SYNOPSIS
use Bio::Seq::SimulatedRead;
use Bio::PrimarySeq;
# Create a reference sequence
my $genome = Bio::PrimarySeq->new( -id => 'human_chr2',
-seq => 'TAAAAAAACCCCTG',
-desc => 'The human genome' );
# A 10-bp error-free read taken from a genome
my $read = Bio::Seq::SimulatedRead->new(
-reference => $genome , # sequence to generate the read from
-id => 'read001', # read ID
-start => 3 , # start of the read on the genome forward strand
-end => 12 , # end of the read on the genome forward strand
-strand => 1 , # genome strand that the read is on
);
# Display the sequence of the read
print $read->seq."\n";
# Add a tag or MID to the beginning of the read
$read->mid('ACGT');
# Add sequencing errors (error positions are 1-based and relative to the
# error-free MID-containing read)
my $errors = {};
$errors->{'8'}->{'+'} = 'AAA'; # insertion of AAA after residue 8
$errors->{'1'}->{'%'} = 'G'; # substitution of residue 1 by a G
$errors->{'4'}->{'-'} = undef; # deletion of residue 4
$read->errors($errors);
# Display the sequence of the read with errors
print $read->seq."\n";
# String representation of where the read came from and its errors
print $read->desc."\n";
DESCRIPTION
This object is a simulated read with sequencing errors. The user can provide a reference sequence to take a read from, the position and orientation of the read on the reference sequence, and the sequencing errors to generate.The sequence of the read is automatically calculated based on this information. By default, the description of the reads contain tracking information and will look like this (Bioperl-style):
reference=human_chr2 start=3 end=12 strand=-1 mid=ACGT errors=1%G,4-,8+AAA description="The human genome"
or Genbank-style:
reference=human_chr2 position=complement(3..12) mid=ACGT errors=1%G,4-,8+AAA description="The human genome"
Creating a simulated read follows these steps:
1/ Define the read start(), end(), strand() and qual_levels() if you want
quality scores to be generated. Do not change these values once set because
the read will not be updated.
2/ Specify the reference sequence that the read should be taken from. Once
this is done, you have a fully functional read. Do not use the reference()
method again after you have gone to the next step.
3/ Use mid() to input a MID (or tag or barcode) to add to the beginning of the
read. You can change the MID until you go to next step.
4/ Give sequencing error specifications using errors() as the last step. You
can do that as many times as you like, and the read will be updated.
AUTHOR
Florent E Angly <florent . angly @ gmail-dot-com>.Copyright (c) 2011 Florent E Angly.
This library is free software; you can redistribute it under the GNU General Public License version 3.
new
Title : new
Function : Create a new simulated read object
Usage : my $read = Bio::Seq::SimulatedRead->new(
-id => 'read001',
-reference => $seq_obj ,
-errors => $errors ,
-start => 10 ,
-end => 135 ,
-strand => 1 ,
);
Arguments: -reference => Bio::SeqI, Bio::PrimarySeqI object representing the
reference sequence to take the read from. See
reference().
-errors => Hashref representing the position of errors in the read
See errors().
-mid => String of a MID to prepend to the read. See mid().
-track => Track where the read came from in the read description?
See track().
-coord_style => Define what coordinate system to use. See coord_style().
All other methods from Bio::LocatableSeq are available.
Returns : new Bio::Seq::SimulatedRead object
qual_levels
Title : qual_levels
Function : Get or set the quality scores to give to the read. By default, if your
reference sequence does not have quality scores, no quality scores
are generated for the simulated read. The generated quality scores
are very basic. If a residue is error-free, it gets the quality score
defined for good residues. If the residue has an error (is an
addition or a mutation), the residue gets the quality score specified
for bad residues. Call the qual_levels() method before using the
reference() method.
Usage : my $qual_levels = $read->qual_levels( );
Arguments: Array reference containing the quality scores to use for:
1/ good residues (e.g. 30)
2/ bad residues (e.g. 10)
Returns : Array reference containing the quality scores to use.
reference
Title : reference
Function : Get or set the reference sequence that the read comes from. Once the
reference has been set, you have a functional simulated read which
supports all the Bio::LocatableSeq methods. This method must be
called after qual_levels() but before mid() or errors().
Usage : my $seq_obj = $read->reference();
Arguments: Bio::SeqI or Bio::PrimarySeqI object
Returns : Bio::SeqI or Bio::PrimarySeqI object
mid
Title : mid
Function : Get or set a multiplex identifier (or MID, or tag, or barcode) to
add to the read. By default, no MID is used. This method must be
called after reference() but before errors().
Usage : my $mid = read->mid();
Arguments: MID sequence string (e.g. 'ACGT')
Returns : MID sequence string
errors
Title : errors
Function : Get or set the sequencing errors and update the read. By default, no
errors are made. This method must be called after the mid() method.
Usage : my $errors = $read->errors();
Arguments: Reference to a hash of the position and nature of sequencing errors.
The positions are 1-based relative to the error-free MID-containing
read (not relative to the reference sequence). For example:
$errors->{34}->{'%'} = 'T' ; # substitution of residue 34 by a T
$errors->{23}->{'+'} = 'GG' ; # insertion of GG after residue 23
$errors->{45}->{'-'} = undef; # deletion of residue 45
Substitutions and deletions are for a single residue, but additions
can be additions of several residues.
An alternative way to specify errors is by using array references
instead of scalar for the hash values. This allows to specify
redundant mutations. For example, the case presented above would
result in the same read sequence as the example below:
$errors->{34}->{'%'} = ['C', 'T'] ; # substitution by a C and then a T
$errors->{23}->{'+'} = ['G', 'G'] ; # insertion of G and then a G
$errors->{45}->{'-'} = [undef, undef]; # deletion of residue, and again
Returns : Reference to a hash of the position and nature of sequencing errors.
track
Title : track
Function : Get or set the tracking status in the read description. By default,
tracking is on. This method can be called at any time.
Usage : my $track = $read->track();
Arguments: 1 for tracking, 0 otherwise
Returns : 1 for tracking, 0 otherwise
coord_style
Title : coord_style
Function : When tracking is on, define which 1-based coordinate system to use
in the read description:
* 'bioperl' uses the start, end and strand keywords (default),
similarly to the GFF3 format. Example:
start=1 end=10 strand=+1
start=1 end=10 strand=-1
* 'genbank' does only provide the position keyword. Example:
position=1..10
position=complement(1..10)
Usage : my $coord_style = $read->track();
Arguments: 'bioperl' or 'genbank'
Returns : 'bioperl' or 'genbank'