;
$hmmout =~ s/\.hmm/\.out/;
## 3) run the hmm search
if (!(-e "$hmmsearch_dir/$hmmout")) {
print "now running $prog using $hmm\n";
# print "$prog $hmm_dir/$hmm $dbfile >$hmmsearch_dir/$hmmout";
!`$prog $hmm_dir/$hmm $dbfile >$hmmsearch_dir/$hmmout` or die "Problem running hmmsearch\n";
}
else {
print "an hmmresult $hmmout already exists. Using this one!\n";
}
## 4) process the hmm search result
my $hitcount = 0;
## 4a) loop through the individual results
## now the modified version for hmmer3 comes
my $hitlimit_local = $hitlimit;
my ($query_name, @results) = parseHmmer3($hmmout, $hmmsearch_dir);
if (! @results) {
print "no hit found for $query_name\n";
next;
}
chomp $query_name;
print "Results for $query_name\n";
my ($check, $refspec_final) = &determineRefspecFinal($query_name, @refspec);
if ($check == 0) {
die "error in retrieving refspec data\n";
}
if (!defined $hitlimit_local or $hitlimit_local > scalar(@results)) {
$hitlimit_local = scalar(@results);
}
for (my $k = 0; $k < $hitlimit_local; $k++) {
my $hitname = $results[$k];
print "$hitname\n";
my $keep = 0;
my $hitseq = '';
$refseq = '';
## 4b) test for the reciprocity criterion fulfilled
($keep, $hitseq) = &check4reciprocity($query_name, $hitname, $refspec_final, @refspec);
if ($keep == 1) {
## blast search with the hmm hit identifies the core-ortholog sequence of the reference species
## check for the taxon from the taxon_file.txt. IN FACT, THIS ROUTINE IS OUTDATED. I ALWAYS USE THE
## GLOBAL TAXON NAME FOR THE HAMSTERED SEQUENCES.
my $taxon = '';
if ($taxon_check){
if ($taxon_check == 1) {
$taxon = &getTaxon($hitname);
}
elsif ($taxon_check == 2) {
$taxon = $taxon_global;
}
}
## put the info about the hits into an object for later post-processing
### HERE COMES THE NEW STUFF THAT DEALS WITH THE DIFFERENT POSSIBILITIES: STRICT, RELAXED OR WHATEVER...
$fileobj = &determineReferences ($fileobj, $taxon, $refspec_final, $hitname, $hitseq, $hitcount);
$hitcount++;
}
else {
print "Reciprocity not fulfilled!\n";
}
}
## 5) do the rest only if at least one hit was obtained
if (defined $fileobj) {
## 5a) if the hits are derived from ESTs, get the best ORF
if ($estflag) {
$fileobj = &predictORF();
}
## 5b) if the user has chosen to postprocess the results
if ($rep) {
&processHits($fileobj);
}
## 6) prepare the output
my @taxa = keys(%$fileobj);
for (my $i = 0; $i< @taxa; $i++) {
if ($rep) {
push @newseqs, ">$query_name|$fileobj->{$taxa[$i]}->{refspec_final}|$taxa[$i]|$fileobj->{$taxa[$i]}->{refid}";
push @newseqs, $fileobj->{$taxa[$i]}->{refprot};
if ($estflag) {
push @newcds, ">$query_name|$fileobj->{$taxa[$i]}->{refspec_final}|$taxa[$i]|$fileobj->{$taxa[$i]}->{refid}";
push @newcds, $fileobj->{$taxa[$i]}->{refcds};
}
}
else {
my $idobj = $fileobj->{$taxa[$i]}->{ids};
my $protobj = $fileobj->{$taxa[$i]}->{prot};
my $cdsobj = $fileobj->{$taxa[$i]}->{cds};
my $refspecobj = $fileobj->{$taxa[$i]}->{refspec};
for (my $j = 0; $j < @$idobj; $j++) {
push @newseqs, ">$query_name|$refspecobj->[$j]|$taxa[$i]|$idobj->[$j]";
push @newseqs, $protobj->[$j];
if ($estflag) {
push @newcds, ">$query_name|$taxa[$i]|$idobj->[$j]|$refspecobj->[$j]";
push @newcds, $cdsobj->[$j];
}
}
}
my $refs = $co_seqs->{$query_name};
for (keys %$refs) {
my $line = ">$query_name|$_|" . $refs->{$_}->{seqid} . "\n" . $refs->{$_}->{seq};
push @seqs, $line;
}
chomp @seqs;
print "\n";
@seqs = (@seqs, @newseqs);
open (OUT, ">$fa_dir_neu/$query_name.fa");
print OUT join "\n", @seqs;
print OUT "\n";
close OUT;
if ($estflag) {
open (OUT, ">$fa_dir_neu/$query_name.cds.fa");
print OUT join "\n", @newcds;
close OUT;
}
for (my $i = 0; $i < @newseqs; $i+= 2) {
my $line = $newseqs[$i] . "|" . $newseqs[$i+1];
$line =~ s/>//;
push @seqs2store, $line;
if ($estflag) {
my $cdsline = $newcds[$i] . "|" . $newcds[$i+1];
$cdsline =~ s/>//;
push @cds2store, $cdsline;
}
}
}
}
}
if (@seqs2store > 0) {
if ($append) {
open (OUT, ">>$seqs2store_file") or die "failed to open output file\n";
}
else {
open (OUT, ">$seqs2store_file") or die "failed to open output file\n";
}
print OUT join "\n", @seqs2store;
print OUT "\n";
close OUT;
if ($estflag) {
if ($append) {
open (OUT, ">>$cds2store_file") or die "failed to open output file\n";
}
else {
open (OUT, ">$cds2store_file") or die "failed to open output file\n";
}
print OUT join "\n", @cds2store;
print OUT "\n";
close OUT;
}
}
else {
print "no hits found\n";
}
exit;
##################### start sub ###############
####### checkInput performs a number of checks whether sufficient information
### and all data are available to run HaMStR
sub checkInput {
######### check a number of flags that only serve for providing the user with some information
if (defined $show_coreortholog_sets) {
## Do nothing but just list all available core ortholog sets in $hmmpath
my @coresets = (`ls $hmmpath`);
chomp @coresets;
print "You can choose from the following core ortholog sets available in $hmmpath:\n\n";
print join "\n", @coresets;
print "\n\n";
exit;
}
my @log;
my $check = 1;
$dbfile_short = $dbfile;
## extract the path from the dbpath if available and prune of trailing '/'
if ($dbfile =~ /(.*\/)/) {
$dbpath = $1;
$dbpath =~ s/\/$//;
}
$dbfile =~ s/.*\///;
$dbfile_short =~ s/\..*//;
##
## 0) Check for presence of the file with the sequences that should be hamstered
if (-e "$dbpath/$dbfile") {
#the file exists
push @log, "INFILE PROCESSING\n";
if (-e "$dbpath/$dbfile.mod") {
push @log, "\tA modified infile $dbfile.mod already exists. Using this one\n\n";
}
else {
print "removing newlines from the infile $dbfile such that a sequence forms a consecutive string\n";
`$path/bin/nentferner.pl -in=$dbpath/$dbfile -out=$dbpath/$dbfile.mod`;
#`nentferner.pl -in=$dbpath/$dbfile -out=$dbpath/$dbfile.mod`;
}
if (! -e "$dbpath/$dbfile.mod") {
push @log, "Problems running the script nentferner.pl\n";
$check = 0;
}
else {
print "nentferner.pl succeeded.\n";
push @log, "\tNewlines from the infile have been removed\n";
$dbfile = $dbfile . '.mod';
if (defined $longhead) {
`$sedprog -i -e "s/[[:space:]]\\+/$idsep/g" -e 's/\\(>.\\{20\\}\\).*/\\1/' $dbfile`;
push @log, "\tOption -longhead was chosen. Replaced whitespaces in the sequence identifier with '$idsep'\n";
}
}
}
else {
#the provided infile does not exist:
push @log, "The specified infile $dbpath/$dbfile does not exist. PLEASE PROVIDE A VALID INFILE!\n";
$check = 0;
return ($check, @log);
}
## 1) check for filetype
print "Checking for filetype:\t";
if (!defined $estflag and !defined $proteinflag) {
push @log, "No file sequence type was determined. HaMStR will guess whether EST or protein sequences are analyzed";
my $seq = `head -n 2 $dbpath/$dbfile |tail -n 1`;
my $orilength = length($seq);
$seq =~ s/[AGCTN]//ig;
if (length($seq) / $orilength >0.1) {
$proteinflag = 1;
print "Guessing sequence type: Protein\n";
push @log, "More than 10% of the first sequence in the file are non-AGCTN. Guessing sequence type: Protein";
}
else {
$estflag = 1;
print "Guessing sequence type: DNA\n";
}
$check = 1;
}
if ($estflag) {
$estfile = $dbfile;
$dbfile = "$dbfile.tc";
push @log, "HaMStR will run on the ESTs in $estfile";
push @log, "Translating ESTs";
if (!(-e "$dbpath/$dbfile")) {
print "translating $estfile, this may take a while\n";
`$path/bin/translate.pl -in=$dbpath/$estfile -out=$dbpath/$dbfile`;
open (LOG, "$dbpath/hamstrsearch.log");
my @info = <LOG>;
@log = (@log, @info);
close LOG;
}
else {
push @log, "Translated file already exists, using this one\n";
}
if (! -e "$dbpath/$dbfile") {
push @log, "The translation of $estfile failed. Check the script translate.pl";
print "failed\n";
$check = 0;
}
else {
## file type is protein
print "succeeded\n";
}
}
## 2) Check for presence of blastall
print "Checking for the blast program\t";
if (`which $blast_prog` =~ / no /) {
push @log, "could not execute $blast_prog. Please check if this program is installed and executable";
print "failed\n";
$check = 0;
}
else {
push @log, "check for $blast_prog succeeded";
print "succeeded\n";
}
## 3) Check for presence of hmmsearch
print "Checking for hmmsearch\t";
my $hmmcheck = `$prog -h |grep -c 'HMMER 3'`;
if (! `$prog -h`) {
push @log, "could not execute $prog. Please check if this program is installed and executable";
print "failed: $prog is not installed or not executable\n";
$check = 0;
}
elsif ($hmmcheck != 1) {
push @log, "It seems that $prog is not from the HMMER 3 package. Please check!";
print "failed: $prog is not from the HMMER 3 package\n";
$check = 0;
}
else {
push @log, "check for $prog succeeded\n";
print "succeeded\n";
}
## 3b) Check for genewise
print "Checking for genewise\t";
if (! `genewise -help`) {
push @log, "Could not execute genewise. Please check if this program is installed and executable";
print "failed: genewise is not executable\n";
$check = 0;
}
else {
my $gwcheck = `echo \$WISECONFIGDIR`;
if (length($gwcheck) < 1) {
push @log, "The environmental variable WISECONFIGDIR has not been set. I am expecting troubles when invoking genewise.
Please consult the installation manual for genewise and set this variable";
print "failed: the environmental variable WISECONFIGDIR has not been set.\n";
$check = 0;
}
else {
print "succeeded\n";
}
}
## 4) Check for presence of the directory structure
print "checking for presence of the hmm files:\t";
if ( ! defined $hmmset or ! -e "$hmmpath/$hmmset") {
push @log, "You need to specify a valid core ortholog set. Make also sure that you provide the path to this set if it is not in the default location $hmmpath.";
print "failed\n";
$check = 0;
}
else {
$hmmpath = "$hmmpath/$hmmset";
$fafile = "$hmmpath/$hmmset" . '.fa';
$hmm_dir = "$hmmpath/$hmm_dir";
$hmmsearch_dir = $dbpath .'/hmm_search_' . $dbfile_short . '_' . $hmmset;
## 4b) check for the presence of the hmm-files and the fasta-file
if (!(-e "$hmm_dir")) {
push @log, "Could not find $hmm_dir";
print "failed\n";
$check = 0;
}
else {
if (defined $hmm) {
@hmms = split ',', $hmm;
chomp @hmms;
### check for the presence of all hmms
for (my $k = 0; $k < @hmms; $k++) {
if (! -e "$hmm_dir/$hmms[$k]") {
push @log, "$hmms[$k] has been defined but could not be found in $hmm_dir/$hmms[$k]";
$check = 0;
last;
}
else {
push @log, "$hmms[$k] has been found\n";
}
}
}
else {
push @log, "running HaMStR with all hmms in $hmm_dir";
@hmms = `ls $hmm_dir`;
}
chomp @hmms;
print "succeeded\n";
}
}
## 6) Test for presence of the fasta file containing the sequences of the core-ortholog cluster
print "checking for presence of the core-ortholog file:\t";
if (defined $fafile) {
if (! -e "$fafile") {
push @log, "Could not find the file $fafile";
print "failed\n";
$check = 0;
}
else {
push @log, "check for $fafile succeeded";
print "succeeded\n";
}
}
else {
push @log, "Please provide path and name of fasta file containing the core-ortholog sequences";
$check = 0;
print "failed\n";
}
## 7) Checks for the taxon_file
print "testing whether the taxon has been determined:\t";
if (!(defined $taxon_file) or (!(-e "$taxon_file"))) {
if (defined $taxon_global) {
push @log, "using default taxon $taxon_global for all sequences";
print "succeeded\n";
$taxon_check = 2;
}
else {
push @log, "No taxon_file found. Please provide a global taxon name using the option -taxon";
print "failed\n";
$check = 0;
}
}
else {
push @log, "using the file $taxon_file as taxon_file";
print "succeeded\n";
$taxon_check = 1;
}
## 8) Check for reference taxon
print "Checking for reference species and blast-dbs\t";
if (!(defined $refspec_string) and (! defined $strict and ! defined $relaxed)) {
push @log, "Please provide a reference species for the reblast!";
print "failed\n";
$check = 0;
}
elsif (defined $strict or defined $relaxed) {
if (! defined $refspec_string) {
## The user has not provided a string of reference taxa. Chose all from the fasta file containing
## the core orthologs.
@refspec = `grep '>' $fafile |cut -d '|' -f 2 |sort |uniq`;
chomp @refspec;
$refspec_string = join ',', @refspec;
}
else {
@refspec = split (/,/, $refspec_string);
}
if ($strict) {
push @log, "Strict flag has been set. Reference species for the reblast: $refspec_string";
}
else {
push @log, "Relaxed flag has been set. Reference species for the reblast: $refspec_string";
}
if (@refspec == 0) {
print "failed\n";
$check = 0;
}
else {
print "succeeded\n";
}
}
else {
push @log, "Reference species for the re-blast: $refspec_string";
@refspec = split(/,/, $refspec_string);
$refspec_name = $refspec[0];
print "succeeded\n";
}
## 9) Check for presence of the required blast dbs
print "checking for blast-dbs:\t";
for (my $i = 0; $i < @refspec; $i++) {
my $blastpathtmp = "$blastpath/$refspec[$i]/$refspec[$i]".'_prot';
if (! (-e "$blastpathtmp.pin")) {
push @log, "please edit the blastpath. Could not find $blastpathtmp";
print "$blastpathtmp failed\n";
$check = 0;
}
else {
push @log, "check for $blastpathtmp succeeded";
print "succeeded\n";
}
}
## 10) Set the file where the matched seqs are found
my $strictstring = '';
if (defined $strict) {
$strictstring = '.strict';
}
$seqs2store_file = $dbpath . '/hamstrsearch_' . $dbfile_short . '_' . $hmmset . $strictstring . '.out';
$cds2store_file = $dbpath . '/hamstrsearch_' . $dbfile_short . '_' . $hmmset . '_cds' . $strictstring . '.out';
## 11) check for filter setting for BLAST
print "checking for low complexity filter setting:\t";
$filter =~ tr/ft/FT/;
if ($filter ne 'T' and $filter ne 'F') {
push @log, "Filter is set to $filter. Please set the low complexity filter either to F or T.";
print "low complexity filter check failed\n";
$check = 0;
}
else {
push @log, "check for low complexity filter setting succeeded. Chosen value is $filter";
print "succeeded\n";
}
## 12) apply the evalue-cut-off to the hmmsearch program
$prog = $prog . " -E $eval";
push @log, "hmmsearch: $prog";
## 12b) hit limit for the re-blast
if ($hitlimit) {
push @log, "re-blast hit_limit: $hitlimit";
}
else {
push @log, "re-blast hit_limit: none applied";
}
## 13) setting up the directories where the output files will be put into.
$fa_dir_neu = $dbpath . '/fa_dir_' . $dbfile_short . '_' . $hmmset . '_' . $refspec[0];
$tmpdir = $dbpath . '/' . $tmpdir;
if ($strict) {
$fa_dir_neu .= '_strict';
}
if ($relaxed) {
$fa_dir_neu .= '_relaxed';
}
if ($check == 1) {
if (!(-e "$hmmsearch_dir")) {
`mkdir "$hmmsearch_dir"`;
}
if (!(-e "$fa_dir_neu")) {
`mkdir "$fa_dir_neu"`;
}
if (!(-e "$tmpdir")) {
`mkdir "$tmpdir"`;
}
}
## 14) determin whether or not the -representative flag has been set
if (defined $rep) {
push @log, "HaMStR will run with the -representative option";
}
else {
push @log, "HaMStR was called without the -representative option. More than one ortholog may be identified per core-ortholog group!";
}
return ($check, @log);
}
#################
## check4reciprocity is the second major part of the program. It checks
## whether the protein sequence that has been identified by the hmmsearch
## identifies in turn the protein from the reference taxon that was used to
## build the hmm.
sub check4reciprocity {
my ($query_name, $hitname, $refspec_final, @refspec) = @_;
my $searchdb;
my $strict_suc = -1; # keeps track of success for all taxa
my $relaxed_suc = 0; # keeps track of success for at least one taxon
## get the sequence that was identified as hit in the pHMM search from the db_file
my $hitseq = `grep -m 1 -A 1 ">$hitname\$" $dbfile | tail -n 1`;
if (!defined $hitseq) {
print "could not retrieve a sequence for $hitname. Skipping...\n";
return(0, '', '', '');
}
## continue with the blast
chomp $hitseq;
## now run the blast
open (OUT, ">$tmpdir/$$.fa") or die "could not open out for writing\n";
print OUT ">$hitname\n$hitseq";
close OUT;
## now comes the new part that does one to many blast searches. We need to iterate through all
## entries in the file $refspec_final and perform the Blast against each reftaxon. Note, unless
## $strict or $relaxed flags are set, there will be only a single reftaxon. If $relaxed is chosen
## then we can stop the blast searches as soon as the reciprocity is fulfilled.
for (my $k = 0; $k < @$refspec_final; $k++) {
my $orthocount = $refspec_final->[$k]->{orthocount};
## 1) Perform the blast search with the k-th reftaxon
print "Reftaxon: $refspec_final->[$k]->{refspec}\n";
if ($blast_prog =~ /blastp/) {
!`$blast_prog -db $refspec_final->[$k]->{searchdb} -num_descriptions 10 -num_alignments 10 -query $tmpdir/$$.fa -out $tmpdir/$$.blast` or die "Problem running blastp\n";
### postprocess the outfile
`$sedprog -i -e 's/Length=\\([0-9]*\\)/ (\\1 letters)/' -e 's/^\\(>*\\)lcl|/\\1/' $tmpdir/$$.blast`;
}
else {
!`blastall -p blastp -d $refspec_final->[$k]->{searchdb} -F $filter -i $tmpdir/$$.fa -o $tmpdir/$$.blast` or die "Problem running blast\n";
}
## 2) now parse the best blast hit
my @hits = &getBestBlasthit("$tmpdir/$$.blast");
if (@hits > 0) {
my $idsref = $refspec_final->[$k]->{refid};
my @original_ids = @$idsref;
print "core_orthologs: ", join "\t", @original_ids , "\n";
## now loop through the best hits with the same evalue and check whether
## among these I find the same seq as in $original
my $i = 0;
my $suc = 0; # keeps track of success for a single taxon
while ($suc == 0 and $i <@hits) {
print "blast-hit: $hits[$i]";
## now loop through all the refspec-sequences in the hmm file
my $j = 0;
while ($suc == 0 and $j < @original_ids) {
if ($original_ids[$j] eq $hits[$i]) {
print "\thitting\n";
$refspec_final->[$k]->{hit} = $j;
$suc = 1;
$relaxed_suc = 1;
}
else {
print "\nnot hitting $original_ids[$j]\n";
$j ++;
}
if ($suc == 1) {
$relaxed_suc = 1;
if ($strict_suc == -1) {
$strict_suc = 1;
}
}
}
$i++;
}
if ($suc == 0) {
$strict_suc = 0; # none of the blast hits matched against the
# the reftaxon seq
}
}
else {
print "no hit obtained\n";
$strict_suc = 0;
}
## when the user has chosen the strict flag, there is no reason to continue when $suc
## has remained 0 (reciprocity criterion not fulfilled). Thus, return to main.
if ($strict and $strict_suc == 0) {
return (0, $hitseq);
}
}
if ($relaxed_suc == 1) {
return (1, $hitseq);
}
else {
return (0, $hitseq);
}
}
#############
sub getBestBlasthit {
my @hits;
my ($file) = @_;
my $searchio = Bio::SearchIO->new(-file => $file,
-format => 'blast',
-report_type => 'blastp') or die "parse failed";
while(my $result = $searchio->next_result){
my $count = 0;
my $sig;
my $sig_old;
while( my $hit = $result->next_hit){
## now I enter all top hits having the same evalue into the result
$sig = $hit->score;
if (!defined $sig_old) {
$sig_old = $sig;
}
if ($sig == $sig_old) {
push @hits, $hit->accession;
}
else {
last;
}
}
}
return(@hits);
}
##################
sub getTaxon {
my ($hitname) = @_;
# my $q = "select name from taxon t, est_project e, est_info i, annotation_neu a where a.id = $hitname and a.contig_id = i.contig_id and i.project_id = e.project_id and e.taxon_id = t.taxon_id";
if ($hitname =~ /\D/) {
$hitname =~ s/_.*//;
}
my $taxon = `grep -m 1 "^$hitname," $taxon_file | $sedprog -e 's/^.*,//'`;
chomp $taxon;
$taxon =~ s/^[0-9]+,//;
$taxon =~ s/\s*$//;
$taxon =~ s/\s/_/g;
if ($taxon) {
return ($taxon);
}
else {
return();
}
}
###############
sub determineReferences {
my ($fileobj, $taxon, $refspec_final, $hitname, $hitseq, $hitcount) = @_;
my $refseq = '';
my $refspec;
## now we have to distinguish between three cases:
## 1) hamstr is running in normal mode and one refspec has been determined. In this case, $refspec_final
## contains data only from a single species.
## 2) hamstr is running in normal mode and alternative refspecs have been determined by the user.
## $refspec_final may contain results from more than one species, but we need to consider only the first
## entry.
## 3) hamstr is running in the strict mode. In this case $refspec_final contains data from several taxa and we need
## to select the taxon and sequence that is most similar to the hamstered sequence.
## 4) hamstr is running in the relaxed mode. In this case $refspec_final may contain data from several taxa and
## we need to select the taxon and the sequence that is most similar to the hamstered sequence.
if (defined $strict or defined $relaxed) {
## more than one refspec. Now find the one that fits best
my $max_score = 0;
for (my $i = 0; $i < @$refspec_final; $i++) {
## first, check whether the reciprocity criterion has been fulfilled
if (defined $refspec_final->[$i]->{hit}) {
my $rcn = $refspec_final->[$i]->{hit};
my $refseq_cand = $refspec_final->[$i]->{sequence}->[$rcn];
my $refspec_cand_id = $refspec_final->[$i]->{refid}->[$rcn];
my $refspec_cand = $refspec_final->[$i]->{refspec};
my $score = &getAlignmentScore($refseq_cand, $hitseq);
if ($score > $max_score) {
$refspec = $refspec_cand;
$refseq = $refseq_cand;
$max_score = $score;
}
}
}
}
else { ## no choice, just one refspec
my $rcn = $refspec_final->[0]->{hit};
$refseq = $refspec_final->[0]->{sequence}->[$rcn];
$refspec = $refspec_final->[0]->{refspec};
}
$fileobj->{$taxon}->{prot}->[$hitcount] = $hitseq;
$fileobj->{$taxon}->{ids}->[$hitcount] = $hitname;
$fileobj->{$taxon}->{refseq}->[$hitcount]= $refseq;
$fileobj->{$taxon}->{refspec}->[$hitcount] = $refspec;
return($fileobj);
}
###############
sub processHits {
my ($fileobj) = @_;
## 1) align all hit sequences for a taxon against the reference species
my @taxa = keys(%$fileobj);
for (my $i = 0; $i < @taxa; $i++) {
&orfRanking($taxa[$i]);
}
}
################
sub predictORF {
my $fileobj_new;
my @taxa = keys(%$fileobj);
for (my $i = 0; $i < @taxa; $i++) {
my $protobj = $fileobj->{$taxa[$i]}->{prot};
my $idobj = $fileobj->{$taxa[$i]}->{ids};
my $refseqobj = $fileobj->{$taxa[$i]}->{refseq};
my $refspecobj = $fileobj->{$taxa[$i]}->{refspec};