avrilcoghlan · February 28, 2013 13:44
diff --git a/make_aln_and_hmm_for_treefam_family.pl b/make_aln_and_hmm_for_treefam_family.pl
 #!/usr/local/bin/perl

 =head1 NAME

    make_aln_and_hmm_for_treefam_family.pl

 =head1 SYNOPSIS
 
    make_aln_and_hmm_for_treefam_family.pl treefam_version output outputdir family hmmer_bin aln_output map_output

 =head1 DESCRIPTION

    For a TreeFam family of interest (<family>), this script reads in the list of sequences
    that belongs to the family from the TreeFam database, and builds a multiple sequence
    alignment for the family using MUSCLE, and then builds a HMM for the alignment using
    HMMER. The HMM is written in the output file <output>. <hmmer_bin> is the directory
    containing the hmmer executables. The multiple sequence alignment is written to file
    <aln_output>. The hmmbuild map output is put into <map_output>.

 =head1 VERSION
  
    Perl script last edited 25-Oct-2012.

 =head1 CONTACT

    [email protected] (Avril Coghlan)

 =cut

 # 
 # Perl script make_aln_and_hmm_for_treefam_family.pl
 # Written by Avril Coghlan ([email protected])
 # 25-Oct-12.
 # Last edited 25-Oct-2012.
 # SCRIPT SYNOPSIS: make_aln_and_hmm_for_treefam_family.pl: for a TreeFam family, gets sequences from the database, and builds an alignment & HMM
 #
 #------------------------------------------------------------------#

 # CHECK IF THERE ARE THE CORRECT NUMBER OF COMMAND-LINE ARGUMENTS:

 use strict;
 use warnings;
 use DBI;
 use Devel::Size qw(size total_size);

 my $num_args               = $#ARGV + 1;
 if ($num_args != 7)
 {
    print "Usage of make_aln_and_hmm_for_treefam_family.pl\n\n";
    print "perl make_aln_and_hmm_for_treefam_family.pl <treefam_version> <output> <outputdir> <family> <hmmer_bin> <aln_output> <map_output>\n";
    print "where <treefam_version> is the version of TreeFam to use (eg. 7),\n";
    print "      <output> is the output file of HMMs,\n";
    print "      <outputdir> is the directory for writing output files,\n";
    print "      <family> is the family of interest,\n";
    print "      <hmmer_bin> is the directory containing the hmmer executable,\n";
    print "      <aln_output> is the output multiple sequence alignment\n";
    print "For example, >perl make_aln_and_hmm_for_treefam_family.pl 7 myhmms\n";
    print "/nfs/users/nfs_a/alc/Documents/StrongyloidesTreeFam TF101000\n";
    print "/software/hmmer/bin/ myaln mymap\n";
    exit;
 }

 # FIND THE TREEFAM VERSION TO USE:                           

 my $treefam_version        = $ARGV[0];

 # FIND THE OUTPUT FILE NAME:

 my $output                 = $ARGV[1];

 # FIND THE DIRECTORY TO WRITE OUTPUT FILES TO:

 my $outputdir              = $ARGV[2];

 # FIND THE FAMILY OF INTEREST:            

 my $family                 = $ARGV[3];

 # FIND THE DIRECTORY WITH THE HMMER EXECUTABLES:

 my $hmmer_bin              = $ARGV[4];

 # FIND THE NAME OF THE OUTPUT MULTIPLE SEQUENCE ALIGNMENT:

 my $aln_output             = $ARGV[5];

 # FIND THE NAME OF THE FILE WITH THE hmmbuild MAP OUTPUT:

 my $map_output             = $ARGV[6];

 # TEST SUBROUTINES: 

 my $PRINT_TEST_DATA        = 0;   # SAYS WHETHER TO PRINT DATA USED DURING TESTING.
 my $PRINT_WARNINGS         = 0;   # SAYS WHETHER TO PRINT WARNINGS ABOUT MISSING DATA. 
 &test_print_error;
 &test_make_hmm($outputdir);

 #------------------------------------------------------------------#

 # RUN THE MAIN PART OF THE CODE:

 &run_main_program($treefam_version,$output,$outputdir,$family,$hmmer_bin,$aln_output,$map_output);

 print STDERR "FINISHED.\n";

 #------------------------------------------------------------------#

 # RUN THE MAIN PART OF THE CODE:

 sub run_main_program
 {
   my $version             = $_[0]; ## THE TREEFAM VERSION TO USE. 
   my $output              = $_[1]; ## THE OUTPUT FILE.     
   my $outputdir           = $_[2]; ## DIRECTORY TO WRITE OUTPUT FILES TO.
   my $family              = $_[3]; ## THE FAMILY OF INTEREST       
   my $hmmer_build         = $_[4]; ## DIRECTORY CONTAINING THE HMMER EXECUTABLES
   my $aln_output          = $_[5]; ## THE MULTIPLE SEQUENCE ALIGNMENT FILE NAME
   my $map_output          = $_[6]; ## THE hmmbuild MAP OUTPUT
   my $errorcode;                   ## RETURNED AS 0 IF THERE IS NO ERROR.
   my $errormsg;                    ## RETURNED AS 'none' IF THERE IS NO ERROR.
   my $fasta;                       ## FASTA FILE OF SEQUENCES IN THE FAMILY 
   my $genelist;                    ## FILE WITH IDENTIFIERS OF SEQUENCES FROM SPECIES THAT WERE ARE INTERESTED IN 
   my $genelist2;                   ## SHORTER FILE WITH IDENTIFIERS OF SEQUENCES FROM SPECIES THAT WERE ARE INTERESTED IN 

   # MAKE A FILE WITH THE IDENTIFIERS OF SEQUENCES FROM SPECIES THAT WE ARE INTERESTED IN:
   ($genelist,$genelist2,$errorcode,$errormsg) = &make_genelist($version,$outputdir); 
   if ($errorcode != 0) { ($errorcode,$errormsg) = &print_error($errormsg,$errorcode,0); }

   # GET THE SEQUENCES FOR THE FAMILY AND WRITE THEM TO A FASTA FILE:   
   ($fasta,$errorcode,$errormsg) = &make_fasta_of_seqs($version,$outputdir,$family,$genelist,$genelist2); 
   if ($errorcode != 0) { ($errorcode,$errormsg) = &print_error($errormsg,$errorcode,0); }

   # USE MUSCLE TO MAKE AN ALIGNMENT FOR THE FAMILY:
   ($errorcode,$errormsg)  = &make_aln($fasta,$aln_output);
   if ($errorcode != 0) { ($errorcode,$errormsg) = &print_error($errormsg,$errorcode,0); }

   # USE HMMER TO MAKE A HMM FOR THE FAMILY:
   ($errorcode,$errormsg)  = &make_hmm($aln_output,$hmmer_bin,$output,$map_output);
   if ($errorcode != 0) { ($errorcode,$errormsg) = &print_error($errormsg,$errorcode,0); }
 }

 #------------------------------------------------------------------#

 # USE MUSCLE TO MAKE AN ALIGNMENT FOR THE FAMILY:
 # SUBROUTINE SYNOPSIS: make_aln(): builds a multiple alignment for a TreeFam family, given a fasta file
 
 sub make_aln
 {
   my $fasta                = $_[0]; # FASTA FILE
   my $mfa                  = $_[1]; # THE MULTIPLE SEQUENCE ALIGNMENT FILE
   my $random_number;                # RANDOM NUMBER TO USE IN TEMPORARY FILE NAMES
   my $cmd;                          # COMMAND TO RUN
   my $errorcode            = 0;     # RETURNED AS 0 IF THERE IS NO ERROR
   my $errormsg             = "none";# RETURNED AS 'none' IF THERE IS NO ERROR 

   # CALL MUSCLE TO MAKE A MULTIPLE ALIGNMENT
   $cmd                     = "/software/pubseq/bin/muscle -in $fasta -out $mfa >& /dev/null";
   system "$cmd";
   # DELETE THE TEMPORARY FASTA FILE:
   system "rm -f $fasta";

   return($errorcode,$errormsg);
 }

 #------------------------------------------------------------------#

 # GET THE SEQUENCES FOR A FAMILY AND WRITE THEM TO A FASTA FILE:
 # SUBROUTINE SYNOPSIS: make_fasta_of_seqs(): retrieve sequences for a TreeFam family and write them to a fasta file

 sub make_fasta_of_seqs
 {
   my $version              = $_[0]; # VERSION OF THE TREEFAM DATABASE TO USE
   my $outputdir            = $_[1]; # DIRECTORY FOR WRITING OUTPUT FILES TO
   my $family               = $_[2]; # FAMILY OF INTEREST
   my $genelist             = $_[3]; # FILE WITH GENES FROM THE SPECIES WE WANT TO TAKE
   my $genelist2            = $_[4]; # SHORTER FILE WITH GENES FROM THE SPECIES WE WANT 
   my $fasta;                        # FASTA FILE FOR THE FAMILY 
   my $random_number;                # RANDOM NUMBER TO USE IN TEMPORARY FILE NAMES 
   my $database;                     # TREEFAM DATABASE TO USE
   my $dbh;                          #  
   my $st;                           #  
   my $sth;                          # 
   my $rv;                           # 
   my @array;                        # 
   my @ids;                          # ARRAY OF IDs TO TAKE 
   my $on_genelist;                  # SAYS WHETHER AN ID IS ON $genelist
   my $line;                         # 
   my @temp;                         # 
   my $no_ids;                       # NUMBER OF IDs IN @ids
   my $id;                           # AN IDENTIFIER FOR A SEQUENCE
   my $i;                            #  
   my $seq;                          # SEQUENCE FOR AN ID.
   my $table_w;                      # DATABASE TABLE OF INTEREST
   my $errorcode            = 0;     # RETURNED AS 0 IF THERE IS NO ERROR
   my $errormsg             = "none";# RETURNED AS 'none' IF THERE IS NO ERROR

   # OPEN A NEW FILE FOR WRITING THE SEQUENCES:
   $random_number           = rand();
   $fasta                   = $outputdir."/tmp".$random_number;
   open(FASTA,">$fasta") || die "ERROR: make_fasta_of_seqs: cannot open $fasta\n";

   # CONNECT TO THE DATABASE AND GET ALL SEQUENCES FOR THE FAMILY: 
   $database                = "treefam_".$version;
   $dbh                     = DBI->connect("dbi:mysql:$database:db.treefam.org:3308", 'anonymous', '') || return;

   # GET THE SEQUENCES FROM THE FAMILY FROM THE DATABASE:
   $table_w                 = "fam_genes";
   $st                      = "SELECT ID from $table_w WHERE AC=\'$family\'"; 
   $sth                     = $dbh->prepare($st) or die "Cannot prepare $st: $dbh->errstr\n";
   $rv                      = $sth->execute or die "Cannot execute the query: $sth->errstr";
   if ($rv >= 1)
   {
      while ((@array) = $sth->fetchrow_array)
      {
         $id                = $array[0];
         # CHECK IF $id APPEARS ON $genelist:
         $on_genelist       = 0;
         open(TMP,"grep $id $genelist |");
         while(<TMP>)
         {
            $line           = $_;
            chomp $line;
            if ($line eq $id) { $on_genelist = 1;}
         }
         close(TMP);
         if ($on_genelist == 1)
         {
            @ids            = (@ids,$id);
         }
      }
   }
  
   # IF THERE ARE MORE THAN 100 SEQUENCES, ONLY TAKE THE SEQUENCES FROM A FEW SPECIES:
   if ($#ids >= 100)
   {
      $no_ids               = $#ids + 1;
      for ($i = 1; $i <= $no_ids; $i++)
      {
         $id                = $ids[($i-1)];
         # CHECK IF THIS IS IN THE SHORTER FILE OF IDs FROM SPECIES TO TAKE:
         # CHECK IF $id APPEARS ON $genelist2:
         $on_genelist       = 0;
         open(TMP,"grep $id $genelist2 |");
         while(<TMP>)
         {
            $line           = $_;
            chomp $line;
            if ($line eq $id) { $on_genelist = 1;}
         }
         close(TMP);
         if ($on_genelist == 0) # RECORD THAT THIS $id IS NOT IN $genelist2:
         {
            $ids[$i]        = "NA";
         }
      }
   } 
 
   # IF THERE ARE <=5 SEQUENCES, TAKE ALL THE SEQUENCES FROM THE FAMILY:
   if ($#ids <= 5)
   {
      @ids                  = ();
      $table_w              = "fam_genes";
      $st                   = "SELECT ID from $table_w WHERE AC=\'$family\'"; 
      $sth                  = $dbh->prepare($st) or die "Cannot prepare $st: $dbh->errstr\n";
      $rv                   = $sth->execute or die "Cannot execute the query: $sth->errstr";
      if ($rv >= 1)
      {
         while ((@array) = $sth->fetchrow_array)
         {
            $id             = $array[0];
            @ids            = (@ids,$id);
         }
      }
   }

   # NOW GET THE SEQUENCES FOR THE IDs:
   $table_w                 = "aa_seq";
   $no_ids                  = $#ids + 1;
   for ($i = 1; $i <= $no_ids; $i++)
   {
      $id                   = $ids[($i-1)];
      if ($id ne 'NA')
      {
         $st                = "SELECT SEQ from $table_w WHERE ID=\'$id\'"; 
         $sth               = $dbh->prepare($st) or die "Cannot prepare $st: $dbh->errstr\n";
         $rv                = $sth->execute or die "Cannot execute the query: $sth->errstr";
         if ($rv >= 1)
         {
            while ((@array) = $sth->fetchrow_array)
            {
               $seq        = $array[0];
               # REMOVE *s FROM THE SEQUENCE:
               $seq        =~ s/\*//g;
               # REMOVE .s FROM THE SEQUENCE:
               $seq        =~ s/\*//g;
               print FASTA ">$id\n";            
               print FASTA "$seq\n";
            }
         }
      }
   }
   close(FASTA);

   # REMOVE THE GENELIST FILE:
   system "rm -f $genelist";
   system "rm -f $genelist2";
 
   $dbh->disconnect();

   return($fasta,$errorcode,$errormsg);

 }

 #------------------------------------------------------------------#

 # MAKE A FILE WITH THE IDENTIFIERS OF SEQUENCES FROM SPECIES THAT WE ARE INTERESTED IN:

 sub make_genelist            
 {
   my $version              = $_[0]; # VERSION OF THE TREEFAM DATABASE TO USE
   my $outputdir            = $_[1]; # DIRECTORY FOR WRITING OUTPUT FILES TO
   my $genelist;                     # FILE FOR WRITING THE LIST OF SEQUENCES TO
   my $random_number;                # RANDOM NUMBER TO USE IN TEMPORARY FILE NAMES
   my $database;                     # TREEFAM DATABASE TO CONNECT TO
   my $dbh;                          # 
   my $table_w;                      # TABLE TO QUERY FROM THE DATABASE
   my @species;                      # ARRAY OF SPECIES WE ARE INTERESTED IN
   my $num_species;                  # NUMBER OF SPECIES WE ARE INTERESTED IN
   my $species;                      # A SPECIES WE ARE INTERESTED IN
   my $i;                            # 
   my $st;                           # 
   my $sth;                          # 
   my $rv;                           # 
   my @array;                        # 
   my $id;                           # IDENTIFIER FOR SEQUENCE 
   my $errorcode            = 0;     # RETURNED AS 0 IF THERE IS NO ERROR
   my $errormsg             = 'none';# RETURNED AS 'none' IF THERE IS NO ERROR
   my $genelist2;                    # A SECOND FILE OF SPECIES TO TAKE, WITH FEWER SPECIES 
 
   # OPEN A NEW FILE FOR WRITING THE LIST OF SEQUENCES:
   $random_number           = rand();
   $genelist                = $outputdir."/tmp".$random_number;
   open(GENELIST,">$genelist") || die "ERROR: make_genelist: cannot open $genelist\n";
   $random_number           = rand();
   $genelist2               = $outputdir."/tmp".$random_number;
   open(GENELIST2,">$genelist2") || die "ERROR: make_genelist2: cannot open $genelist2\n";

   # CONNECT TO THE DATABASE AND GET ALL SEQUENCES OF PARTICULAR SPECIES: 
   $database                = "treefam_".$version;
   $dbh                     = DBI->connect("dbi:mysql:$database:db.treefam.org:3308", 'anonymous', '') || return;

   # GET THE SEQUENCES FROM CERTAIN SPECIES FROM THE DATABASE:
   $table_w                 = "genes";
   @species                 = ("6239", # CAENORHABDITIS ELEGANS
                               "6238", # CAENOBHABDITIS BRIGGSAE
                               "6279", # BRUGIA MALAYI
                               "7227", # DROSOPHILA MELANOGASTER
                               "7234", # DROSOPHILA PERSIMILIS
                               "7165", # ANOPHELES GAMBIAE
                               "7159", # AEDES AEGYPTI
                               "6183", # SCHISTOSOMA MANSONI
                               "45351",# NEMATOSTELLA VECTENSIS
                               "7719", # CIONA INTESTINALIS
                               "9606", # HUMAN
                               "9598", # CHIMP
                               "9544", # MACAQUE 
                               "10116",# RAT
                               "10090",# MOUSE 
                               "9913", # COW
                               "9615", # DOG
                               "13616",# MONODELPHIS DOMESTICA
                               "9031", # CHICKEN
                               "7955", # ZEBRAFISH
                               "8364", # XENOPUS TROPICALIS  
                               "99883",# TETRAODON NIGROVIRIDIS
                               "69293",# GASTEROSTEUS ACULEATUS (STICKLEBACK)
                               "4932", # SACCHAROMYCES CEREVISIAE
                               "4896", # SCHIZOSACCHAROMYCES POMBE
                               "3702", # ARABIDOPSIS THALIANA
                               "7668", # STRONGYLOCENTROTUS PURPURATUS
                               "44689");# DICTYOSTELIUM DISCOIDEUM
   $num_species             = $#species + 1; 
   for ($i = 1; $i <= $num_species; $i++)
   {
      $species              = $species[($i-1)];
      $st                   = "SELECT ID from $table_w WHERE TAX_ID=\'$species\'"; 
      $sth                  = $dbh->prepare($st) or die "Cannot prepare $st: $dbh->errstr\n";
      $rv                   = $sth->execute or die "Cannot execute the query: $sth->errstr";
      if ($rv >= 1)
      {
         while ((@array) = $sth->fetchrow_array)
   {
 	    $id             = $array[0];
            print GENELIST "$id\n";
            if ($species eq '6329' || $species eq '7227' || $species eq '6183' ||  # C. ELEGANS, D. MELANOGASTER, S. MANSONI
                $species eq '45351'|| $species eq '7719' || $species eq '9606' ||  # N. VECTENSIS, C. INTESTINALIS, HUMAN
                $species eq '4932' ||                                              # S. CEREVISIAE
                $species eq '3702' || $species eq '7668' || $species eq '44689')   # A. THALIANA, S. PURPURATUS, D. DICOIDEUM
            {
               print GENELIST2 "$id\n";
            }
         }
      }
   }
   close(GENELIST);
   close(GENELIST2);
   $dbh->disconnect();

   return($genelist,$genelist2,$errorcode,$errormsg);

 }

 #------------------------------------------------------------------#

 # TEST &make_hmm

 sub test_make_hmm
 {
   my $outputdir            = $_[0]; ## DIRECTORY FOR WRITING OUTPUT FILES TO 
   my $errorcode;                    ## RETURNED AS 0 BY A FUNCTION IF THERE IS NO ERROR
   my $errormsg;                     ## RETURNED AS 'none' BY A FUNCTION IF THERE IS NO ERROR
   my $random_number;                ## RANDOM NUMBER FOR TEMPORARY FILE NAME
   my $mfafile;                      ## FILE FOR WRITING MULTIPLE ALIGNMENT TO 
   my $hmmfile;                      ## FILE FOR WRITING THE HMM TO 
   my $map_output;                   ## hmmbuild MAP OUTPUT FILE 

   $random_number           = rand();
   $mfafile                 = $outputdir."/tmp".$random_number;
   $random_number           = rand();
   $hmmfile                 = $outputdir."/tmp".$random_number;
   $random_number           = rand();
   $map_output              = $outputdir."/tmp".$random_number;
   open(MFAFILE,">$mfafile") || die "ERROR: cannot open $mfafile\n";
   print MFAFILE ">seq1\nACGTACC\n";
   print MFAFILE ">seq2\n-CCTACC\n";
   print MFAFILE ">seq3\nAC-TACC\n";
   close(MFAFILE);
   ($errorcode,$errormsg) = &make_hmm($mfafile,"/software/hmmer/bin/",$hmmfile,$map_output);
   if ($errorcode != 0) { print STDERR "ERROR: test_make_hmm: failed test1\n"; exit;}
   system "rm -f $mfafile";
   system "rm -f $hmmfile";
   system "rm -f $map_output";
 }

 #------------------------------------------------------------------#

 # MAKE A HMM FOR THIS FAMILY:
 # SUBROUTINE SYNOPSIS: make_hmm(): build a HMM for a TreeFam family, given a multiple alignment

 sub make_hmm
 {
   my $mfafile              = $_[0]; ## FILE CONTAINING THE MULTIPLE ALIGNMENT FOR THE FAMILY
   my $hmmer_bin            = $_[1]; ## DIRECTORY CONTAINING THE HMMER EXECUTABLES
   my $output               = $_[2]; ## NAME OF THE OUTPUT HMM FILE 
   my $map_output           = $_[3]; ## NAME OF THE HMMBUILD MAP OUTPUT FILE
   my $errorcode            = 0;     ## RETURNED AS 0 IF THERE IS NO ERROR
   my $errormsg             = "none";## RETURNED AS 'none' IF THERE IS NO ERROR
   my $cmd;                          ## COMMAND TO RUN 

   # RUN HMMER2 TO MAKE THE HMM:
   
   $cmd                      = $hmmer_bin."/hmmbuild -o $map_output $output $mfafile >& /dev/null";
   system "$cmd";
   $cmd                      = $hmmer_bin."/hmmcalibrate $output >& /dev/null";
   system "$cmd";

   return($errorcode,$errormsg); 
 }

 #------------------------------------------------------------------#

 # TEST &print_error

 sub test_print_error
 {
   my $errormsg;                    # RETURNED AS 'none' FROM A FUNCTION IF THERE WAS NO ERROR
   my $errorcode;                   # RETURNED AS 0 FROM A FUNCTION IF THERE WAS NO ERROR

   ($errormsg,$errorcode)  = &print_error(45,45,1);
   if ($errorcode != 12) { print STDERR "ERROR: test_print_error: failed test1\n"; exit;}

   ($errormsg,$errorcode)  = &print_error('My error message','My error message',1);
   if ($errorcode != 11) { print STDERR "ERROR: test_print_error: failed test2\n"; exit;}

   ($errormsg,$errorcode)  = &print_error('none',45,1);
   if ($errorcode != 13) { print STDERR "ERROR: test_print_error: failed test3\n"; exit;} 

   ($errormsg,$errorcode)  = &print_error('My error message', 0, 1);
   if ($errorcode != 13) { print STDERR "ERROR: test_print_error: failed test4\n"; exit;}
 }

 #------------------------------------------------------------------#

 # PRINT OUT AN ERROR MESSAGE AND EXIT.
 
 sub print_error
 {
   my $errormsg            = $_[0]; # THIS SHOULD BE NOT 'none' IF AN ERROR OCCURRED.
   my $errorcode           = $_[1]; # THIS SHOULD NOT BE 0 IF AN ERROR OCCURRED.
   my $called_from_test    = $_[2]; # SAYS WHETHER THIS WAS CALLED FROM test_print_error OR NOT

   if ($errorcode =~ /[A-Z]/ || $errorcode =~ /[a-z]/) 
   { 
      if ($called_from_test == 1)
      {
         $errorcode = 11; $errormsg = "ERROR: print_error: the errorcode is $errorcode, should be a number.\n"; # ERRORCODE=11
         return($errormsg,$errorcode);
      }
      else 
      { 
         print STDERR "ERROR: print_error: the errorcode is $errorcode, should be a number.\n"; 
         exit;
      }
   }

   if (!($errormsg =~ /[A-Z]/ || $errormsg =~ /[a-z]/)) 
   { 
      if ($called_from_test == 1)
      {
         $errorcode = 12; $errormsg = "ERROR: print_error: the errormessage $errormsg does not seem to contain text.\n"; # ERRORCODE=12
         return($errormsg,$errorcode);
      }
      else
      {
         print STDERR "ERROR: print_error: the errormessage $errormsg does not seem to contain text.\n"; 
         exit;
      }
   }

   if    ($errormsg eq 'none' || $errorcode == 0) 
   { 
      if ($called_from_test == 1)
      {
         $errorcode = 13; $errormsg = "ERROR: print_error: errormsg $errormsg, errorcode $errorcode.\n"; # ERRORCODE=13
         return($errormsg,$errorcode);
      }
      else 
      {
         print STDERR "ERROR: print_error: errormsg $errormsg, errorcode $errorcode.\n"; 
         exit;
      }
   }
   else                                           
   { 
      print STDERR "$errormsg"; 
      exit;                                                      
   } 

   return($errormsg,$errorcode);
 }

 #------------------------------------------------------------------#
	#!/usr/local/bin/perl

	=head1 NAME

	make_aln_and_hmm_for_treefam_family.pl

	=head1 SYNOPSIS

	make_aln_and_hmm_for_treefam_family.pl treefam_version output outputdir family hmmer_bin aln_output map_output

	=head1 DESCRIPTION

	For a TreeFam family of interest (<family>), this script reads in the list of sequences
	that belongs to the family from the TreeFam database, and builds a multiple sequence
	alignment for the family using MUSCLE, and then builds a HMM for the alignment using
	HMMER. The HMM is written in the output file <output>. <hmmer_bin> is the directory
	containing the hmmer executables. The multiple sequence alignment is written to file
	<aln_output>. The hmmbuild map output is put into <map_output>.

	=head1 VERSION

	Perl script last edited 25-Oct-2012.

	=head1 CONTACT

	[email protected] (Avril Coghlan)

	=cut

	#
	# Perl script make_aln_and_hmm_for_treefam_family.pl
	# Written by Avril Coghlan ([email protected])
	# 25-Oct-12.
	# Last edited 25-Oct-2012.
	# SCRIPT SYNOPSIS: make_aln_and_hmm_for_treefam_family.pl: for a TreeFam family, gets sequences from the database, and builds an alignment & HMM
	#
	#------------------------------------------------------------------#

	# CHECK IF THERE ARE THE CORRECT NUMBER OF COMMAND-LINE ARGUMENTS:

	use strict;
	use warnings;
	use DBI;
	use Devel::Size qw(size total_size);

	my $num_args = $#ARGV + 1;
	if ($num_args != 7)
	{
	print "Usage of make_aln_and_hmm_for_treefam_family.pl\n\n";
	print "perl make_aln_and_hmm_for_treefam_family.pl <treefam_version> <output> <outputdir> <family> <hmmer_bin> <aln_output> <map_output>\n";
	print "where <treefam_version> is the version of TreeFam to use (eg. 7),\n";
	print " <output> is the output file of HMMs,\n";
	print " <outputdir> is the directory for writing output files,\n";
	print " <family> is the family of interest,\n";
	print " <hmmer_bin> is the directory containing the hmmer executable,\n";
	print " <aln_output> is the output multiple sequence alignment\n";
	print "For example, >perl make_aln_and_hmm_for_treefam_family.pl 7 myhmms\n";
	print "/nfs/users/nfs_a/alc/Documents/StrongyloidesTreeFam TF101000\n";
	print "/software/hmmer/bin/ myaln mymap\n";
	exit;
	}

	# FIND THE TREEFAM VERSION TO USE:

	my $treefam_version = $ARGV[0];

	# FIND THE OUTPUT FILE NAME:

	my $output = $ARGV[1];

	# FIND THE DIRECTORY TO WRITE OUTPUT FILES TO:

	my $outputdir = $ARGV[2];

	# FIND THE FAMILY OF INTEREST:

	my $family = $ARGV[3];

	# FIND THE DIRECTORY WITH THE HMMER EXECUTABLES:

	my $hmmer_bin = $ARGV[4];

	# FIND THE NAME OF THE OUTPUT MULTIPLE SEQUENCE ALIGNMENT:

	my $aln_output = $ARGV[5];

	# FIND THE NAME OF THE FILE WITH THE hmmbuild MAP OUTPUT:

	my $map_output = $ARGV[6];

	# TEST SUBROUTINES:

	my $PRINT_TEST_DATA = 0; # SAYS WHETHER TO PRINT DATA USED DURING TESTING.
	my $PRINT_WARNINGS = 0; # SAYS WHETHER TO PRINT WARNINGS ABOUT MISSING DATA.
	&test_print_error;
	&test_make_hmm($outputdir);

	#------------------------------------------------------------------#

	# RUN THE MAIN PART OF THE CODE:

	&run_main_program($treefam_version,$output,$outputdir,$family,$hmmer_bin,$aln_output,$map_output);

	print STDERR "FINISHED.\n";

	#------------------------------------------------------------------#

	# RUN THE MAIN PART OF THE CODE:

	sub run_main_program
	{
	my $version = $_[0]; ## THE TREEFAM VERSION TO USE.
	my $output = $_[1]; ## THE OUTPUT FILE.
	my $outputdir = $_[2]; ## DIRECTORY TO WRITE OUTPUT FILES TO.
	my $family = $_[3]; ## THE FAMILY OF INTEREST
	my $hmmer_build = $_[4]; ## DIRECTORY CONTAINING THE HMMER EXECUTABLES
	my $aln_output = $_[5]; ## THE MULTIPLE SEQUENCE ALIGNMENT FILE NAME
	my $map_output = $_[6]; ## THE hmmbuild MAP OUTPUT
	my $errorcode; ## RETURNED AS 0 IF THERE IS NO ERROR.
	my $errormsg; ## RETURNED AS 'none' IF THERE IS NO ERROR.
	my $fasta; ## FASTA FILE OF SEQUENCES IN THE FAMILY
	my $genelist; ## FILE WITH IDENTIFIERS OF SEQUENCES FROM SPECIES THAT WERE ARE INTERESTED IN
	my $genelist2; ## SHORTER FILE WITH IDENTIFIERS OF SEQUENCES FROM SPECIES THAT WERE ARE INTERESTED IN

	# MAKE A FILE WITH THE IDENTIFIERS OF SEQUENCES FROM SPECIES THAT WE ARE INTERESTED IN:
	($genelist,$genelist2,$errorcode,$errormsg) = &make_genelist($version,$outputdir);
	if ($errorcode != 0) { ($errorcode,$errormsg) = &print_error($errormsg,$errorcode,0); }

	# GET THE SEQUENCES FOR THE FAMILY AND WRITE THEM TO A FASTA FILE:
	($fasta,$errorcode,$errormsg) = &make_fasta_of_seqs($version,$outputdir,$family,$genelist,$genelist2);
	if ($errorcode != 0) { ($errorcode,$errormsg) = &print_error($errormsg,$errorcode,0); }

	# USE MUSCLE TO MAKE AN ALIGNMENT FOR THE FAMILY:
	($errorcode,$errormsg) = &make_aln($fasta,$aln_output);
	if ($errorcode != 0) { ($errorcode,$errormsg) = &print_error($errormsg,$errorcode,0); }

	# USE HMMER TO MAKE A HMM FOR THE FAMILY:
	($errorcode,$errormsg) = &make_hmm($aln_output,$hmmer_bin,$output,$map_output);
	if ($errorcode != 0) { ($errorcode,$errormsg) = &print_error($errormsg,$errorcode,0); }
	}

	#------------------------------------------------------------------#

	# USE MUSCLE TO MAKE AN ALIGNMENT FOR THE FAMILY:
	# SUBROUTINE SYNOPSIS: make_aln(): builds a multiple alignment for a TreeFam family, given a fasta file

	sub make_aln
	{
	my $fasta = $_[0]; # FASTA FILE
	my $mfa = $_[1]; # THE MULTIPLE SEQUENCE ALIGNMENT FILE
	my $random_number; # RANDOM NUMBER TO USE IN TEMPORARY FILE NAMES
	my $cmd; # COMMAND TO RUN
	my $errorcode = 0; # RETURNED AS 0 IF THERE IS NO ERROR
	my $errormsg = "none";# RETURNED AS 'none' IF THERE IS NO ERROR

	# CALL MUSCLE TO MAKE A MULTIPLE ALIGNMENT
	$cmd = "/software/pubseq/bin/muscle -in $fasta -out $mfa >& /dev/null";
	system "$cmd";
	# DELETE THE TEMPORARY FASTA FILE:
	system "rm -f $fasta";

	return($errorcode,$errormsg);
	}

	#------------------------------------------------------------------#

	# GET THE SEQUENCES FOR A FAMILY AND WRITE THEM TO A FASTA FILE:
	# SUBROUTINE SYNOPSIS: make_fasta_of_seqs(): retrieve sequences for a TreeFam family and write them to a fasta file

	sub make_fasta_of_seqs
	{
	my $version = $_[0]; # VERSION OF THE TREEFAM DATABASE TO USE
	my $outputdir = $_[1]; # DIRECTORY FOR WRITING OUTPUT FILES TO
	my $family = $_[2]; # FAMILY OF INTEREST
	my $genelist = $_[3]; # FILE WITH GENES FROM THE SPECIES WE WANT TO TAKE
	my $genelist2 = $_[4]; # SHORTER FILE WITH GENES FROM THE SPECIES WE WANT
	my $fasta; # FASTA FILE FOR THE FAMILY
	my $random_number; # RANDOM NUMBER TO USE IN TEMPORARY FILE NAMES
	my $database; # TREEFAM DATABASE TO USE
	my $dbh; #
	my $st; #
	my $sth; #
	my $rv; #
	my @array; #
	my @ids; # ARRAY OF IDs TO TAKE
	my $on_genelist; # SAYS WHETHER AN ID IS ON $genelist
	my $line; #
	my @temp; #
	my $no_ids; # NUMBER OF IDs IN @ids
	my $id; # AN IDENTIFIER FOR A SEQUENCE
	my $i; #
	my $seq; # SEQUENCE FOR AN ID.
	my $table_w; # DATABASE TABLE OF INTEREST
	my $errorcode = 0; # RETURNED AS 0 IF THERE IS NO ERROR
	my $errormsg = "none";# RETURNED AS 'none' IF THERE IS NO ERROR

	# OPEN A NEW FILE FOR WRITING THE SEQUENCES:
	$random_number = rand();
	$fasta = $outputdir."/tmp".$random_number;
	open(FASTA,">$fasta") \|\| die "ERROR: make_fasta_of_seqs: cannot open $fasta\n";

	# CONNECT TO THE DATABASE AND GET ALL SEQUENCES FOR THE FAMILY:
	$database = "treefam_".$version;
	$dbh = DBI->connect("dbi:mysql:$database:db.treefam.org:3308", 'anonymous', '') \|\| return;

	# GET THE SEQUENCES FROM THE FAMILY FROM THE DATABASE:
	$table_w = "fam_genes";
	$st = "SELECT ID from $table_w WHERE AC=\'$family\'";
	$sth = $dbh->prepare($st) or die "Cannot prepare $st: $dbh->errstr\n";
	$rv = $sth->execute or die "Cannot execute the query: $sth->errstr";
	if ($rv >= 1)
	{
	while ((@array) = $sth->fetchrow_array)
	{
	$id = $array[0];
	# CHECK IF $id APPEARS ON $genelist:
	$on_genelist = 0;
	open(TMP,"grep $id $genelist \|");
	while(<TMP>)
	{
	$line = $_;
	chomp $line;
	if ($line eq $id) { $on_genelist = 1;}
	}
	close(TMP);
	if ($on_genelist == 1)
	{
	@ids = (@ids,$id);
	}
	}
	}

	# IF THERE ARE MORE THAN 100 SEQUENCES, ONLY TAKE THE SEQUENCES FROM A FEW SPECIES:
	if ($#ids >= 100)
	{
	$no_ids = $#ids + 1;
	for ($i = 1; $i <= $no_ids; $i++)
	{
	$id = $ids[($i-1)];
	# CHECK IF THIS IS IN THE SHORTER FILE OF IDs FROM SPECIES TO TAKE:
	# CHECK IF $id APPEARS ON $genelist2:
	$on_genelist = 0;
	open(TMP,"grep $id $genelist2 \|");
	while(<TMP>)
	{
	$line = $_;
	chomp $line;
	if ($line eq $id) { $on_genelist = 1;}
	}
	close(TMP);
	if ($on_genelist == 0) # RECORD THAT THIS $id IS NOT IN $genelist2:
	{
	$ids[$i] = "NA";
	}
	}
	}

	# IF THERE ARE <=5 SEQUENCES, TAKE ALL THE SEQUENCES FROM THE FAMILY:
	if ($#ids <= 5)
	{
	@ids = ();
	$table_w = "fam_genes";
	$st = "SELECT ID from $table_w WHERE AC=\'$family\'";
	$sth = $dbh->prepare($st) or die "Cannot prepare $st: $dbh->errstr\n";
	$rv = $sth->execute or die "Cannot execute the query: $sth->errstr";
	if ($rv >= 1)
	{
	while ((@array) = $sth->fetchrow_array)
	{
	$id = $array[0];
	@ids = (@ids,$id);
	}
	}
	}

	# NOW GET THE SEQUENCES FOR THE IDs:
	$table_w = "aa_seq";
	$no_ids = $#ids + 1;
	for ($i = 1; $i <= $no_ids; $i++)
	{
	$id = $ids[($i-1)];
	if ($id ne 'NA')
	{
	$st = "SELECT SEQ from $table_w WHERE ID=\'$id\'";
	$sth = $dbh->prepare($st) or die "Cannot prepare $st: $dbh->errstr\n";
	$rv = $sth->execute or die "Cannot execute the query: $sth->errstr";
	if ($rv >= 1)
	{
	while ((@array) = $sth->fetchrow_array)
	{
	$seq = $array[0];
	# REMOVE *s FROM THE SEQUENCE:
	$seq =~ s/\*//g;
	# REMOVE .s FROM THE SEQUENCE:
	$seq =~ s/\*//g;
	print FASTA ">$id\n";
	print FASTA "$seq\n";
	}
	}
	}
	}
	close(FASTA);

	# REMOVE THE GENELIST FILE:
	system "rm -f $genelist";
	system "rm -f $genelist2";

	$dbh->disconnect();

	return($fasta,$errorcode,$errormsg);

	}

	#------------------------------------------------------------------#

	# MAKE A FILE WITH THE IDENTIFIERS OF SEQUENCES FROM SPECIES THAT WE ARE INTERESTED IN:

	sub make_genelist
	{
	my $version = $_[0]; # VERSION OF THE TREEFAM DATABASE TO USE
	my $outputdir = $_[1]; # DIRECTORY FOR WRITING OUTPUT FILES TO
	my $genelist; # FILE FOR WRITING THE LIST OF SEQUENCES TO
	my $random_number; # RANDOM NUMBER TO USE IN TEMPORARY FILE NAMES
	my $database; # TREEFAM DATABASE TO CONNECT TO
	my $dbh; #
	my $table_w; # TABLE TO QUERY FROM THE DATABASE
	my @species; # ARRAY OF SPECIES WE ARE INTERESTED IN
	my $num_species; # NUMBER OF SPECIES WE ARE INTERESTED IN
	my $species; # A SPECIES WE ARE INTERESTED IN
	my $i; #
	my $st; #
	my $sth; #
	my $rv; #
	my @array; #
	my $id; # IDENTIFIER FOR SEQUENCE
	my $errorcode = 0; # RETURNED AS 0 IF THERE IS NO ERROR
	my $errormsg = 'none';# RETURNED AS 'none' IF THERE IS NO ERROR
	my $genelist2; # A SECOND FILE OF SPECIES TO TAKE, WITH FEWER SPECIES

	# OPEN A NEW FILE FOR WRITING THE LIST OF SEQUENCES:
	$random_number = rand();
	$genelist = $outputdir."/tmp".$random_number;
	open(GENELIST,">$genelist") \|\| die "ERROR: make_genelist: cannot open $genelist\n";
	$random_number = rand();
	$genelist2 = $outputdir."/tmp".$random_number;
	open(GENELIST2,">$genelist2") \|\| die "ERROR: make_genelist2: cannot open $genelist2\n";

	# CONNECT TO THE DATABASE AND GET ALL SEQUENCES OF PARTICULAR SPECIES:
	$database = "treefam_".$version;
	$dbh = DBI->connect("dbi:mysql:$database:db.treefam.org:3308", 'anonymous', '') \|\| return;

	# GET THE SEQUENCES FROM CERTAIN SPECIES FROM THE DATABASE:
	$table_w = "genes";
	@species = ("6239", # CAENORHABDITIS ELEGANS
	"6238", # CAENOBHABDITIS BRIGGSAE
	"6279", # BRUGIA MALAYI
	"7227", # DROSOPHILA MELANOGASTER
	"7234", # DROSOPHILA PERSIMILIS
	"7165", # ANOPHELES GAMBIAE
	"7159", # AEDES AEGYPTI
	"6183", # SCHISTOSOMA MANSONI
	"45351",# NEMATOSTELLA VECTENSIS
	"7719", # CIONA INTESTINALIS
	"9606", # HUMAN
	"9598", # CHIMP
	"9544", # MACAQUE
	"10116",# RAT
	"10090",# MOUSE
	"9913", # COW
	"9615", # DOG
	"13616",# MONODELPHIS DOMESTICA
	"9031", # CHICKEN
	"7955", # ZEBRAFISH
	"8364", # XENOPUS TROPICALIS
	"99883",# TETRAODON NIGROVIRIDIS
	"69293",# GASTEROSTEUS ACULEATUS (STICKLEBACK)
	"4932", # SACCHAROMYCES CEREVISIAE
	"4896", # SCHIZOSACCHAROMYCES POMBE
	"3702", # ARABIDOPSIS THALIANA
	"7668", # STRONGYLOCENTROTUS PURPURATUS
	"44689");# DICTYOSTELIUM DISCOIDEUM
	$num_species = $#species + 1;
	for ($i = 1; $i <= $num_species; $i++)
	{
	$species = $species[($i-1)];
	$st = "SELECT ID from $table_w WHERE TAX_ID=\'$species\'";
	$sth = $dbh->prepare($st) or die "Cannot prepare $st: $dbh->errstr\n";
	$rv = $sth->execute or die "Cannot execute the query: $sth->errstr";
	if ($rv >= 1)
	{
	while ((@array) = $sth->fetchrow_array)
	{
	$id = $array[0];
	print GENELIST "$id\n";
	if ($species eq '6329' \|\| $species eq '7227' \|\| $species eq '6183' \|\| # C. ELEGANS, D. MELANOGASTER, S. MANSONI
	$species eq '45351'\|\| $species eq '7719' \|\| $species eq '9606' \|\| # N. VECTENSIS, C. INTESTINALIS, HUMAN
	$species eq '4932' \|\| # S. CEREVISIAE
	$species eq '3702' \|\| $species eq '7668' \|\| $species eq '44689') # A. THALIANA, S. PURPURATUS, D. DICOIDEUM
	{
	print GENELIST2 "$id\n";
	}
	}
	}
	}
	close(GENELIST);
	close(GENELIST2);
	$dbh->disconnect();

	return($genelist,$genelist2,$errorcode,$errormsg);

	}

	#------------------------------------------------------------------#

	# TEST &make_hmm

	sub test_make_hmm
	{
	my $outputdir = $_[0]; ## DIRECTORY FOR WRITING OUTPUT FILES TO
	my $errorcode; ## RETURNED AS 0 BY A FUNCTION IF THERE IS NO ERROR
	my $errormsg; ## RETURNED AS 'none' BY A FUNCTION IF THERE IS NO ERROR
	my $random_number; ## RANDOM NUMBER FOR TEMPORARY FILE NAME
	my $mfafile; ## FILE FOR WRITING MULTIPLE ALIGNMENT TO
	my $hmmfile; ## FILE FOR WRITING THE HMM TO
	my $map_output; ## hmmbuild MAP OUTPUT FILE

	$random_number = rand();
	$mfafile = $outputdir."/tmp".$random_number;
	$random_number = rand();
	$hmmfile = $outputdir."/tmp".$random_number;
	$random_number = rand();
	$map_output = $outputdir."/tmp".$random_number;
	open(MFAFILE,">$mfafile") \|\| die "ERROR: cannot open $mfafile\n";
	print MFAFILE ">seq1\nACGTACC\n";
	print MFAFILE ">seq2\n-CCTACC\n";
	print MFAFILE ">seq3\nAC-TACC\n";
	close(MFAFILE);
	($errorcode,$errormsg) = &make_hmm($mfafile,"/software/hmmer/bin/",$hmmfile,$map_output);
	if ($errorcode != 0) { print STDERR "ERROR: test_make_hmm: failed test1\n"; exit;}
	system "rm -f $mfafile";
	system "rm -f $hmmfile";
	system "rm -f $map_output";
	}

	#------------------------------------------------------------------#

	# MAKE A HMM FOR THIS FAMILY:
	# SUBROUTINE SYNOPSIS: make_hmm(): build a HMM for a TreeFam family, given a multiple alignment

	sub make_hmm
	{
	my $mfafile = $_[0]; ## FILE CONTAINING THE MULTIPLE ALIGNMENT FOR THE FAMILY
	my $hmmer_bin = $_[1]; ## DIRECTORY CONTAINING THE HMMER EXECUTABLES
	my $output = $_[2]; ## NAME OF THE OUTPUT HMM FILE
	my $map_output = $_[3]; ## NAME OF THE HMMBUILD MAP OUTPUT FILE
	my $errorcode = 0; ## RETURNED AS 0 IF THERE IS NO ERROR
	my $errormsg = "none";## RETURNED AS 'none' IF THERE IS NO ERROR
	my $cmd; ## COMMAND TO RUN

	# RUN HMMER2 TO MAKE THE HMM:

	$cmd = $hmmer_bin."/hmmbuild -o $map_output $output $mfafile >& /dev/null";
	system "$cmd";
	$cmd = $hmmer_bin."/hmmcalibrate $output >& /dev/null";
	system "$cmd";

	return($errorcode,$errormsg);
	}

	#------------------------------------------------------------------#

	# TEST &print_error

	sub test_print_error
	{
	my $errormsg; # RETURNED AS 'none' FROM A FUNCTION IF THERE WAS NO ERROR
	my $errorcode; # RETURNED AS 0 FROM A FUNCTION IF THERE WAS NO ERROR

	($errormsg,$errorcode) = &print_error(45,45,1);
	if ($errorcode != 12) { print STDERR "ERROR: test_print_error: failed test1\n"; exit;}

	($errormsg,$errorcode) = &print_error('My error message','My error message',1);
	if ($errorcode != 11) { print STDERR "ERROR: test_print_error: failed test2\n"; exit;}

	($errormsg,$errorcode) = &print_error('none',45,1);
	if ($errorcode != 13) { print STDERR "ERROR: test_print_error: failed test3\n"; exit;}

	($errormsg,$errorcode) = &print_error('My error message', 0, 1);
	if ($errorcode != 13) { print STDERR "ERROR: test_print_error: failed test4\n"; exit;}
	}

	#------------------------------------------------------------------#

	# PRINT OUT AN ERROR MESSAGE AND EXIT.

	sub print_error
	{
	my $errormsg = $_[0]; # THIS SHOULD BE NOT 'none' IF AN ERROR OCCURRED.
	my $errorcode = $_[1]; # THIS SHOULD NOT BE 0 IF AN ERROR OCCURRED.
	my $called_from_test = $_[2]; # SAYS WHETHER THIS WAS CALLED FROM test_print_error OR NOT

	if ($errorcode =~ /[A-Z]/ \|\| $errorcode =~ /[a-z]/)
	{
	if ($called_from_test == 1)
	{
	$errorcode = 11; $errormsg = "ERROR: print_error: the errorcode is $errorcode, should be a number.\n"; # ERRORCODE=11
	return($errormsg,$errorcode);
	}
	else
	{
	print STDERR "ERROR: print_error: the errorcode is $errorcode, should be a number.\n";
	exit;
	}
	}

	if (!($errormsg =~ /[A-Z]/ \|\| $errormsg =~ /[a-z]/))
	{
	if ($called_from_test == 1)
	{
	$errorcode = 12; $errormsg = "ERROR: print_error: the errormessage $errormsg does not seem to contain text.\n"; # ERRORCODE=12
	return($errormsg,$errorcode);
	}
	else
	{
	print STDERR "ERROR: print_error: the errormessage $errormsg does not seem to contain text.\n";
	exit;
	}
	}

	if ($errormsg eq 'none' \|\| $errorcode == 0)
	{
	if ($called_from_test == 1)
	{
	$errorcode = 13; $errormsg = "ERROR: print_error: errormsg $errormsg, errorcode $errorcode.\n"; # ERRORCODE=13
	return($errormsg,$errorcode);
	}
	else
	{
	print STDERR "ERROR: print_error: errormsg $errormsg, errorcode $errorcode.\n";
	exit;
	}
	}
	else
	{
	print STDERR "$errormsg";
	exit;
	}

	return($errormsg,$errorcode);
	}

	#------------------------------------------------------------------#