SYNOPSIS
# analyze a DNA sequence for restriction enzymes
use Bio::Restriction::Analysis;
use Bio::PrimarySeq;
use Data::Dumper;
# get a DNA sequence from somewhere
my $seq = Bio::PrimarySeq->new
(-seq =>'AGCTTAATTCATTAGCTCTGACTGCAACGGGCAATATGTCTC',
-primary_id => 'synopsis',
-molecule => 'dna');
# now start an analysis.
# this is using the default set of enzymes
my $ra = Bio::Restriction::Analysis->new(-seq=>$seq);
# find unique cutters. This returns a
# Bio::Restriction::EnzymeCollection object
my $enzymes = $ra->unique_cutters;
print "Unique cutters: ", join (', ',
map {$_->name} $enzymes->unique_cutters), "\n";
# AluI is one them. Where does it cut?
# This is will return an array of the sequence strings
my $enz = 'AluI';
my @frags = $ra->fragments($enz);
# how big are the fragments?
print "AluI fragment lengths: ", join(' & ', map {length $_} @frags), "\n";
# You can also bypass fragments and call sizes directly:
# to see all the fragment sizes
print "All sizes: ", join " ", $ra->sizes($enz), "\n";
# to see all the fragment sizes sorted by size like on a gel
print "All sizes, sorted ", join (" ", $ra->sizes($enz, 0, 1)), "\n";
# how many times does each enzyme cut
my $cuts = $ra->cuts_by_enzyme('BamHI');
print "BamHI cuts $cuts times\n";
# How many enzymes do not cut at all?
print "There are ", scalar $ra->zero_cutters->each_enzyme,
" enzymes that do not cut\n";
# what about enzymes that cut twice?
my $two_cutters = $ra->cutters(2);
print join (" ", map {$_->name} $two_cutters->each_enzyme),
" cut the sequence twice\n";
# what are all the enzymes that cut, and how often do they cut
printf "\n%-10s%s\n", 'Enzyme', 'Number of Cuts';
my $all_cutters = $ra->cutters;
map {
printf "%-10s%s\n", $_->name, $ra->cuts_by_enzyme($_->name)
} $all_cutters->each_enzyme;
# Finally, we can interact the restriction enzyme object by
# retrieving it from the collection object see the docs for
# Bio::Restriction::Enzyme.pm
my $enzobj = $enzymes->get_enzyme($enz);
DESCRIPTION
Bio::Restriction::Analysis describes the results of cutting a DNA sequence with restriction enzymes.To use this module you can pass a sequence object and optionally a Bio::Restriction::EnzymeCollection that contains the enzyme(s) to cut the sequences with. There is a default set of enzymes that will be loaded if you do not pass in a Bio::Restriction::EnzymeCollection.
To cut a sequence, set up a Restriction::Analysis object with a sequence like this:
use Bio::Restriction::Analysis; my $ra = Bio::Restriction::Analysis->new(-seq=>$seqobj);
or
my $ra = Bio::Restriction::Analysis->new
(-seq=>$seqobj, -enzymes=>$enzs);
Then, to get the fragments for a particular enzyme use this:
@fragments = $ra->fragments('EcoRI');
Note that the naming of restriction enzymes is that the last numbers are usually Roman numbers (I, II, III, etc). You may want to use something like this:
# get a reference to an array of unique (single) cutters
$singles = $re->unique_cutters;
foreach my $enz ($singles->each_enzyme) {
@fragments = $re->fragments($enz);
... do something here ...
}
Note that if your sequence is circular, the first and last fragment will be joined so that they are the appropriate length and sequence for further analysis. This fragment will also be checked for cuts by the enzyme(s). However, this will change the start of the sequence!
There are two separate algorithms used depending on whether your enzyme has ambiguity. The non-ambiguous algorithm is a lot faster, and if you are using very large sequences you should try and use this algorithm. If you have a large sequence (e.g. genome) and want to use ambgiuous enzymes you may want to make separate Bio::Restriction::Enzyme objects for each of the possible alternatives and make sure that you do not set is_ambiguous!
This version should correctly deal with overlapping cut sites in both ambiguous and non-ambiguous enzymes.
I have tried to write this module with speed and memory in mind so that it can be effectively used for large (e.g. genome sized) sequence. This module only stores the cut positions internally, and calculates everything else on an as-needed basis. Therefore when you call fragment_maps (for example), there may be another delay while these are generated.
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 one of the Bioperl mailing lists. Your participation is much appreciated.
[email protected] - General discussion http://bioperl.org/wiki/Mailing_lists - About the mailing lists
Support
Please direct usage questions or support issues to the mailing list: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 the bugs and their resolution. Bug reports can be submitted via the web:
https://github.com/bioperl/bioperl-live/issues
CONTRIBUTORS
Heikki Lehvaslaiho, heikki-at-bioperl-dot-org Mark A. Jensen, maj-at-fortinbras-dot-usCOPYRIGHT
Copyright (c) 2003 Rob Edwards. Some of this work is Copyright (c) 1997-2002 Steve A. Chervitz. All Rights Reserved.This module is free software; you can redistribute it and/or modify it under the same terms as Perl itself.
APPENDIX
Methods beginning with a leading underscore are considered private and are intended for internal use by this module. They are not considered part of the public interface and are described here for documentation purposes only.new
Title : new
Function : Initializes the restriction enzyme object
Returns : The Restriction::Analysis object
Arguments :
$re_anal->new(-seq=$seqobj,
-enzymes=>Restriction::EnzymeCollection object)
-seq requires a Bio::PrimarySeq object
-enzymes is optional.
If omitted it will use the default set of enzymes
This is the place to start. Pass in a sequence, and you will be able to get the fragments back out. Several other things are available like the number of zero cutters or single cutters.
Methods to set parameters
seq
Title : seq Usage : $ranalysis->seq($newval); Function : get/set method for the sequence to be cut Example : $re->seq($seq); Returns : value of seq Args : A Bio::PrimarySeqI dna object (optional)
enzymes
Title : enzymes
Usage : $re->enzymes($newval)
Function : gets/Set the restriction enzyme enzymes
Example : $re->enzymes('EcoRI')
Returns : reference to the collection
Args : an array of Bio::Restriction::EnzymeCollection and/or
Bio::Restriction::Enzyme objects
The default object for this method is Bio::Restriction::EnzymeCollection. However, you can also pass it a list of Bio::Restriction::Enzyme objects - even mixed with Collection objects. They will all be stored into one collection.
Perform the analysis
cut
Title : cut
Usage : $re->cut()
Function : Cut the sequence with the enzymes
Example : $re->cut(); $re->cut('single'); or $re->cut('multiple', $enzymecollection);
Returns : $self
Args : 'single' (optional), 'multiple' with enzyme collection.
An explicit cut method is needed to pass arguments to it.
There are two varieties of cut. Single is the default, and need not be explicitly called. This cuts the sequence with each enzyme separately.
Multiple cuts a sequence with more than one enzyme. You must pass it a Bio::Restriction::EnzymeCollection object of the set of enzymes that you want to use in the double digest. The results will be stored as an enzyme named ``multiple_digest'', so you can use all the retrieval methods to get the data.
If you want to use the default setting there is no need to call cut directly. Every method in the class that needs output checks the object's internal status and recalculates the cuts if needed.
Note: cut doesn't now re-initialize everything before figuring out cuts. This is so that you can do multiple digests, or add more data or whatever. You'll have to use new to reset everything.
See also the comments in above about ambiguous and non-ambiguous sequences.
mulitple_digest
Title : multiple_digest
Function : perform a multiple digest on a sequence
Returns : $self so you can go and get any of the other methods
Arguments : An enzyme collection
Multiple digests can use 1 or more enzymes, and the data is stored
in as if it were an enzyme called multiple_digest. You can then
retrieve information about multiple digests from any of the other
methods.
You can use this method in place of $re->cut('multiple', $enz_coll);
Query the results of the analysis
positions
Title : positions
Function : Retrieve the positions that an enzyme cuts at
Returns : An array of the positions that an enzyme cuts at
: or an empty array if the enzyme doesn't cut
Arguments: An enzyme name to retrieve the positions for
Comments : The cut occurs after the base specified.
fragments
Title : fragments Function : Retrieve the fragments that we cut Returns : An array of the fragments retrieved. Arguments: An enzyme name to retrieve the fragments for
For example this code will retrieve the fragments for all enzymes that cut your sequence
my $all_cutters = $analysis->cutters;
foreach my $enz ($$all_cutters->each_enzyme}) {
@fragments=$analysis->fragments($enz);
}
fragment_maps
Title : fragment_maps
Function : Retrieves fragment sequences with start and end
points. Useful for feature construction.
Returns : An array containing a hash reference for each fragment,
containing the start point, end point and DNA
sequence. The hash keys are 'start', 'end' and
'seq'. Returns an empty array if not defined.
Arguments : An enzyme name, enzyme object,
or enzyme collection to retrieve the fragments for.
If passes an enzyme collection it will return the result of a multiple digest. This : will also cause the special enzyme 'multiple_digest' to be created so you can get : other information about this multiple digest. (TMTOWTDI).
There is a minor problem with this and $self->fragments that I haven't got a good answer for (at the moment). If the sequence is not cut, do we return undef, or the whole sequence?
For linear fragments it would be good to return the whole sequence. For circular fragments I am not sure.
At the moment it returns the whole sequence with start of 1 and end of length of the sequence. For example:
use Bio::Restriction::Analysis;
use Bio::Restriction::EnzymeCollection;
use Bio::PrimarySeq;
my $seq = Bio::PrimarySeq->new
(-seq =>'AGCTTAATTCATTAGCTCTGACTGCAACGGGCAATATGTCTCTGTGTGGATCCAAAAAAGAGTGAGCTTCTGAT',
-primary_id => 'synopsis',
-molecule => 'dna');
my $ra = Bio::Restriction::Analysis->new(-seq=>$seq);
my @gel;
my @bam_maps = $ra->fragment_maps('BamHI');
foreach my $i (@bam_maps) {
my $start = $i->{start};
my $end = $i->{end};
my $sequence = $i->{seq};
push @gel, "$start--$sequence--$end";
@gel = sort {length $b <=> length $a} @gel;
}
print join("\n", @gel) . "\n";
sizes
Title : sizes
Function : Retrieves an array with the sizes of the fragments
Returns : Array that has the sizes of the fragments ordered from
largest to smallest like they would appear in a gel.
Arguments: An enzyme name to retrieve the sizes for is required and
kilobases to the nearest 0.1 kb, else it will be in
bp. If the optional third entry is set the results will
be sorted.
This is designed to make it easy to see what fragments you should get on a gel!
You should be able to do these:
# to see all the fragment sizes, print join "\n", $re->sizes($enz), "\n"; # to see all the fragment sizes sorted print join "\n", $re->sizes($enz, 0, 1), "\n"; # to see all the fragment sizes in kb sorted print join "\n", $re->sizes($enz, 1, 1), "\n";
How many times does enzymes X cut?
cuts_by_enzyme
Title : cuts_by_enzyme
Function : Return the number of cuts for an enzyme
Returns : An integer with the number of times each enzyme cuts.
Returns 0 if doesn't cut or undef if not defined
Arguments : An enzyme name string
Which enzymes cut the sequence N times?
cutters
Title : cutters
Function : Find enzymes that cut a given number of times
Returns : a Bio::Restriction::EnzymeCollection
Arguments : 1. exact time or lower limit,
non-negative integer, optional
2. upper limit, non-negative integer,
larger or equalthan first, optional
If no arguments are given, the method returns all enzymes that do cut the sequence. The argument zero, '0', is same as method zero_cutters(). The argument one, '1', corresponds to unique_cutters. If either of the limits is larger than number of cuts any enzyme cuts the sequence, the that limit is automagically lowered. The method max_cuts() gives the largest number of cuts.
See Also : unique_cutters, zero_cutters, max_cuts
unique_cutters
Title : unique_cutters Function : A special case if cutters() where enzymes only cut once Returns : a Bio::Restriction::EnzymeCollection Arguments : -
See also: cutters, zero_cutters
zero_cutters
Title : zero_cutters Function : A special case if cutters() where enzymes don't cut the sequence Returns : a Bio::Restriction::EnzymeCollection Arguments : -
See also: cutters, unique_cutters
max_cuts
Title : max_cuts Function : Find the most number of cuts Returns : The number of times the enzyme that cuts most cuts. Arguments : None
This is not a very practical method, but if you are curious...
Internal methods
_cuts
Title : _cuts
Function : Figures out which enzymes we know about and cuts the sequence.
Returns : Nothing.
Arguments : None.
Comments : An internal method. This will figure out where the sequence
should be cut, and provide the appropriate results.
_enzyme_sites
Title : _enzyme_sites
Function : An internal method to figure out the two sides of an enzyme
Returns : The sequence before the cut and the sequence after the cut
Arguments : A Bio::Restriction::Enzyme object,
$comp : boolean, calculate based on $enz->complementary_cut()
if true, $enz->cut() if false
Status : NOW DEPRECATED - maj
_non_pal_enz
Title : _non_pal_enz
Function : Analyses non_palindromic enzymes for cuts in both ways
(in fact, delivers only minus strand cut positions in the
plus strand coordinates/maj)
Returns : A reference to an array of cut positions
Arguments: The sequence to check and the enzyme object
NOW DEPRECATED/maj
_ambig_cuts
Title : _ambig_cuts
Function : An internal method to localize the cuts in the sequence
Returns : A reference to an array of cut positions
Arguments : The separated enzyme site, the target sequence, and the enzyme object
Comments : This is a slow implementation but works for ambiguous sequences.
Whenever possible, _nonambig_cuts should be used as it is a lot faster.
_nonambig_cuts
Title : _nonambig_cuts Function : Figures out which enzymes we know about and cuts the sequence. Returns : Nothing. Arguments : The separated enzyme site, the target sequence, and the enzyme object
An internal method. This will figure out where the sequence should be cut, and provide the appropriate results. This is a much faster implementation because it doesn't use a regexp, but it can not deal with ambiguous sequences
_make_cuts
Title : _make_cuts
Usage : $an->_make_cuts( $target_sequence, $enzyme, $complement_q )
Function: Returns an array of cut sites on target seq, using enzyme
on the plus strand ($complement_q = 0) or minus strand
($complement_q = 1); follows Enzyme objects in
$enzyme->others()
Returns : array of scalar integers
Args : sequence string, B:R:Enzyme object, boolean
_multiple_cuts
Title : _multiple_cuts
Function : Figures out multiple digests
Returns : An array of the cut sites for multiply digested DNA
Arguments : A Bio::Restriction::EnzymeCollection object
Comments : Double digests is one subset of this, but you can use
as many enzymes as you want.
_circular
Title : _circular
Function : Identifies cuts at the join of the end of the target with
the beginning of the target
Returns : array of scalar integers ( cut sites near join, if any )
Arguments : scalar string (target sequence), Bio::Restriction::Enzyme obj
_expanded_string
Title : _expanded_string Function : Expand nucleotide ambiguity codes to their representative letters Returns : The full length string Arguments : The string to be expanded.
Stolen from the original RestrictionEnzyme.pm