Bio::Seq - Sequence object, with features # This is the main sequence object in Bioperl # gets a sequence from a file $seqio = Bio::SeqIO->new( '-format' => 'embl' , -file => 'myfile.dat'); $seqobj = $seqio->next_seq(); # SeqIO can both read and write sequences; see Bio::SeqIO # for more information and examples # get from database $db = Bio::DB::GenBank->new(); $seqobj = $db->get_Seq_by_acc('X78121'); # make from strings in script $seqobj = Bio::Seq->new( -display_id => 'my_id', -seq => $sequence_as_string); # gets sequence as a string from sequence object $seqstr = $seqobj->seq(); # actual sequence as a string $seqstr = $seqobj->subseq(10,50); # slice in biological coordinates # retrieves information from the sequence # features must implement Bio::SeqFeatureI interface @features = $seqobj->get_SeqFeatures(); # just top level foreach my $feat ( @features ) { print "Feature ",$feat->primary_tag," starts ",$feat->start," ends ", $feat->end," strand ",$feat->strand,"\n"; # features retain link to underlying sequence object print "Feature sequence is ",$feat->seq->seq(),"\n" } # sequences may have a species if( defined $seq->species ) { print "Sequence is from ",$species->binomial_name," [",$species->common_name,"]\n"; } # annotation objects are Bio::AnnotationCollectionI's $ann = $seqobj->annotation(); # annotation object # references is one type of annotations to get. Also get # comment and dblink. Look at Bio::AnnotationCollection for # more information foreach my $ref ( $ann->get_Annotations('reference') ) { print "Reference ",$ref->title,"\n"; } # you can get truncations, translations and reverse complements, these # all give back Bio::Seq objects themselves, though currently with no # features transfered my $trunc = $seqobj->trunc(100,200); my $rev = $seqobj->revcom(); # there are many options to translate - check out the docs my $trans = $seqobj->translate(); # these functions can be chained together my $trans_trunc_rev = $seqobj->trunc(100,200)->revcom->translate(); A Seq object is a sequence with sequence features placed on it. The Seq object contains a PrimarySeq object for the actual sequence and also implements its interface. In Bioperl we have 3 main players that people are going to use frequently Bio::PrimarySeq - just the sequence and its names, nothing else. Bio::SeqFeatureI - a location on a sequence, potentially with a sequence and annotation. Bio::Seq - A sequence and a collection of sequence features (an aggregate) with its own annotation. Although Bioperl is not tied heavily to file formats these distinctions do map to file formats sensibly and for some bioinformaticians this might help Bio::PrimarySeq - Fasta file of a sequence Bio::SeqFeatureI - A single entry in an EMBL/GenBank/DDBJ feature table Bio::Seq - A single EMBL/GenBank/DDBJ entry By having this split we avoid a lot of nasty circular references (sequence features can hold a reference to a sequence without the sequence holding a reference to the sequence feature). See Bio::PrimarySeq and Bio::SeqFeatureI for more information. Ian Korf really helped in the design of the Seq and SeqFeature system.

Examples
A simple and fundamental block of code: use Bio::SeqIO; my $seqIOobj = Bio::SeqIO->new(-file=>"1.fa"); # create a SeqIO object my $seqobj = $seqIOobj->next_seq; # get a Seq object With the Seq object in hand one has access to a powerful set of Bioperl methods and related Bioperl objects. This next script will take a file of sequences in EMBL format and create a file of the reverse-complemented sequences in Fasta format using Seq objects. It also prints out details about the exons it finds as sequence features in Genbank Flat File format. use Bio::Seq; use Bio::SeqIO; $seqin = Bio::SeqIO->new( -format => 'EMBL' , -file => 'myfile.dat'); $seqout= Bio::SeqIO->new( -format => 'Fasta', -file => '>output.fa'); while((my $seqobj = $seqin->next_seq())) { print "Seen sequence ",$seqobj->display_id,", start of seq ", substr($seqobj->seq,1,10),"\n"; if( $seqobj->alphabet eq 'dna') { $rev = $seqobj->revcom; $id = $seqobj->display_id(); $id = "$id.rev"; $rev->display_id($id); $seqout->write_seq($rev); } foreach $feat ( $seqobj->get_SeqFeatures() ) { if( $feat->primary_tag eq 'exon' ) { print STDOUT "Location ",$feat->start,":", $feat->end," GFF[",$feat->gff_string,"]\n"; } } } Let's examine the script. The lines below import the Bioperl modules. Seq is the main Bioperl sequence object and SeqIO is the Bioperl support for reading sequences from files and to files use Bio::Seq; use Bio::SeqIO; These two lines create two SeqIO streams: one for reading in sequences and one for outputting sequences: $seqin = Bio::SeqIO->new( -format => 'EMBL' , -file => 'myfile.dat'); $seqout= Bio::SeqIO->new( -format => 'Fasta', -file => '>output.fa'); Notice that in the "$seqout" case there is a greater-than sign, indicating the file is being opened for writing. Using the '-argument' => value syntax is common in Bioperl. The file argument is like an argument to open() . You can also pass in filehandles or FileHandle objects by using the -fh argument (see Bio::SeqIO documentation for details). Many formats in Bioperl are handled, including Fasta, EMBL, GenBank, Swissprot (swiss), PIR, and GCG. $seqin = Bio::SeqIO->new( -format => 'EMBL' , -file => 'myfile.dat'); $seqout= Bio::SeqIO->new( -format => 'Fasta', -file => '>output.fa'); This is the main loop which will loop progressively through sequences in a file, and each call to $seqio->next_seq() provides a new Seq object from the file: while((my $seqobj = $seqio->next_seq())) { This print line below accesses fields in the Seq object directly. The $seqobj->display_id is the way to access the display_id attribute of the Seq object. The $seqobj->seq method gets the actual sequence out as string. Then you can do manipulation of this if you want to (there are however easy ways of doing truncation, reverse-complement and translation). print "Seen sequence ",$seqobj->display_id,", start of seq ", substr($seqobj->seq,1,10),"\n"; Bioperl has to guess the alphabet of the sequence, being either 'dna', 'rna', or 'protein'. The alphabet attribute is one of these three possibilities. if( $seqobj->alphabet eq 'dna') { The $seqobj->revcom method provides the reverse complement of the Seq object as another Seq object. Thus, the $rev variable is a reference to another Seq object. For example, one could repeat the above print line for this Seq object (putting $rev in place of $seqobj). In this case we are going to output the object into the file stream we built earlier on. $rev = $seqobj->revcom; When we output it, we want the id of the outputted object to be changed to "$id.rev", ie, with .rev on the end of the name. The following lines retrieve the id of the sequence object, add .rev to this and then set the display_id of the rev sequence object to this. Notice that to set the display_id attribute you just need call the same method, display_id(), with the new value as an argument. Getting and setting values with the same method is common in Bioperl. $id = $seqobj->display_id(); $id = "$id.rev"; $rev->display_id($id); The write_seq method on the SeqIO output object, $seqout, writes the $rev object to the filestream we built at the top of the script. The filestream knows that it is outputting in fasta format, and so it provides fasta output. $seqout->write_seq($rev); This block of code loops over sequence features in the sequence object, trying to find ones who have been tagged as 'exon'. Features have start and end attributes and can be outputted in Genbank Flat File format, GFF, a standarized format for sequence features. foreach $feat ( $seqobj->get_SeqFeatures() ) { if( $feat->primary_tag eq 'exon' ) { print STDOUT "Location ",$feat->start,":", $feat->end," GFF[",$feat->gff_string,"]\n"; } } The code above shows how a few Bio::Seq methods suffice to read, parse, reformat and analyze sequences from a file. A full list of methods available to Bio::Seq objects is shown below. Bear in mind that some of these methods come from PrimarySeq objects, which are simpler than Seq objects, stripped of features (see Bio::PrimarySeq for more information). # these methods return strings, and accept strings in some cases: $seqobj->seq(); # string of sequence $seqobj->subseq(5,10); # part of the sequence as a string $seqobj->accession_number(); # when there, the accession number $seqobj->alphabet(); # one of 'dna','rna',or 'protein' $seqobj->seq_version() # when there, the version $seqobj->keywords(); # when there, the Keywords line $seqobj->length() # length $seqobj->desc(); # description $seqobj->primary_id(); # a unique id for this sequence regardless # of its display_id or accession number $seqobj->display_id(); # the human readable id of the sequence Some of these values map to fields in common formats. For example, The display_id() method returns the LOCUS name of a Genbank entry, the (\S+) following the > character in a Fasta file, the ID from a SwissProt file, and so on. The desc() method will return the DEFINITION line of a Genbank file, the description following the display_id in a Fasta file, and the DE field in a SwissProt file. # the following methods return new Seq objects, but # do not transfer features across to the new object: $seqobj->trunc(5,10) # truncation from 5 to 10 as new object $seqobj->revcom # reverse complements sequence $seqobj->translate # translation of the sequence # if new() can be called this method returns 1, else 0 $seqobj->can_call_new # the following method determines if the given string will be accepted # by the seq() method - if the string is acceptable then validate() # returns 1, or 0 if not $seqobj->validate_seq($string) # the following method returns or accepts a Species object: $seqobj->species(); Please see Bio::Species for more information on this object. # the following method returns or accepts an Annotation object # which in turn allows access to Annotation::Reference # and Annotation::Comment objects: $seqobj->annotation(); These annotations typically refer to entire sequences, unlike features. See Bio::AnnotationCollectionI, Bio::Annotation::Collection, Bio::Annotation::Reference, and Bio::Annotation::Comment for details. It is also important to be able to describe defined portions of a sequence. The combination of some description and the corresponding sub-sequence is called a feature - an exon and its coordinates within a gene is an example of a feature, or a domain within a protein. # the following methods return an array of SeqFeatureI objects: $seqobj->get_SeqFeatures # The 'top level' sequence features $seqobj->get_all_SeqFeatures # All sequence features, including sub-seq # features, such as features in an exon # to find out the number of features use: $seqobj->feature_count Here are just some of the methods available to SeqFeatureI objects: # these methods return numbers: $feat->start # start position (1 is the first base) $feat->end # end position (2 is the second base) $feat->strand # 1 means forward, -1 reverse, 0 not relevant # these methods return or accept strings: $feat->primary_tag # the name of the sequence feature, eg # 'exon', 'glycoslyation site', 'TM domain' $feat->source_tag # where the feature comes from, eg, 'EMBL_GenBank', # or 'BLAST' # this method returns the more austere PrimarySeq object, not a # Seq object - the main difference is that PrimarySeq objects do not # themselves contain sequence features $feat->seq # the sequence between start,end on the # correct strand of the sequence See Bio::PrimarySeq for more details on PrimarySeq objects. # useful methods for feature comparisons, for start/end points $feat->overlaps($other) # do $feat and $other overlap? $feat->contains($other) # is $other completely within $feat? $feat->equals($other) # do $feat and $other completely agree? # one can also add features $seqobj->add_SeqFeature($feat) # returns 1 if successful $seqobj->add_SeqFeature(@features) # returns 1 if successful # sub features. For complex join() statements, the feature # is one sequence feature with many sub SeqFeatures $feat->sub_SeqFeature # returns array of sub seq features Please see Bio::SeqFeatureI and Bio::SeqFeature::Generic, for more information on sequence features. It is worth mentioning that one can also retrieve the start and end positions of a feature using a Bio::LocationI object: $location = $feat->location # $location is a Bio::LocationI object $location->start; # start position $location->end; # end position This is useful because one needs a Bio::Location::SplitLocationI object in order to retrieve the coordinates inside the Genbank or EMBL join() statements (e.g. "CDS join(51..142,273..495,1346..1474)"): if ( $feat->location->isa('Bio::Location::SplitLocationI') && $feat->primary_tag eq 'CDS' ) { foreach $loc ( $feat->location->sub_Location ) { print $loc->start . ".." . $loc->end . "\n"; } } See Bio::LocationI and Bio::Location::SplitLocationI for more information. This class implements the following interfaces. Note that this includes implementing Bio::PrimarySeqI.

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. bioperl-l@bioperl.org - General discussion http://bio.perl.org/MailList.html - About the mailing lists

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 email or the web: bioperl-bugs@bioperl.org http://bugzilla.bioperl.org/ Email birney@ebi.ac.uk Jason Stajich <jason@bioperl.org> The rest of the documentation details each of the object methods. Internal methods are usually preceded with a "_".

new
Title : new Usage : $seq = Bio::Seq->new( -seq => 'ATGGGGGTGGTGGTACCCT', -id => 'human_id', -accession_number => 'AL000012', ); Function: Returns a new Seq object from basic constructors, being a string for the sequence and strings for id and accession_number Returns : a new Bio::Seq object The PrimarySeq interface provides the basic sequence getting and setting methods for on all sequences. These methods implement the Bio::PrimarySeq interface by delegating to the primary_seq inside the object. This means that you can use a Seq object wherever there is a PrimarySeq, and of course, you are free to use these functions anyway.

seq
Title : seq Usage : $string = $obj->seq() Function: Get/Set the sequence as a string of letters. The case of the letters is left up to the implementer. Suggested cases are upper case for proteins and lower case for DNA sequence (IUPAC standard), but implementations are suggested to keep an open mind about case (some users... want mixed case!) Returns : A scalar Args : Optionally on set the new value (a string). An optional second argument presets the alphabet (otherwise it will be guessed). Both parameters may also be given in named paramater style with -seq and -alphabet being the names.

validate_seq
Title : validate_seq Usage : if(! $seq->validate_seq($seq_str) ) { print "sequence $seq_str is not valid for an object of type ", ref($seq), "\n"; } Function: Validates a given sequence string. A validating sequence string must be accepted by seq(). A string that does not validate will lead to an exception if passed to seq(). The implementation provided here does not take alphabet() into account. Allowed are all letters (A-Z) and '-','.', and '*'. Example : Returns : 1 if the supplied sequence string is valid for the object, and 0 otherwise. Args : The sequence string to be validated.

length
Title : length Usage : $len = $seq->length() Function: Example : Returns : Integer representing the length of the sequence. Args : None

subseq
Title : subseq Usage : $substring = $obj->subseq(10,40); Function: Returns the subseq from start to end, where the first base is 1 and the number is inclusive, ie 1-2 are the first two bases of the sequence Start cannot be larger than end but can be equal Returns : A string Args : 2 integers

display_id
Title : display_id Usage : $id = $obj->display_id or $obj->display_id($newid); Function: Gets or sets the display id, also known as the common name of the Seq object. The semantics of this is that it is the most likely string to be used as an identifier of the sequence, and likely to have "human" readability. The id is equivalent to the LOCUS field of the GenBank/EMBL databanks and the ID field of the Swissprot/sptrembl database. In fasta format, the >(\S+) is presumed to be the id, though some people overload the id to embed other information. Bioperl does not use any embedded information in the ID field, and people are encouraged to use other mechanisms (accession field for example, or extending the sequence object) to solve this. Notice that $seq->id() maps to this function, mainly for legacy/convenience issues. Returns : A string Args : None or a new id

accession_number
Title : accession_number Usage : $unique_biological_key = $obj->accession_number; Function: Returns the unique biological id for a sequence, commonly called the accession_number. For sequences from established databases, the implementors should try to use the correct accession number. Notice that primary_id() provides the unique id for the implemetation, allowing multiple objects to have the same accession number in a particular implementation. For sequences with no accession number, this method should return "unknown". Can also be used to set the accession number. Example : $key = $seq->accession_number or $seq->accession_number($key) Returns : A string Args : None or an accession number

desc
Title : desc Usage : $seqobj->desc($string) or $seqobj->desc() Function: Sets or gets the description of the sequence Example : Returns : The description Args : The description or none

primary_id
Title : primary_id Usage : $unique_implementation_key = $obj->primary_id; Function: Returns the unique id for this object in this implementation. This allows implementations to manage their own object ids in a way the implementation can control clients can expect one id to map to one object. For sequences with no natural id, this method should return a stringified memory location. Can also be used to set the primary_id. Also notice that this method is not delegated to the internal Bio::PrimarySeq object [Note this method name is likely to change in 1.3] Example : $id = $seq->primary_id or $seq->primary_id($id) Returns : A string Args : None or an id

can_call_new
Title : can_call_new Usage : if ( $obj->can_call_new ) { $newobj = $obj->new( %param ); } Function: can_call_new returns 1 or 0 depending on whether an implementation allows new constructor to be called. If a new constructor is allowed, then it should take the followed hashed constructor list. $myobject->new( -seq => $sequence_as_string, -display_id => $id -accession_number => $accession -alphabet => 'dna', ); Example : Returns : 1 or 0 Args : None

alphabet
Title : alphabet Usage : if ( $obj->alphabet eq 'dna' ) { /Do Something/ } Function: Returns the type of sequence being one of 'dna', 'rna' or 'protein'. This is case sensitive. This is not called <type> because this would cause upgrade problems from the 0.5 and earlier Seq objects. Returns : A string either 'dna','rna','protein'. NB - the object must make a call of the type - if there is no type specified it has to guess. Args : None

is_circular
Title : is_circular Usage : if( $obj->is_circular) { /Do Something/ } Function: Returns true if the molecule is circular Returns : Boolean value Args : none

object_id
Title : object_id Usage : $string = $obj->object_id() Function: a string which represents the stable primary identifier in this namespace of this object. For DNA sequences this is its accession_number, similarly for protein sequences This is aliased to accession_number(). Returns : A scalar

version
Title : version Usage : $version = $obj->version() Function: a number which differentiates between versions of the same object. Higher numbers are considered to be later and more relevant, but a single object described the same identifier should represent the same concept Returns : A number

authority
Title : authority Usage : $authority = $obj->authority() Function: a string which represents the organisation which granted the namespace, written as the DNS name for organisation (eg, wormbase.org) Returns : A scalar

namespace
Title : namespace Usage : $string = $obj->namespace() Function: A string representing the name space this identifier is valid in, often the database name or the name describing the collection Returns : A scalar

display_name
Title : display_name Usage : $string = $obj->display_name() Function: A string which is what should be displayed to the user the string should have no spaces (ideally, though a cautious user of this interface would not assumme this) and should be less than thirty characters (though again, double checking this is a good idea) This is aliased to display_id(). Returns : A scalar

description
Title : description Usage : $string = $obj->description() Function: A text string suitable for displaying to the user a description. This string is likely to have spaces, but should not have any newlines or formatting - just plain text. The string should not be greater than 255 characters and clients can feel justified at truncating strings at 255 characters for the purposes of display This is aliased to desc(). Returns : A scalar

annotation
Title : annotation Usage : $ann = $seq->annotation or $seq->annotation($annotation) Function: Gets or sets the annotation Returns : L<Bio::AnnotationCollectionI> object Args : None or L<Bio::AnnotationCollectionI> object See Bio::AnnotationCollectionI and Bio::Annotation::Collection for more information This includes methods for retrieving, adding, and removing features.

get_SeqFeatures
Title : get_SeqFeatures Usage : Function: Get the feature objects held by this feature holder. Features which are not top-level are subfeatures of one or more of the returned feature objects, which means that you must traverse the subfeature arrays of each top-level feature object in order to traverse all features associated with this sequence. Use get_all_SeqFeatures() if you want the feature tree flattened into one single array. Example : Returns : an array of Bio::SeqFeatureI implementing objects Args : none At some day we may want to expand this method to allow for a feature filter to be passed in.

get_all_SeqFeatures
Title : get_all_SeqFeatures Usage : @feat_ary = $seq->get_all_SeqFeatures(); Function: Returns the tree of feature objects attached to this sequence object flattened into one single array. Top-level features will still contain their subfeature-arrays, which means that you will encounter subfeatures twice if you traverse the subfeature tree of the returned objects. Use get_SeqFeatures() if you want the array to contain only the top-level features. Returns : An array of Bio::SeqFeatureI implementing objects. Args : None

feature_count
Title : feature_count Usage : $seq->feature_count() Function: Return the number of SeqFeatures attached to a sequence Returns : integer representing the number of SeqFeatures Args : None

add_SeqFeature
Title : add_SeqFeature Usage : $seq->add_SeqFeature($feat); $seq->add_SeqFeature(@feat); Function: Adds the given feature object (or each of an array of feature objects to the feature array of this sequence. The object passed is required to implement the Bio::SeqFeatureI interface. Returns : 1 on success Args : A Bio::SeqFeatureI implementing object, or an array of such objects.

remove_SeqFeatures
Title : remove_SeqFeatures Usage : $seq->remove_SeqFeatures(); Function: Flushes all attached SeqFeatureI objects. To remove individual feature objects, delete those from the returned array and re-add the rest. Example : Returns : The array of Bio::SeqFeatureI objects removed from this seq. Args : None These methods are inherited from the PrimarySeq interface and work as one expects, building new Bio::Seq objects or other information as expected. See Bio::PrimarySeq for more information. Sequence Features are not transfered to the new objects. This is possibly a mistake. Anyone who feels the urge in dealing with this is welcome to give it a go.

revcom
Title : revcom Usage : $rev = $seq->revcom() Function: Produces a new Bio::Seq object which is the reversed complement of the sequence. For protein sequences this throws an exception of "Sequence is a protein. Cannot revcom" The id is the same id as the original sequence, and the accession number is also identical. If someone wants to track that this sequence has be reversed, it needs to define its own extensions To do an in-place edit of an object you can go: $seq = $seq->revcom(); This of course, causes Perl to handle the garbage collection of the old object, but it is roughly speaking as efficient as an in-place edit. Returns : A new (fresh) Bio::Seq object Args : None

trunc
Title : trunc Usage : $subseq = $myseq->trunc(10,100); Function: Provides a truncation of a sequence Example : Returns : A fresh Seq object Args : A Seq object

id
Title : id Usage : $id = $seq->id() Function: This is mapped on display_id Returns : value of display_id() Args : [optional] value to update display_id These methods are specific to the Bio::Seq object, and not found on the Bio::PrimarySeq object

primary_seq
Title : primary_seq Usage : $seq->primary_seq or $seq->primary_seq($newval) Function: Get or set a PrimarySeq object Example : Returns : PrimarySeq object Args : None or PrimarySeq object

species
Title : species Usage : $species = $seq->species() or $seq->species($species) Function: Gets or sets the species Returns : L<Bio::Species> object Args : None or L<Bio::Species> object See Bio::Species for more information