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->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
  # 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.

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://bioperl.org/wiki/Mailing_lists  - About the mailing lists

Support

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 the bugs and their resolution. Bug reports can be submitted via the web:

  https://github.com/bioperl/bioperl-live/issues

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

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 parameter style
           with -seq and -alphabet being the names.

validate_seq

 Title   : validate_seq
 Usage   : if(! $seqobj->validate_seq($seq_str) ) {
                print "sequence $seq_str is not valid for an object of
                alphabet ",$seqobj->alphabet, "\n";
           }
 Function: Test that the given sequence is valid, i.e. contains only valid
           characters. The allowed characters are all letters (A-Z) and '-','.',
           '*','?','=' and '~'. Spaces are not valid. Note that this
           implementation does not take alphabet() into account.
 Returns : 1 if the supplied sequence string is valid, 0 otherwise.
 Args    : - Sequence string to be validated
           - Boolean to throw an error if the sequence is invalid

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 implementation, 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 (or unset to undef).
           [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, or undef to unset the primary 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: Get/Set 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    : optional string to set : 'dna' | 'rna' | 'protein'

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 assume 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 
           $seq->annotation($ann)
 Function: Gets or sets the annotation
 Returns : Bio::AnnotationCollectionI object
 Args    : None or Bio::AnnotationCollectionI object

See Bio::AnnotationCollectionI and Bio::Annotation::Collection for more information

get_Annotations()

 Usage   : my @annotations = $seq->get_Annotations('key')
 Function: Retrieves all the Bio::AnnotationI objects for a specific key
           for this object
 Returns : list of Bio::AnnotationI - empty if no objects stored for a key
 Args    : string which is key for annotations

add_Annotation()

 Usage   : $seq->add_Annotation('reference',$object);
           $seq->add_Annotation($object,'Bio::MyInterface::DiseaseI');
           $seq->add_Annotation($object);
           $seq->add_Annotation('disease',$object,'Bio::MyInterface::DiseaseI');
 Function: Adds an annotation for a specific key for this sequence object.
           If the key is omitted, the object to be added must provide a value
           via its tagname().
           If the archetype is provided, this and future objects added under
           that tag have to comply with the archetype and will be rejected
           otherwise.
 Returns : none
 Args    : annotation key ('disease', 'dblink', ...)
           object to store (must be Bio::AnnotationI compliant)
           [optional] object archetype to map future storage of object
           of these types to

remove_Annotations()

 Usage   : $seq->remove_Annotations()
 Function: Remove the annotations for the specified key from this sequence 
           object
 Returns : an list of Bio::AnnotationI compliant objects which were stored
           under the given key(s) for this sequence object
 Args    : the key(s) (tag name(s), one or more strings) for which to
           remove annotations (optional; if none given, flushes all
           annotations)

get_num_of_annotations()

 Usage   : my $count = $seq->get_num_of_annotations()
 Alias   : num_Annotations
 Function: Returns the count of all annotations stored for this sequence
           object      
 Returns : integer
 Args    : none

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.
           Specific features can be obtained by primary tag, specified in 
           the argument.
           Use get_all_SeqFeatures() if you want the feature tree
           flattened into one single array.
 Example : my @feats = $seq->get_SeqFeatures or
           my @genefeats = $seq->get_SeqFeatures('gene')
 Returns : an array of Bio::SeqFeatureI implementing objects
 Args    : [optional] string (feature tag)

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);
 Function: Adds the given feature object to the feature array of this
           sequence. The object passed is required to implement the
           Bio::SeqFeatureI interface.
           The 'EXPAND' qualifier (see L<Bio::FeatureHolderI>) is supported, but
           has no effect,
 Returns : 1 on success
 Args    : A Bio::SeqFeatureI implementing object.

remove_SeqFeatures

 Title   : remove_SeqFeatures
 Usage   : $seq->remove_SeqFeatures();
 Function: Removes all attached SeqFeatureI objects or those with the 
           specified primary tag
 Example : my @gene_feats = $seq->remove_seqFeatures('gene') or
           my @feats = $seq->remove_seqFeatures()
 Returns : The array of Bio::SeqFeatureI objects removed from the sequence
 Args    : None, or a feature primary tag

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

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