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 # # BioPerl module for Bio::Tools::Run::Phylo::SLR # # Please direct questions and support issues to # # Cared for by Albert Vilella # # Copyright Albert Vilella # # You may distribute this module under the same terms as perl itself # POD documentation - main docs before the code =head1 NAME Bio::Tools::Run::Phylo::SLR - Wrapper around the SLR program =head1 SYNOPSIS use Bio::Tools::Run::Phylo::SLR; use Bio::AlignIO; use Bio::TreeIO; use Bio::SimpleAlign; my alignio = Bio::AlignIO->new (-format => 'fasta', -file => 't/data/219877.cdna.fasta'); myaln = alignio->next_aln; mytreeio = Bio::TreeIO->new (-format => 'newick', -file => 't/data/219877.tree'); my $tree =$treeio->next_tree; my $slr = Bio::Tools::Run::Phylo::SLR->new();$slr->alignment($aln);$slr->tree($tree); #$rc = 1 for success, 0 for errors my ($rc,$results) = $slr->run(); my$positive_sites = $results->{'positive'}; print "# Site\tNeutral\tOptimal\tOmega\t", "lower\tupper\tLRT_Stat\tPval\tAdj.Pval\tResult\tNote\n"; foreach my$positive_site (@$positive_sites) { print$positive_site->[0], "\t", $positive_site->[1], "\t",$positive_site->[2], "\t", $positive_site->[3], "\t",$positive_site->[4], "\t", $positive_site->[5], "\t",$positive_site->[6], "\t", $positive_site->[7], "\t",$positive_site->[8], "\t", "positive\n"; } =head1 DESCRIPTION This is a wrapper around the SLR program. See http://www.ebi.ac.uk/goldman/SLR/ for more information. This module is more about generating the proper ctl file and will run the program in a separate temporary directory to avoid creating temp files all over the place. =head1 FEEDBACK =head2 Mailing Lists User feedback is an integral part of the evolution of this and other Bioperl modules. Send your comments and suggestions preferably to the Bioperl mailing list. Your participation is much appreciated. bioperl-l@bioperl.org - General discussion http://bioperl.org/wiki/Mailing_lists - About the mailing lists =head2 Support Please direct usage questions or support issues to the mailing list: I rather than to the module maintainer directly. Many experienced and reponsive experts will be able look at the problem and quickly address it. Please include a thorough description of the problem with code and data examples if at all possible. =head2 Reporting Bugs Report bugs to the Bioperl bug tracking system to help us keep track of the bugs and their resolution. Bug reports can be submitted via the web: http://redmine.open-bio.org/projects/bioperl/ =head1 AUTHOR - Albert Vilella Email avilella-at-gmail-dot-com =head1 CONTRIBUTORS Additional contributors names and emails here =head1 APPENDIX The rest of the documentation details each of the object methods. Internal methods are usually preceded with a _ =cut #' keep my emacs happy # Let the code begin... package Bio::Tools::Run::Phylo::SLR; use vars qw(@ISA %VALIDVALUES $MINNAMELEN$PROGRAMNAME PROGRAM); use strict; use Bio::Root::Root; use Bio::AlignIO; use Bio::TreeIO; use Bio::SimpleAlign; use Bio::Tools::Run::WrapperBase; use Cwd; use File::Spec; @ISA = qw(Bio::Root::Root Bio::Tools::Run::WrapperBase); =head2 Default Values INCOMPLETE DOCUMENTATION OF ALL METHODS seqfile [incodon] File from which to read alignment of codon sequences. The file should be in PAML format. treefile [intree] File from which tree should be read. The tree should be in Nexus format outfile [slr.res] File to which results are written. If the file already exists, it will be overwritten. reoptimise [1] Should the branch lengths, omega and kappa be reoptimized? 0 - no 1 - yes. kappa [2.0] Value for kappa. If 'reoptimise' is specified, the value given will be used as am initial estimate, omega [0.1] Value for omega (dN/dS). If 'reoptimise' is specified, the value given will be used as an initial estimate. codonf [0] How codon frequencies are estimated: 0: F61/F60 Estimates used are the empirical frequencies from the data. 1: F3x4 The frequencies of nucleotides at each codon position are estimated from the data and then multiplied together to get the frequency of observing a given codon. The frequency of stop codons is set to zero, and all other frequencies scaled appropriately. 2: F1x4 Nucleotide frequencies are estimated from the data (not taking into account at which position in the codon it occurs). The nucleotide frequencies are multiplied together to get the frequency of observing and then corrected for stop codons. freqtype [0] How codon frequencies are incorporated into the substitution matrix. 0: q_{ij} = pi_{j} s_{ij} 1: q_{ij} = \sqrt(pi_j/pi_i) s_{ij} 2: q_{ij} = \pi_{n} s_{ij}, where n is the nucleotide that the subsitution is to. 3: q_{ij} = s_{ij} / pi_i Option 0 is the tradition method of incorporating equilibrium frequencies into subsitution matrices (Felsenstein 1981; Goldman and Yang, 1994) Option 1 is described by Goldman and Whelan (2002), in this case with the additional parameter set to 0.5. Option 2 was suggested by Muse and Gaut (1994). Option 3 is included as an experiment, originally suggested by Bret Larget. it does not appear to describe evolution very successfully and should not be used for analyses. Kosakovsky-Pond has repeatedly stated that he finds incorporating codon frequencies in the manner of option 2 to be superior to option 0. We find that option 1 tends to perform better than either of these options. positive_only [0] If only positively selected sites are of interest, set this to "1". Calculation will be slightly faster, but information about sites under purifying selection is lost. gencode [universal] Which genetic code to use when determining whether a given mutation is synonymous or nonsynonymous. Currently only "universal" and "mammalian" mitochondrial are supported. nucleof [0] Allow for empirical exchangabilities for nucleotide substitution. 0: No adjustment. All nucleotides treated the same, modulo transition / transversion. 1: The rate at which a substitution caused a mutation from nucleotide a to nucleotide b is adjust by a constant N_{ab}. This adjustment is in addition to other adjustments (e.g. transition / transversion or base frequencies). aminof [0] Incorporate amino acid similarity parameters into substitution matrix, adjusting omega for a change between amino acid i and amino acid j. A_{ij} is a symmetric matrix of constants representing amino acid similarities. 0: Constant omega for all amino acid changes 1: omega_{ij} = omega^{A_{ij}} 2: omega_{ij} = a_{ij} log(omega) / [ 1 - exp(-a_{ij} log(omega)) ] Option 1 has the same form as the original codon subsitution model proposed by Goldman and Yang (but with potentially different constants). Option 2 has a more population genetic derivtion, with omega being interpreted as the ratio of fixation probabilities. nucfile [nuc.dat] If nucleof is non-zero, read nucleotide substitution constants from nucfile. If this file does not exist, hard coded constants are used. aminofile [amino.dat] If aminof is non-zero, read amino acid similarity constants from aminofile. If this file does not exist, hard coded constants are used. timemem [0] Print summary of real time and CPU time used. Will eventually print summary of memory use as well. ldiff [3.841459] Twice log-likelihood difference used as a threshold for calculating support (confidence) intervals for sitewise omega estimates. This value should be the quantile from a chi-square distribution with one degree of freedom corresponding to the support required. E.g. qchisq(0.95,1) = 3.841459 0.4549364 = 50% support 1.323304 = 75% support 2.705543 = 90% support 3.841459 = 95% support 6.634897 = 99% support 7.879439 = 99.5% support 10.82757 = 99.9% support paramin [] If not blank, read in parameters from file given by the argument. paramout [] If not blank, write out parameter estimates to file given. skipsitewise [0] Skip sitewise estimation of omega. Depending on other options given, either calculate maximum likelihood or likelihood fixed at parameter values given. seed [0] Seed for random number generator. If seed is 0, then previously produced seed file (~/.rng64) is used. If this does not exist, the random number generator is initialised using the clock. saveseed [1] If non-zero, save finial seed in file (~/.rng64) to be used as initial seed in future runs of program. =head2 Results Format Results file (default: slr.res) ------------ Results are presented in nine columns Site Number of sites in alignment Neutral (minus) Log-probability of observing site given that it was evolving neutrally (omega=1) Optimal (minus) Log-probability of observing site given that it was evolving at the optimal value of omega. Omega The value of omega which maximizes the log-probability of observing LRT_Stat Log-likelihood ratio statistic for non-neutral selection (or positive selection if the positive_only option is set to 1). LRT_Stat = 2 * (Neutral-Optimal) Pval P-value for non-neutral (or positive) selection at a site, unadjusted for multiple comparisons. Adj. Pval P-value for non-neutral (or positive) selection at a site, after adjusting for multiple comparisons using the Hochberg procedure (see the file "MultipleComparisons.txt" in the doc directory). Result A simple visual guide to the result. Sites detected as having been under positive selection are marked with a '+', sites under purifying selection are marked with '-'. The number of symbols Number symbols Threshold 1 95% 2 99% 3 95% after adjustment 4 99% after adjustment Occasionally the result may also contain an exclamation mark. This indicates that the observation at a site is not significantly different from random (equivalent to infinitely strong positive selection). This may indicate that the alignment at that site is bad Note The following events are flagged: Synonymous All codons at a site code for the same amino acid. Single character Only one sequence at the site is ungapped, the result of a recent insertion for example. All gaps All sequences at a site contain a gap character. Sites marked "Single character" or "All gaps" are not counted towards the number of sites for the purposes of correcting for multiple comparisons since it is not possible to detect selection from none or one observation under the assumptions made by the sitewise likelihood ratio test. =cut #' keep my emacs happy BEGIN {MINNAMELEN = 25; $PROGRAMNAME = 'Slr_Linux_static'; if ($^O =~ /darwin/i) { $PROGRAMNAME = 'Slr_osx'; } elsif ($^O =~ /mswin/i) { $PROGRAMNAME = 'Slr_windows.exe'; } if( defined$ENV{'SLRDIR'} ) { $PROGRAM = Bio::Root::IO->catfile($ENV{'SLRDIR'},$PROGRAMNAME). ($^O =~ /mswin/i ?'_windows.exe':'');; } # valid values for parameters, the default one is always # the first one in the array # example file provided with the package %VALIDVALUES = ( 'outfile' => 'slr.res', 'reoptimise' => [ 1,0], 'kappa' => '2.0', 'omega' => '0.1', 'codonf' => [ 0, 1,2], 'freqtype' => [ 0, 1,2,3], 'positive_only' => [ 0, 1], 'gencode' => [ "universal", "mammalian"], 'nucleof' => [ 0, 1], 'aminof' => [ 0, 1,2], 'nucfile' => '', 'aminofile' => '', 'timemem' => [ 0, 1], 'ldiff' => [ 3.841459, 0.4549364,1.323304,2.705543,6.634897,7.879439,10.82757], 'paramin' => '', 'paramout' => '', 'skipsitewise' => [ 0, 1], 'seed' => [0], 'saveseed' => [ 1, 0] ); } =head2 program_name Title : program_name Usage : $factory->program_name() Function: holds the program name Returns: string Args : None =cut sub program_name { return$PROGRAMNAME; } =head2 program_dir Title : program_dir Usage : ->program_dir() Function: returns the program directory, obtained from ENV variable. Returns: string Args : =cut sub program_dir { return Bio::Root::IO->catfile($ENV{SLRDIR}) if$ENV{SLRDIR}; } =head2 new Title : new Usage : my obj = Bio::Tools::Run::Phylo::SLR->new(); Function: Builds a new Bio::Tools::Run::Phylo::SLR object Returns : Bio::Tools::Run::Phylo::SLR Args : -alignment => the Bio::Align::AlignI object -save_tempfiles => boolean to save the generated tempfiles and NOT cleanup after onesself (default FALSE) -tree => the Bio::Tree::TreeI object -params => a hashref of SLR parameters (all passed to set_parameter) -executable => where the SLR executable resides See also: L, L =cut sub new { my(class,@args) = @_; my $self =$class->SUPER::new(@args); my ($aln,$tree, $st,$params, $exe,$ubl) = $self->_rearrange([qw(ALIGNMENT TREE SAVE_TEMPFILES PARAMS EXECUTABLE)], @args); defined$aln && self->alignment(aln); defined $tree &&$self->tree($tree); defined$st && $self->save_tempfiles($st); defined $exe &&$self->executable($exe);$self->set_default_parameters(); if( defined $params ) { if( ref($params) !~ /HASH/i ) { $self->warn("Must provide a valid hash ref for parameter -FLAGS"); } else { map {$self->set_parameter($_,$$params{$_}) } keys %$params; } } return$self; } =head2 prepare Title : prepare Usage : my $rundir =$slr->prepare(aln); Function: prepare the SLR analysis using the default or updated parameters the alignment parameter must have been set Returns : value of rundir Args : L object, L object =cut sub prepare{ my (self,$aln,$tree) = @_; unless ( $self->save_tempfiles ) { # brush so we don't get plaque buildup ;)$self->cleanup(); } $tree =$self->tree unless $tree;$aln = self->alignment unlessaln; if( ! $aln ) {$self->warn("must have supplied a valid alignment file in order to run SLR"); return 0; } if( ! $tree ) {$self->warn("must have supplied a valid tree file in order to run SLR"); return 0; } my ($tempdir) =$self->tempdir(); my ($tempseqFH,$tempseqfile); # Reorder the alignment according to the tree my $ct = 1; my %order; foreach my$node ($tree->get_leaf_nodes) {$order{$node->id_output} =$ct++; } my @seq; my @ids; foreach my $seq ($aln->each_seq() ) { push @seq, $seq; push @ids,$seq->display_id; } # use the map-sort-map idiom: my @sorted = map { $_->[1] } sort {$a->[0] <=> $b->[0] } map { [$order{$_->id()},$_] } @seq; my $sorted_aln = Bio::SimpleAlign->new(); foreach (@sorted) {$sorted_aln->add_seq($_); } # Rename the leaf nodes in the tree from 1 to n$ct = 1; foreach my $node ($tree->get_leaf_nodes) { $node->id($ct++); } ($tempseqFH,$tempseqfile) = $self->io->tempfile ('-dir' =>$tempdir, UNLINK => ($self->save_tempfiles ? 0 : 1)); my$alnout = Bio::AlignIO->new('-format' => 'phylip', '-fh' => $tempseqFH, '-interleaved' => 0, '-idlinebreak' => 1, '-idlength' =>$MINNAMELEN > $aln->maxdisplayname_length() ?$MINNAMELEN : $aln->maxdisplayname_length() +1);$alnout->write_aln($sorted_aln);$alnout->close(); undef $alnout; close($tempseqFH); my ($temptreeFH,$temptreefile); ($temptreeFH,$temptreefile) = $self->io->tempfile ('-dir' =>$tempdir, UNLINK => ($self->save_tempfiles ? 0 : 1)); my$treeout = Bio::TreeIO->new('-format' => 'newick', '-fh' => $temptreeFH); # We need to add a line with the num of leaves ($ct-1) and the # num of trees (1) $treeout->_print(sprintf("%d 1\n",($ct-1))); $treeout->write_tree($tree); $treeout->close(); close($temptreeFH); # now let's print the ctl file. # many of the these programs are finicky about what the filename is # and won't even run without the properly named file. my ($treevolume,$treedirectories,$treefile) = File::Spec->splitpath($temptreefile ); my ($alnvolume,$alndirectories,$alnfile) = File::Spec->splitpath($tempseqfile ); my $slr_ctl = "$tempdir/slr.ctl"; open(SLR, ">$slr_ctl") or$self->throw("cannot open $slr_ctl for writing"); print SLR "seqfile\:$alnfile\n"; print SLR "treefile\: $treefile\n"; my$outfile = $self->outfile_name; print SLR "outfile\:$outfile\n"; my %params = $self->get_parameters; while( my ($param,$val) = each %params ) { next if$param eq 'outfile'; print SLR "$param\:$val\n"; } close(SLR); return $tempdir; } =head2 run Title : run Usage : my ($rc,$parser) =$slr->run($aln,$tree); Function: run the SLR analysis using the default or updated parameters the alignment parameter must have been set Returns : Return code, L Args : L object, L object =cut sub run { my ($self) = shift;; my$outfile = $self->outfile_name; my$tmpdir = $self->prepare(@_); #my ($rc,$parser) = (1); my ($rc,$results) = (1); { my$cwd = cwd(); my $exit_status; chdir($tmpdir); my $slrexe =$self->executable(); $self->throw("unable to find or run executable for SLR") unless$slrexe && -e $slrexe && -x _; my$run; open($run, "$slrexe |") or $self->throw("Cannot open exe$slrexe"); my @output = <$run>;$exit_status = close($run);$self->error_string(join('',@output)); if( (grep { /\berr(or)?: /io } @output) || !$exit_status) {$self->warn("There was an error - see error_string for the program output"); $rc = 0; } eval { open RESULTS, "$tmpdir/$outfile" or die "couldnt open results file:$!\n"; my $okay = 0; my$sites; my $type = 'default'; while () { chomp$_; if ( /^\#/ ) {next;} if ( /\!/ ) {$type = 'random';} # random is last elsif ( /\+/ ) {$type = 'positive';} elsif ( /\-\s+/ ) {$type = 'negative';} elsif ( /Constant/ ) {$type = 'constant';} elsif ( /All gaps/ ) {$type = 'all_gaps';} elsif ( /Single character/ ) {$type = 'single_character';} elsif ( /Synonymous/ ) {$type = 'synonymous';} else {$type = 'default'} if ( /^\s+(\d+)\s+(\S+)\s+(\S+)\s+(\S+)\s+(\S+)\s+(\S+)\s+(\S+)\s+(\S+)\s+(\S+)/ ) { push @{$sites->{$type}}, [$1,$2,$3,$4,$5,$6,$7,$8,$9]; } else {$DB::single=1;1; } } $results =$sites; close RESULTS; # TODO: we could have a proper parser object # $parser = Bio::Tools::Phylo::SLR->new(-file => "$tmpdir/$outfile", # -dir => "$tmpdir"); }; if( $@ ) {$self->warn($self->error_string); } chdir($cwd); } # return ($rc,$parser); return ($rc,$results); } =head2 error_string Title : error_string Usage : $obj->error_string($newval) Function: Where the output from the last analysus run is stored. Returns : value of error_string Args : newvalue (optional) =cut sub error_string{ my ($self,$value) = @_; if( defined $value) {$self->{'error_string'} = $value; } return$self->{'error_string'}; } =head2 alignment Title : alignment Usage : slr->align(aln); Function: Get/Set the L object Returns : L object Args : [optional] L Comment : We could potentially add support for running directly on a file but we shall keep it simple See also: L =cut sub alignment{ my ($self,$aln) = @_; if( defined $aln ) { if( -e$aln ) { self->{'_alignment'} =aln; } elsif( !ref($aln) || !$aln->isa('Bio::Align::AlignI') ) { self->warn("Must specify a valid Bio::Align::AlignI object to the alignment function notaln"); return undef; } else { self->{'_alignment'} =aln; } } return self->{'_alignment'}; } =head2 tree Title : tree Usage :slr->tree($tree, %params); Function: Get/Set the L object Returns : L Args : [optional]$tree => L, Comment : We could potentially add support for running directly on a file but we shall keep it simple See also: L =cut sub tree { my ($self,$tree, %params) = @_; if( defined $tree ) { if( ! ref($tree) || ! $tree->isa('Bio::Tree::TreeI') ) {$self->warn("Must specify a valid Bio::Tree::TreeI object to the alignment function"); } $self->{'_tree'} =$tree; } return $self->{'_tree'}; } =head2 get_parameters Title : get_parameters Usage : my %params =$self->get_parameters(); Function: returns the list of parameters as a hash Returns : associative array keyed on parameter names Args : none =cut sub get_parameters{ my ($self) = @_; # we're returning a copy of this return %{$self->{'_slrparams'} }; } =head2 set_parameter Title : set_parameter Usage : $slr->set_parameter($param,$val); Function: Sets a SLR parameter, will be validated against the valid values as set in the %VALIDVALUES class variable. The checks can be ignored if one turns off param checks like this:$slr->no_param_checks(1) Returns : boolean if set was success, if verbose is set to -1 then no warning will be reported Args : $param => name of the parameter$value => value to set the parameter to See also: L =cut sub set_parameter{ my ($self,$param,$value) = @_; unless (defined$self->{'no_param_checks'} && $self->{'no_param_checks'} == 1) { if ( ! defined$VALIDVALUES{$param} ) {$self->warn("unknown parameter $param will not be set unless you force by setting no_param_checks to true"); return 0; } if ( ref($VALIDVALUES{$param}) =~ /ARRAY/i && scalar @{$VALIDVALUES{$param}} > 0 ) { unless ( grep {$value eq $_ } @{$VALIDVALUES{$param} } ) {$self->warn("parameter $param specified value$value is not recognized, please see the documentation and the code for this module or set the no_param_checks to a true value"); return 0; } } } $self->{'_slrparams'}->{$param} = $value; return 1; } =head2 set_default_parameters Title : set_default_parameters Usage :$slr->set_default_parameters(0); Function: (Re)set the default parameters from the defaults (the first value in each array in the %VALIDVALUES class variable) Returns : none Args : boolean: keep existing parameter values =cut sub set_default_parameters{ my ($self,$keepold) = @_; $keepold = 0 unless defined$keepold; while( my ($param,$val) = each %VALIDVALUES ) { # skip if we want to keep old values and it is already set next if( defined $self->{'_slrparams'}->{$param} && $keepold); if(ref($val)=~/ARRAY/i ) { $self->{'_slrparams'}->{$param} = $val->[0]; } else {$self->{'_slrparams'}->{$param} =$val; } } } =head1 Bio::Tools::Run::WrapperBase methods =cut =head2 no_param_checks Title : no_param_checks Usage : $obj->no_param_checks($newval) Function: Boolean flag as to whether or not we should trust the sanity checks for parameter values Returns : value of no_param_checks Args : newvalue (optional) =cut sub no_param_checks{ my ($self,$value) = @_; if( defined $value) {$self->{'no_param_checks'} = $value; } return$self->{'no_param_checks'}; } =head2 save_tempfiles Title : save_tempfiles Usage : $obj->save_tempfiles($newval) Function: Returns : value of save_tempfiles Args : newvalue (optional) =cut =head2 outfile_name Title : outfile_name Usage : my $outfile =$slr->outfile_name(); Function: Get/Set the name of the output file for this run (if you wanted to do something special) Returns : string Args : [optional] string to set value to =cut sub outfile_name { my $self = shift; if( @_ ) { return$self->{'_slrparams'}->{'outfile'} = shift @_; } unless (defined $self->{'_slrparams'}->{'outfile'}) {$self->{'_slrparams'}->{'outfile'} = 'out.res'; } return $self->{'_slrparams'}->{'outfile'}; } =head2 tempdir Title : tempdir Usage : my$tmpdir = $self->tempdir(); Function: Retrieve a temporary directory name (which is created) Returns : string which is the name of the temporary directory Args : none =cut =head2 cleanup Title : cleanup Usage :$slr->cleanup(); Function: Will cleanup the tempdir directory after an SLR run Returns : none Args : none =cut =head2 io Title : io Usage : $obj->io($newval) Function: Gets a L object Returns : L Args : none =cut sub DESTROY { my $self= shift; unless ($self->save_tempfiles ) { $self->cleanup(); }$self->SUPER::DESTROY(); } 1;