NAME
Bio::Tools::Run::Alignment::Clustalw - Object for the calculation of a multiple sequence alignment from a set of unaligned sequences or alignments using the Clustalw program
VERSION
version 1.7.3
SYNOPSIS
# Build a clustalw alignment factory
@params = ('ktuple' => 2, 'matrix' => 'BLOSUM');
$factory = Bio::Tools::Run::Alignment::Clustalw->new(@params);
# Pass the factory a list of sequences to be aligned.
$inputfilename = 't/data/cysprot.fa';
$aln = $factory->align($inputfilename); # $aln is a SimpleAlign object.
# or
$seq_array_ref = \@seq_array;
# where @seq_array is an array of Bio::Seq objects
$aln = $factory->align($seq_array_ref);
# Or one can pass the factory a pair of (sub)alignments
#to be aligned against each other, e.g.:
$aln = $factory->profile_align($aln1,$aln2);
# where $aln1 and $aln2 are Bio::SimpleAlign objects.
# Or one can pass the factory an alignment and one or more unaligned
# sequences to be added to the alignment. For example:
$aln = $factory->profile_align($aln1,$seq); # $seq is a Bio::Seq object.
# Get a tree of the sequences
$tree = $factory->tree(\@seq_array);
# Get both an alignment and a tree
($aln, $tree) = $factory->run(\@seq_array);
# Do a footprinting analysis on the supplied sequences, getting back the
# most conserved sub-alignments
my @results = $factory->footprint(\@seq_array);
foreach my $result (@results) {
print $result->consensus_string, "\n";
}
# There are various additional options and input formats available.
# See the DESCRIPTION section that follows for additional details.DESCRIPTION
Note: this DESCRIPTION only documents the Bioperl interface to Clustalw. Clustalw, itself, is a large & complex program - for more information regarding clustalw, please see the clustalw documentation which accompanies the clustalw distribution. Clustalw is available from (among others) ftp://ftp.ebi.ac.uk/pub/software/. Clustalw.pm has only been tested using version 1.8 of clustalw. Compatibility with earlier versions of the clustalw program is currently unknown. Before running Clustalw successfully it will be necessary: to install clustalw on your system, and to ensure that users have execute privileges for the clustalw program.
Helping the module find your executable
You will need to enable Clustalw to find the clustalw program. This can be done in (at least) three ways:
1. Make sure the clustalw executable is in your path so that
which clustalw
returns a clustalw executable on your system.
2. Define an environmental variable CLUSTALDIR which is a
directory which contains the 'clustalw' application:
In bash:
export CLUSTALDIR=/home/username/clustalw1.8
In csh/tcsh:
setenv CLUSTALDIR /home/username/clustalw1.8
3. Include a definition of an environmental variable CLUSTALDIR in
every script that will use this Clustalw wrapper module, e.g.:
BEGIN { $ENV{CLUSTALDIR} = '/home/username/clustalw1.8/' }
use Bio::Tools::Run::Alignment::Clustalw;If you are running an application on a webserver make sure the webserver environment has the proper PATH set or use the options 2 or 3 to set the variables.
How it works
Bio::Tools::Run::Alignment::Clustalw is an object for performing a multiple sequence alignment from a set of unaligned sequences and/or sub-alignments by means of the clustalw program.
Initially, a clustalw "factory object" is created. Optionally, the factory may be passed most of the parameters or switches of the clustalw program, e.g.:
@params = ('ktuple' => 2, 'matrix' => 'BLOSUM');
$factory = Bio::Tools::Run::Alignment::Clustalw->new(@params);Any parameters not explicitly set will remain as the defaults of the clustalw program. Additional parameters and switches (not available in clustalw) may also be set. Currently, the only such parameter is "quiet", which when set to a non-zero value, suppresses clustalw terminal output. Not all clustalw parameters are supported at this stage.
By default, Clustalw output is returned solely in a the form of a Bio::SimpleAlign object which can then be printed and/or saved in multiple formats using the AlignIO.pm module. Optionally the raw clustalw output file can be saved if the calling script specifies an output file (with the clustalw parameter OUTFILE). Currently only the GCG-MSF output file formats is supported.
Not all parameters and features have been implemented yet in Perl format.
Alignment parameters can be changed and/or examined at any time after the factory has been created. The program checks that any parameter/switch being set/read is valid. However, currently no additional checks are included to check that parameters are of the proper type (eg string or numeric) or that their values are within the proper range. As an example, to change the value of the clustalw parameter ktuple to 3 and subsequently to check its value one would write:
$ktuple = 3;
$factory->ktuple($ktuple);
$get_ktuple = $factory->ktuple();Once the factory has been created and the appropriate parameters set, one can call the method align() to align a set of unaligned sequences, or call profile_align() to add one or more sequences or a second alignment to an initial alignment.
Input to align() may consist of a set of unaligned sequences in the form of the name of file containing the sequences. For example,
$inputfilename = 't/data/cysprot.fa';
$aln = $factory-E<gt>align($inputfilename);Alternately one can create an array of Bio::Seq objects somehow
$str = Bio::SeqIO->new(-file=> 't/data/cysprot.fa', -format => 'Fasta');
@seq_array =();
while ( my $seq = $str->next_seq() ) {push (@seq_array, $seq) ;}and pass the factory a reference to that array
$seq_array_ref = \@seq_array;
$aln = $factory->align($seq_array_ref);In either case, align() returns a reference to a SimpleAlign object which can then used (see Bio::SimpleAlign).
Once an initial alignment exists, one can pass the factory additional sequence(s) to be added (ie aligned) to the original alignment. The alignment can be passed as either an alignment file or a Bio:SimpleAlign object. The unaligned sequence(s) can be passed as a filename or as an array of BioPerl sequence objects or as a single BioPerl Seq object. For example (to add a single sequence to an alignment),
$str = Bio::AlignIO->new(-file=> 't/data/cysprot1a.msf');
$aln = $str->next_aln();
$str1 = Bio::SeqIO->new(-file=> 't/data/cysprot1b.fa');
$seq = $str1->next_seq();
$aln = $factory->profile_align($aln,$seq);In either case, profile_align() returns a reference to a SimpleAlign object containing a new SimpleAlign object of the alignment with the additional sequence(s) added in.
Finally one can pass the factory a pair of (sub)alignments to be aligned against each other. The alignments can be passed in the form of either a pair of alignment files or a pair of Bio:SimpleAlign objects. For example,
$profile1 = 't/data/cysprot1a.msf';
$profile2 = 't/data/cysprot1b.msf';
$aln = $factory->profile_align($profile1,$profile2);or
$str1 = Bio::AlignIO->new(-file=> 't/data/cysprot1a.msf');
$aln1 = $str1->next_aln();
$str2 = Bio::AlignIO->new(-file=> 't/data/cysprot1b.msf');
$aln2 = $str2->next_aln();
$aln = $factory->profile_align($aln1,$aln2);In either case, profile_align() returns a reference to a SimpleAlign object containing an (super)alignment of the two input alignments.
For more examples of syntax and use of Clustalw, the user is encouraged to look at the script Clustalw.t in the t/ directory.
Note: Clustalw is still under development. Various features of the clustalw program have not yet been implemented. If you would like that a specific clustalw feature be added to this perl contact bioperl-l@bioperl.org.
These can be specified as parameters when instantiating a new Clustalw object, or through get/set methods of the same name (lowercase).
INTERNAL METHODS
_run
Title : _run
Usage : Internal function, not to be called directly
Function: makes actual system call to clustalw program
Returns : nothing; clustalw output is written to a
temporary file
Args : Name of a file containing a set of unaligned fasta sequences
and hash of parameters to be passed to clustalw_setinput()
Title : _setinput
Usage : Internal function, not to be called directly
Function: Create input file for clustalw program
Returns : name of file containing clustalw data input
Args : Seq or Align object reference or input file name_setparams()
Title : _setparams
Usage : Internal function, not to be called directly
Function: Create parameter inputs for clustalw program
Returns : parameter string to be passed to clustalw
during align or profile_align
Args : name of calling objectEXAMPLE
You will need to have installed clustalw and to ensure that Clustalw.pm can find it. This can be done in different ways (bash syntax):
export PATH=$PATH:/home/peter/clustalw1.8or define an environmental variable CLUSTALDIR:
export CLUSTALDIR=/home/peter/clustalw1.8or include a definition of an environmental variable CLUSTALDIR in every script that will use Clustal.pm:
BEGIN {$ENV{CLUSTALDIR} = '/home/peter/clustalw1.8/'; }We are going to demonstrate 3 possible applications of Clustalw.pm:
Test effect of varying clustalw alignment parameter(s) on resulting alignment
Test effect of changing the order that sequences are added to the alignment on the resulting alignment
Test effect of incorporating an "anchor point" in the alignment process
Before we can do any tests, we need to set up the environment, create the factory and read in the unaligned sequences.
use Getopt::Long;
use Bio::Tools::Run::Alignment::Clustalw;
use Bio::SimpleAlign;
use Bio::AlignIO;
use Bio::SeqIO;
use strict;
# set some default values
my $infile = 't/data/cysprot1a.fa';
my @params = ('quiet' => 1 );
my $do_only = '123'; # string listing examples to be executed. Default is to
# execute all tests (ie 1,2 and 3)
my $param = 'ktuple'; # parameter to be varied in example 1
my $startvalue = 1; # initial value for parameter $param
my $stopvalue = 3; # final value for parameter $param
my $regex = 'W[AT]F'; # regular expression for 'anchoring' alignment in example 3
my $extension = 30; # distance regexp anchor should be extended in each direction
# for local alignment in example 3
my $helpflag = 0; # Flag to show usage info.
# get user options
my @argv = @ARGV; # copy ARGV before GetOptions() massacres it.
&GetOptions("h!" => \$helpflag, "help!" => \$helpflag,
"in=s" => \$infile,
"param=s" => \$param,
"do=s" => \$do_only,
"start=i" => \$startvalue,
"stop=i" => \$stopvalue,
"ext=i" => \$extension,
"regex=s" => \$regex,) ;
if ($helpflag) { &clustalw_usage(); exit 0;}
# create factory & set user-specified global clustalw parameters
foreach my $argv (@argv) {
unless ($argv =~ /^(.*)=>(.*)$/) { next;}
push (@params, $1 => $2);
}
my $factory = Bio::Tools::Run::Alignment::Clustalw->new(@params);
# put unaligned sequences in a Bio::Seq array
my $str = Bio::SeqIO->new(-file=> $infile, '-format' => 'Fasta');
my ($paramvalue, $aln, $subaln, @consensus, $seq_num, $string, $strout, $id);
my @seq_array =();
while ( my $seq = $str->next_seq() ) { push (@seq_array, $seq) ;}
# Do each example that has digit present in variable $do_only
$_= $do_only;
/1/ && &vary_params();
/2/ && &vary_align_order();
/3/ && &anchored_align();
## End of "main"
#################################################
# vary_params(): Example demonstrating varying of clustalw parameter
#
sub vary_params {
print "Beginning parameter-varying example... \n";
# Now we'll create several alignments, 1 for each value of the selected
# parameter. We also compute a simple consensus string for each alignment.
# (In the default case, we vary the "ktuple" parameter, creating 3
# alignments using ktuple values from 1 to 3.)
my $index =0;
for ($paramvalue = $startvalue; $paramvalue < ($stopvalue + 1); $paramvalue++) {
$factory->$param($paramvalue); # set parameter value
print "Performing alignment with $param = $paramvalue \n";
$aln = $factory->align(\@seq_array);
$string = $aln->consensus_string(); # Get consensus of alignment
# convert '?' to 'X' at non-consensus positions
$string =~ s/\?/X/g;
$consensus[$index] = Bio::Seq->new(-id=>"$param=$paramvalue",-seq=>$string);
$index++;
}
# Compare consensus strings for alignments with different $param values by
# making an alignment of the different consensus strings
# $factory->ktuple(1); # set ktuple parameter
print "Performing alignment of $param consensus sequences \n";
$aln = $factory->align(\@consensus);
$strout = Bio::AlignIO->newFh('-format' => 'msf');
print $strout $aln;
return 1;
}
#################################################
# vary_align_order():
#
# For our second example, we'll test the effect of changing the order
# that sequences are added to the alignment
sub vary_align_order {
print "\nBeginning alignment-order-changing example... \n";
@consensus = (); # clear array
for ($seq_num = 0; $seq_num < scalar(@seq_array); $seq_num++) {
my $obj_out = shift @seq_array; # remove one Seq object from array and save
$id = $obj_out->display_id;
# align remaining sequences
print "Performing alignment with sequence $id left out \n";
$subaln = $factory->align(\@seq_array);
# add left-out sequence to subalignment
$aln = $factory->profile_align($subaln,$obj_out);
$string = $aln->consensus_string(); # Get consensus of alignment
# convert '?' to 'X' for non-consensus positions
$string =~ s/\?/X/g;
$consensus[$seq_num] = Bio::Seq->new(-id=>"$id left out",-seq=>$string);
push @seq_array, $obj_out; # return Seq object for next (sub) alignment
}
# Compare consensus strings for alignments created in different orders
# $factory->ktuple(1); # set ktuple parameter
print "\nPerforming alignment of consensus sequences for different reorderings \n";
print "Each consensus is labeled by the sequence which was omitted in the initial alignment\n";
$aln = $factory->align(\@consensus);
$strout = Bio::AlignIO->newFh('-format' => 'msf');
print $strout $aln;
return 1;
}
#################################################
# anchored_align()
#
# For our last example, we'll test a way to perform a local alignment by
# "anchoring" the alignment to a regular expression. This is similar
# to the approach taken in the recent dbclustal program.
# In principle, we could write a script to search for a good regular expression
# to use. Instead, here we'll simply choose one manually after looking at the
# previous alignments.
sub anchored_align {
my @local_array = ();
my @seqs_not_matched = ();
print "\n Beginning anchored-alignment example... \n";
for ($seq_num = 0; $seq_num < scalar(@seq_array); $seq_num++) {
my $seqobj = $seq_array[$seq_num];
my $seq = $seqobj->seq();
my $id = $seqobj->id();
# if $regex is not found in the sequence, save sequence id name and set
# array value =0 for later
unless ($seq =~/$regex/) {
$local_array[$seq_num] = 0;
push (@seqs_not_matched, $id) ;
next;
}
# find positions of start and of subsequence to be aligned
my $match_start_pos = length($`);
my $match_stop_pos = length($`) + length($&);
my $start = ($match_start_pos - $extension) > 1 ?
($match_start_pos - $extension) +1 : 1;
my $stop = ($match_stop_pos + $extension) < length($seq) ?
($match_stop_pos + $extension) : length($seq);
my $string = $seqobj->subseq($start, $stop);
$local_array[$seq_num] = Bio::Seq->new(-id=>$id, -seq=>$string);
}
@local_array = grep $_ , @local_array; # remove array entries with no match
# Perform alignment on the local segments of the sequences which match "anchor"
$aln = $factory->align(\@local_array);
my $consensus = $aln->consensus_string(); # Get consensus of local alignment
if (scalar(@seqs_not_matched) ) {
print " Sequences not matching $regex : @seqs_not_matched \n"
} else {
print " All sequences match $regex : @seqs_not_matched \n"
}
print "Consensus sequence of local alignment: $consensus \n";
return 1;
}
#----------------
sub clustalw_usage {
#----------------
#-----------------------
# Prints usage information for general parameters.
print STDERR <<"QQ_PARAMS_QQ";
Command-line accessible script variables and commands:
-------------------------------
-h : Display this usage info and exit.
-in <str> : File containing input sequences in fasta format (default = $infile) .
-do <str> : String listing examples to be executed. Default is to execute
all tests (ie default = '123')
-param <str> : Parameter to be varied in example 1. Any clustalw parameter
which takes inteer values can be varied (default = 'ktuple')
-start <int> : Initial value for varying parameter in example 1 (default = 1)
-stop <int> : Final value for varying parameter (default = 3)
-regex <str> : Regular expression for 'anchoring' alignment in example 3
(default = $regex)
-ext <int> : Distance regexp anchor should be extended in each direction
for local alignment in example 3 (default = 30)
In addition, any valid Clustalw parameter can be set using the syntax
"parameter=>value" as in "ktuple=>3"
So a typical command lines might be:
> clustalw.pl -param=pairgap -start=2 -stop=3 -do=1 "ktuple=>3"
or
> clustalw.pl -ext=10 -regex='W[AST]F' -do=23 -in='t/cysprot1a.fa'
QQ_PARAMS_QQ
}PARAMETER FOR ALIGNMENT COMPUTATION
KTUPLE
Title : KTUPLE
Description : (optional) set the word size to be used in the alignment
This is the size of exactly matching fragment that is used.
INCREASE for speed (max= 2 for proteins; 4 for DNA),
DECREASE for sensitivity.
For longer sequences (e.g. >1000 residues) you may
need to increase the defaultTOPDIAGS
Title : TOPDIAGS
Description : (optional) number of best diagonals to use
The number of k-tuple matches on each diagonal
(in an imaginary dot-matrix plot) is calculated.
Only the best ones (with most matches) are used in
the alignment. This parameter specifies how many.
Decrease for speed; increase for sensitivity.WINDOW
Title : WINDOW
Description : (optional) window size
This is the number of diagonals around each of the 'best'
diagonals that will be used. Decrease for speed;
increase for sensitivity.PAIRGAP
Title : PAIRGAP
Description : (optional) gap penalty for pairwise alignments
This is a penalty for each gap in the fast alignments.
It has little affect on the speed or sensitivity except
for extreme values.FIXEDGAP
Title : FIXEDGAP
Description : (optional) fixed length gap penaltyFLOATGAP
Title : FLOATGAP
Description : (optional) variable length gap penaltyMATRIX
Title : MATRIX
Default : PAM100 for DNA - PAM250 for protein alignment
Description : (optional) substitution matrix used in the multiple
alignments. Depends on the version of clustalw as to
what default matrix will be used
PROTEIN WEIGHT MATRIX leads to a new menu where you are
offered a choice of weight matrices. The default for
proteins in version 1.8 is the PAM series derived by
Gonnet and colleagues. Note, a series is used! The
actual matrix that is used depends on how similar the
sequences to be aligned at this alignment step
are. Different matrices work differently at each
evolutionary distance.
DNA WEIGHT MATRIX leads to a new menu where a single
matrix (not a series) can be selected. The default is
the matrix used by BESTFIT for comparison of nucleic
acid sequences.TYPE
Title : TYPE
Description : (optional) sequence type: protein or DNA. This allows
you to explicitly overide the programs attempt at
guessing the type of the sequence. It is only useful
if you are using sequences with a VERY strange
composition.OUTPUT
Title : OUTPUT
Description : (optional) clustalw supports GCG or PHYLIP or PIR or
Clustal format. See the Bio::AlignIO modules for
which formats are supported by bioperl.OUTFILE
Title : OUTFILE
Description : (optional) Name of clustalw output file. If not set
module will erase output file. In any case alignment will
be returned in the form of SimpleAlign objectsTRANSMIT
Title : TRANSMIT
Description : (optional) transitions not weighted. The default is to
weight transitions as more favourable than other
mismatches in DNA alignments. This switch makes all
nucleotide mismatches equally weighted.program_name
Title : program_name
Usage : $factory>program_name()
Function: holds the program name
Returns: string
Args : Noneprogram_dir
Title : program_dir
Usage : $factory->program_dir(@params)
Function: returns the program directory, obtained from ENV variable.
Returns: string
Args :version
Title : version
Usage : exit if $prog->version() < 1.8
Function: Determine the version number of the program
Example :
Returns : float or undef
Args : nonerun
Title : run
Usage : ($aln, $tree) = $factory->run($inputfilename);
($aln, $tree) = $factory->run($seq_array_ref);
Function: Perform a multiple sequence alignment, generating a tree at the same
time. (Like align() and tree() combined.)
Returns : A SimpleAlign object containing the sequence alignment and a
Bio::Tree::Tree object with the tree relating the sequences.
Args : Name of a file containing a set of unaligned fasta sequences
or else an array of references to Bio::Seq objects.align
Title : align
Usage : $inputfilename = 't/data/cysprot.fa';
$aln = $factory->align($inputfilename);
or
$seq_array_ref = \@seq_array; # @seq_array is array of Seq objs
$aln = $factory->align($seq_array_ref);
Function: Perform a multiple sequence alignment
Returns : Reference to a SimpleAlign object containing the
sequence alignment.
Args : Name of a file containing a set of unaligned fasta sequences
or else an array of references to Bio::Seq objects.
Throws an exception if argument is not either a string (eg a
filename) or a reference to an array of Bio::Seq objects. If
argument is string, throws exception if file corresponding to string
name can not be found. If argument is Bio::Seq array, throws
exception if less than two sequence objects are in array.profile_align
Title : profile_align
Usage : $aln = $factory->profile_align(@simple_aligns);
or
$aln = $factory->profile_align(@subalignment_filenames);
Function: Perform an alignment of 2 (sub)alignments
Returns : Reference to a SimpleAlign object containing the (super)alignment.
Args : Names of 2 files containing the subalignments
or references to 2 Bio::SimpleAlign objects.Throws an exception if arguments are not either strings (eg filenames) or references to SimpleAlign objects.
add_sequences
Title : add_sequences
Usage :
Function: Align and add sequences into an alignment
Example :
Returns : Reference to a SimpleAlign object containing the (super)alignment.
Args : Names of 2 files, the first one containing an alignment and the second one containing sequences to be added
or references to 2 Bio::SimpleAlign objects.Throws an exception if arguments are not either strings (eg filenames) or references to SimpleAlign objects.
tree
Title : tree
Usage : @params = ('bootstrap' => 1000,
'tossgaps' => 1,
'kimura' => 1,
'seed' => 121,
'bootlabels'=> 'nodes',
'quiet' => 1);
$factory = Bio::Tools::Run::Alignment::Clustalw->new(@params);
$tree_obj = $factory->tree($aln_obj);
or
$tree_obj = $factory->tree($treefilename);
Function: Retrieve a tree corresponding to the input
Returns : Bio::TreeIO object
Args : Bio::SimpleAlign or filename of a treefootprint
Title : footprint
Usage : @alns = $factory->footprint($treefilename, $window_size, $diff);
@alns = $factory->footprint($seqs_array_ref);
Function: Aligns all the supplied sequences and slices out of the alignment
those regions along a sliding window who's tree length differs
significantly from the total average tree length.
Returns : list of Bio::SimpleAlign objects
Args : first argument as per run(), optional second argument to specify
the size of the sliding window (default 5 bp) and optional third
argument to specify the % difference from the total tree length
needed for a window to be considered a footprint (default 33%).FEEDBACK
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/Support.html - About the mailing listsSupport
Please direct usage questions or support issues to the mailing list: bioperl-l@bioperl.org
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.
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:
https://github.com/bioperl/bio-tools-run-alignment-clustalw/issuesAUTHORS
Peter Schattner <schattner@alum.mit.edu>
Jason Stajich <jason@bioperl.org>
Sendu Bala <bix@sendu.me.uk>
COPYRIGHT
This software is copyright (c) by Peter Schattner <schattner@alum.mit.edu>.
This software is available under the same terms as the perl 5 programming language system itself.