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Copyright [2015-2018] EMBL-European Bioinformatics Institute
Licensed under the Apache License, Version 2.0 (the "License"); you may not use this file except in
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NAME
Bio::DB::HTS -- Read files using HTSlib including BAM/CRAM, Tabix and BCF database files
SYNOPSIS
use Bio::DB::HTS;
# high level API
# Note that the high level API does not reset the CRAM file pointer to the start
# of the file as the method to do so is (at time or writing) not easily accessible.
# Therefore a new HTS object may be needed to repeat a query.
my $hts = Bio::DB::HTS->new(-bam =>"data/ex1.bam",
-fasta=>"data/ex1.fa",
);
my @targets = $hts->seq_ids;
my @alignments = $hts->get_features_by_location(-seq_id => 'seq2',
-start => 500,
-end => 800);
for my $a (@alignments) {
# where does the alignment start in the reference sequence
my $seqid = $a->seq_id;
my $start = $a->start;
my $end = $a->end;
my $strand = $a->strand;
my $cigar = $a->cigar_str;
my $paired = $a->get_tag_values('PAIRED');
# where does the alignment start in the query sequence
my $query_start = $a->query->start;
my $query_end = $a->query->end;
my $ref_dna = $a->dna; # reference sequence bases
my $query_dna = $a->query->dna; # query sequence bases
my @scores = $a->qscore; # per-base quality scores
my $match_qual= $a->qual; # quality of the match
}
my @pairs = $hts->get_features_by_location(-type => 'read_pair',
-seq_id => 'seq2',
-start => 500,
-end => 800);
for my $pair (@pairs)
{
my $length = $pair->length; # insert length
my ($first_mate,$second_mate) = $pair->get_SeqFeatures;
my $f_start = $first_mate->start;
my $s_start = $second_mate->start;
}
# low level API
my $hfile = Bio::DB::HTSfile->open('/path/to/alignment_file');
my $header = $hfile->header_read;
my $target_count = $header->n_targets;
my $target_names = $header->target_name;
while (my $align = $hfile->read1($header))
{
my $seqid = $target_names->[$align->tid];
my $start = $align->pos+1;
my $end = $align->calend;
my $cigar = $align->cigar_str;
}
Bio::DB::HTSfile->index_build($bamfile);
my $index = Bio::DB::HTSfile->index_load($hfile);
my $index = Bio::DB::HTSfile->index_open_in_safewd($hfile);
my $callback = sub {
my $alignment = shift;
my $start = $alignment->start;
my $end = $alignment->end;
my $seqid = $target_names->[$alignment->tid];
print $alignment->qname," aligns to $seqid:$start..$end\n";
}
my $header = $index->header;
$index->fetch($hfile,$header->parse_region('seq2'),$callback);
DESCRIPTION
This module provides a Perl interface to the HTSlib library for indexed and unindexed SAM/BAM/CRAM
sequence alignment databases. It provides support for retrieving information on individual alignments,
read pairs, and alignment coverage information across large regions. It also provides callback
functionality for calling SNPs and performing other base-by-base functions.
The high-level API
The high-level API provides a BioPerl-compatible interface to indexed BAM and CRAM files. The alignment
file database is treated as a collection of Bio::SeqFeatureI features, and can be searched for features
by name, location, type and combinations of feature tags such as whether the alignment is part of a mate-
pair.
When opening a alignment database using the high-level API, you provide the pathnames of two files: the
FASTA file that contains the reference genome sequence, and the BAM file that contains the query
sequences and their alignments. If either of the two files needs to be indexed, the indexing will need to
be built. You can then query the database for alignment features by combinations of name, position, type,
and feature tag.
The high-level API provides access to up to four feature "types":
* "match": The "raw" unpaired alignment between a read and the
reference sequence.
* "read_pair": Paired alignments; a single composite
feature that contains two subfeatures for the alignments of each
of the mates in a mate pair.
* "coverage": A feature that spans a region of interest that contains
numeric information on the coverage of reads across the region.
* "region": A way of retrieving information about the reference
sequence. Searching for features of type "region" will return a
list of chromosomes or contigs in the reference sequence, rather
than read alignments.
* "chromosome": A synonym for "region".
Features can be en masse in a single call, retrieved in a memory-efficient streaming basis using an
iterator, or interrogated using a filehandle that return a series of SAM-format lines.
SAM alignment flags can be retrieved using BioPerl's feature "tag" mechanism. For example, to interrogate
the FIRST_MATE flag, one fetches the "FIRST_MATE" tag:
warn "aye aye captain!" if $alignment->get_tag_values('FIRST_MATE');
The Bio::SeqFeatureI interface has been extended to retrieve all flags as a compact human-readable
string, and to return the CIGAR alignment in a variety of formats.
Split alignments, such as reads that cover introns, are dealt with in one of two ways. The default is to
leave split alignments alone: they can be detected by one or more "N" operations in the CIGAR string.
Optionally, you can choose to have the API split these alignments across two or more subfeatures; the
CIGAR strings of these split alignments will be adjusted accordingly.
Interface to the pileup routines The API provides you with access to the samtools "pileup" API. This
gives you the ability to write a callback that will be invoked on every column of the alignment for the
purpose of calculating coverage, quality score metrics, or SNP calling.
Access to the reference sequence When you create the Bio::DB::HTS object, you can pass the path to a
FASTA file containing the reference sequence. Alternatively, you may pass an object that knows how to
retrieve DNA sequences across a range via the seq() or fetch_seq() methods, as described under new().
If the SAM/BAM file has MD tags, then these tags will be used to reconstruct the reference sequence when
necessary, in which case you can completely omit the -fasta argument. Note that not all SAM/BAM files
have MD tags, and those that do may not use them correctly due to the newness of this part of the SAM
spec. You may wish to populate these tags using samtools' "calmd" command.
If the -fasta argument is omitted and no MD tags are present, then the reference sequence will be
returned as 'N'.
The main object classes that you will be dealing with in the high-level API are as follows:
* Bio::DB::HTS -- A collection of alignments and reference sequences.
* Bio::DB::HTS::Alignment -- The alignment between a query and the reference.
* Bio::DB::HTS::Query -- An object corresponding to the query sequence in
which both (+) and (-) strand alignments are
shown in the reference (+) strand.
* Bio::DB::HTS::Target -- An interface to the query sequence in which
(-) strand alignments are shown in reverse
complement
You may encounter other classes as well. These include:
* Bio::DB::HTS::Segment -- This corresponds to a region on the reference
sequence.
* Bio::DB::HTS::Constants -- This defines CIGAR symbol constants and flags.
* Bio::DB::HTS::AlignWrapper -- An alignment helper object that adds split
alignment functionality. See Bio::DB::HTS::Alignment
for the documentation on using it.
* Bio::DB::HTS::ReadIterator -- An iterator that mediates the one-feature-at-a-time
retrieval mechanism.
* Bio::DB::HTS::FetchIterator -- Another iterator for feature-at-a-time retrieval.
The low-level API
The low-level API closely mirrors that of the HTSlib library. It provides the ability to open and read
SAM, BAM and CRAM files, build indexes, and perform searches across them.
The classes you will be interacting with in the low-level API are as follows:
* Bio::DB::HTS -- Methods that read and write SAM, BAM and CRAM files.
* Bio::DB::HTS::Header -- Methods for manipulating the BAM file header.
* Bio::DB::HTS::Alignment -- Methods for manipulating alignment data.
* Bio::DB::HTS::Pileup -- Methods for manipulating the pileup data structure.
* Bio::DB::HTS::Fai -- Methods for creating and reading from indexed Fasta
files.
METHODS
We cover the high-level API first. The high-level API code can be found in the files Bio/DB/HTS.pm and
Bio/DB/HTS/*.pm.
Bio::DB::HTS Constructor and basic accessors
$sam = Bio::DB::HTS->new(%options)
The Bio::DB::HTS object combines a Fasta file of the reference sequences with an SAM/BAM/CRAM
alignment file to allow for convenient retrieval of human-readable sequence IDs and reference
sequences. The new() constructor accepts a -name=>value style list of options as follows:
Option Description
------ -------------
-bam Path to the SAM/BAM/CRAM alignment file that contains the
alignments (required). A http: or ftp: URL is accepted.
-fasta Path to the Fasta file that contains
the reference sequences (optional). Alternatively,
you may pass any object that supports a seq()
or fetch_seq() method and takes the three arguments
($seq_id,$start,$end).
-expand_flags A boolean value. If true then the standard
alignment flags will be broken out as
individual tags such as 'M_UNMAPPED' (default false).
-split_splices A boolean value. If true, then alignments that
are split across splices will be broken out
into a single alignment containing two sub-
alignments (default false).
-split The same as -split_splices.
-force_refseq Always use the reference sequence file to derive the
reference sequence, even when the sequence can be
derived from the MD tag. This is slower, but safer
when working with BAM files derived from buggy aligners
or when the reference contains non-canonical (modified)
bases.
-autoindex Create an alignment index file if one does not exist
or the current one has a modification date
earlier than the alignment file.
An example of a typical new() constructor invocation is:
$hts = Bio::DB::HTS->new(-fasta => '/home/projects/genomes/hu17.fa',
-bam => '/home/projects/alignments/ej88.bam',
-expand_flags => 1,
-split_splices => 1);
If the -fasta argument is present, then you will be able to use the interface to fetch the reference
sequence's bases. Otherwise, calls that return the reference sequence will return sequences
consisting entirely of "N".
-expand_flags option, if true, has the effect of turning each of the standard SAM flags into a
separately retrievable tag in the Bio::SeqFeatureI interface. Otherwise, the standard flags will be
concatenated in easily parseable form as a tag named "FLAGS". See get_all_tags() and get_tag_values()
for more information.
Any two-letter extension flags, such as H0 or H1, will always appear as separate tags regardless of
the setting.
-split_splices has the effect of breaking up alignments that contain an "N" operation into subparts
for more convenient manipulation. For example, if you have both paired reads and spliced alignments
in the BAM file, the following code shows the subpart relationships:
$pair = $hts->get_feature_by_name('E113:01:01:23');
@mates = $pair->get_SeqFeatures;
@mate1_parts = $mates[0]->get_SeqFeatures;
@mate2_parts = $mates[1]->get_SeqFeatures;
Because there is some overhead to splitting up the spliced alignments, this option is false by
default.
Remote access to alignment files located on an HTTP or FTP server is possible. Simply replace the
path to the BAM file with the appropriate URL. Note that incorrect URLs may lead to a core dump.
It is not currently possible to refer to a remote FASTA file. These will have to be downloaded
locally and indexed before using.
$flag = $hts->expand_flags([$new_value])
Get or set the expand_flags option. This can be done after object creation and will have an immediate
effect on all alignments fetched from the alignment file.
$flag = $hts->split_splices([$new_value])
Get or set the split_splices option. This can be done after object creation and will affect all
alignments fetched from the alignment file subsequently.
$header = $hts->header
Return the Bio::DB::HTS::Header object associated with the alignment file. You can manipulate the
header using the low-level API.
$hts_path = $hts->hts_path
Return the path of the alignment file used to create the hts object. This makes the object more
portable.
$hts_file = $hts->$hts_file
Returns the low-level Bio::DB::HTSfile object associated with the opened file.
$fai = $hts->fai
Returns the Bio::DB::HTS::Fai object associated with the Fasta file. You can then manipulate this
object with the low-level API.
The index can be built automatically for you if it does not already exist. If index building is
necessarily, the process will need write privileges to the same directory in which the Fasta file
resides.> If the process does not have write permission, then the call will fail.
$hts_idx = $hts->hts_index
Return the Bio::DB::HTS::Index object associated with the alignment file.
The index is not automatically built.
$hts->clone
Bio::DB::HTS objects are not stable across fork() operations. If you fork, you must call clone()
either in the parent or the child process before attempting to call any methods.
Getting information about reference sequences
The Bio::DB::HTS object provides the following methods for getting information about the reference
sequence(s) contained in the associated Fasta file.
@seq_ids = $hts->seq_ids
Returns an unsorted list of the IDs of the reference sequences (known elsewhere in this document as
seq_ids). This is the same as the identifier following the ">" sign in the Fasta file (e.g. "chr1").
$num_targets = $hts->n_targets
Return the number of reference sequences.
$length = $hts->length('seqid')
Returns the length of the reference sequence named "seqid".
$seq_id = $hts->target_name($tid)
Translates a numeric target ID (TID) returned by the low-level API into a seq_id used by the high-
level API.
$length = $hts->target_len($tid)
Translates a numeric target ID (TID) from the low-level API to a sequence length.
$dna = $hts->seq($seqid,$start,$end)
Returns the DNA across the region from start to end on reference seqid. Note that this is a string,
not a Bio::PrimarySeq object. If no -fasta path was passed when the sam object was created, then you
will receive a series of N nucleotides of the requested length.
Creating and querying segments
Bio::DB::HTS::Segment objects refer regions on the reference sequence. They can be used to retrieve the
sequence of the reference, as well as alignments that overlap with the region.
$segment = $hts->segment($seqid,$start,$end);
$segment = $hts->segment(-seq_id=>'chr1',-start=>5000,-end=>6000);
Segments are created using the Bio:DB::HTS->segment() method. It can be called using one to three
positional arguments corresponding to the seq_id of the reference sequence, and optionally the start
and end positions of a subregion on the sequence. If the start and/or end are undefined, they will be
replaced with the beginning and end of the sequence respectively.
Alternatively, you may call segment() with named -seq_id, -start and -end arguments.
All coordinates are 1-based.
$seqid = $segment->seq_id
Return the segment's sequence ID.
$start = $segment->start
Return the segment's start position.
$end = $segment->end
Return the segment's end position.
$strand = $segment->strand
Return the strand of the segment (always 0).
$length = $segment->length
Return the length of the segment.
$dna = $segment->dna
Return the DNA string for the reference sequence under this segment.
$seq = $segment->seq
Return a Bio::PrimarySeq object corresponding to the sequence of the reference under this segment.
You can get the actual DNA string in this redundant-looking way:
$dna = $segment->seq->seq
The advantage of working with a Bio::PrimarySeq object is that you can perform operations on it,
including taking its reverse complement and subsequences.
@alignments = $segment->features(%args)
Return alignments that overlap the segment in the associated alignment file. The optional %args list
allows you to filter features by name, tag or other attributes. See the documentation of the
Bio::DB::HTS->features() method for the full list of options. Here are some typical examples:
# get all the overlapping alignments
@all_alignments = $segment->features;
# get an iterator across the alignments
my $iterator = $segment->features(-iterator=>1);
while (my $align = $iterator->next_seq) { do something }
# get a SAM filehandle across the alignments
my $fh = $segment->features(-fh=>1);
while (<$fh>) { print }
# get only the alignments with unmapped mates
my @unmapped = $segment->features(-flags=>{M_UNMAPPED=>1});
# get coverage across this region
my ($coverage) = $segment->features('coverage');
my @data_points = $coverage->coverage;
# grep through features using a coderef
my @reverse_alignments = $segment->features(
-filter => sub {
my $a = shift;
return $a->strand < 0;
});
$tag = $segment->primary_tag
$tag = $segment->source_tag
Return the strings "region" and "sam/bam" respectively. These methods allow the segment to be passed
to BioPerl methods that expect Bio::SeqFeatureI objects.
$segment->name, $segment->display_name, $segment->get_SeqFeatures, $segment->get_tag_values
These methods are provided for Bio::SeqFeatureI compatibility and don't do anything of interest.
Retrieving alignments, mate pairs and coverage information
The features() method is an all-purpose tool for retrieving alignment information from the SAM/BAM/CRAM
alignment file database. In addition, the methods get_features_by_name(), get_features_by_location() and
others provide convenient shortcuts to features().
These methods either return a list of features, an iterator across a list of features, or a filehandle
opened on a pseudo-SAM file.
@features = $hts->features(%options)
$iterator = $hts->features(-iterator=>1,%more_options)
$filehandle = $hts->features(-fh=>1,%more_options)
@features = $hts->features('type1','type2'...)
This is the all-purpose interface for fetching alignments and other types of features from the
database. Arguments are a -name=>value option list selected from the following list of options:
Option Description
------ -------------
-type Filter on features of a given type. You may provide
either a scalar typename, or a reference to an
array of desired feature types. Valid types are
"match", "read_pair", "coverage" and "chromosome."
See below for a full explanation of feature types.
-name Filter on reads with the designated name. Note that
this can be a slow operation unless accompanied by
the feature location as well.
-seq_id Filter on features that align to seq_id between start
-start and end. -start and -end must be used in conjunction
-end with -seq_id. If -start and/or -end are absent, they
will default to 1 and the end of the reference
sequence, respectively.
-flags Filter features that match a list of one or more
flags. See below for the format.
-attributes The same as -flags, for compatibility with other
-tags APIs.
-filter Filter on features with a coderef. The coderef will
receive a single argument consisting of the feature
and should return true to keep the feature, or false
to discard it.
-iterator Instead of returning a list of features, return an
iterator across the results. To retrieve the results,
call the iterator's next_seq() method repeatedly
until it returns undef to indicate that no more
matching features remain.
-fh Instead of returning a list of features, return a
filehandle. Read from the filehandle to retrieve
each of the results in TAM format, one alignment
per line read. This only works for features of type
"match."
The high-level API introduces the concept of a feature "type" in order to provide several convenience
functions. You specify types by using the optional -type argument. The following types are currently
supported:
match. The "match" type corresponds to the unprocessed SAM alignment. It will retrieve single reads,
either mapped or unmapped. Each match feature's primary_tag() method will return the string "match."
The features returned by this call are of type Bio::DB::HTS::AlignWrapper.
read_pair. The "paired_end" type causes the sam interface to find and merge together mate pairs.
Fetching this type of feature will yield a series of Bio::SeqFeatureI objects, each as long as the
total distance on the reference sequence spanned by the mate pairs. The top-level feature is of type
Bio::SeqFeature::Lite; it contains two Bio::DB::HTS::AlignWrapper subparts.
Call get_SeqFeatures() to get the two individual reads. Example:
my @pairs = $hts->features(-type=>'read_pair');
my $p = $pairs[0];
my $i_length = $p->length;
my @ends = $p->get_SeqFeatures;
my $left = $ends[0]->start;
my $right = $ends[1]->end;
coverage. The "coverage" type causes the sam interface to calculate coverage across the designated
region. It only works properly if accompanied by the desired location of the coverage graph; -seq_id
is a mandatory argument for coverage calculation, and -start and -end are optional. The call will
return a single Bio::SeqFeatureI object whose primary_tag() is "coverage." To recover the coverage
data, call the object's coverage() method to obtain an array (list context) or arrayref (scalar
context) of coverage counts across the region of interest:
my ($coverage) = $hts->features(-type=>'coverage',-seq_id=>'seq1');
my @data = $coverage->coverage;
my $total;
for (@data) { $total += $_ }
my $average_coverage = $total/@data;
By default the coverage graph will be at the base pair level. So for a region 5000 bp wide,
coverage() will return an array or arrayref with exactly 5000 elements. However, you also have the
option of calculating the coverage across larger bins. Simply append the number of intervals you are
interested to the "coverage" typename. For example, fetching "coverage:500" will return a feature
whose coverage() method will return the coverage across 500 intervals.
chromosome or region. The "chromosome" or "region" type are interchangeable. They ask the sam
interface to construct Bio::DB::HTS::Segment representing the reference sequences. These two calls
give similar results:
my $segment = $hts->segment('seq2',1=>500);
my ($seg) = $hts->features(-type=>'chromosome',
-seq_id=>'seq2',-start=>1,-end=>500);
Due to an unresolved bug, you cannot fetch chromosome features in the same call with matches and
other feature types call. Specifically, this works as expected:
my @chromosomes = $hts->features (-type=>'chromosome');
But this doesn't (as of 18 June 2009):
my @chromosomes_and_matches = $hts->features(-type=>['match','chromosome']);
If no -type argument is provided, then features() defaults to finding features of type "match."
You may call features() with a plain list of strings (positional arguments, not -type=>value
arguments). This will be interpreted as a list of feature types to return:
my ($coverage) = $hts->features('coverage')
For a description of the methods available in the features returned from this call, please see
Bio::SeqfeatureI and Bio::DB::HTS::Alignment.
You can filter "match" and "read_pair" features by name, location and/or flags. The name and flag
filters are not very efficient. Unless they are combined with a location filter, they will initiate
an exhaustive search of the BAM database.
Name filters are case-insensitive, and allow you to use shell-style "*" and "?" wildcards. Flag
filters created with the -flag, -attribute or -tag options have the following syntax:
-flag => { FLAG_NAME_1 => ['list','of','possible','values'],
FLAG_NAME_2 => ['list','of','possible','values'],
...
}
The value of -flag is a hash reference in which the keys are flag names and the values are array
references containing lists of acceptable values. The list of values are OR'd with each other, and
the flag names are AND'd with each other.
The -filter option provides a completely generic filtering interface. Provide a reference to a
subroutine. It will be called once for each potential feature. Return true to keep the feature, or
false to discard it. Here is an example of how to find all matches whose alignment quality scores are
greater than 80.
@features = $hts->features(-filter=>sub {shift->qual > 80} );
By default, features() returns a list of all matching features. You may instead request an iterator
across the results list by passing -iterator=>1. This will give you an object that has a single
method, next_seq():
my $high_qual = $hts->features(-filter => sub {shift->qual > 80},
-iterator=> 1 );
while (my $feature = $high_qual->next_seq) {
# do something with the alignment
}
Similarly, by passing a true value to the argument -fh, you can obtain a filehandle to a virtual SAM
file. This only works with the "match" feature type:
my $high_qual = $hts->features(-filter => sub {shift->qual > 80},
-fh => 1 );
while (my $tam_line = <$high_qual>) {
chomp($tam_line);
# do something with it
}
@features = $hts->get_features_by_name($name)
Convenience method. The same as calling $hts->features(-name=>$name);
$feature = $hts->get_feature_by_name($name)
Convenience method. The same as ($hts->features(-name=>$name))[0].
@features = $hts->get_features_by_location($seqid,$start,$end)
Convenience method. The same as calling $hts->features(-seq_id=>$seqid,-start=>$start,-end=>$end).
@features = $hts->get_features_by_flag(%flags)
Convenience method. The same as calling $hts->features(-flags=>\%flags). This method is also called
get_features_by_attribute() and get_features_by_tag(). Example:
@features = $hts->get_features_by_flag(H0=>1)
$feature = $hts->get_feature_by_id($id)
The high-level API assigns each feature a unique ID composed of its read name, position and strand
and returns it when you call the feature's primary_id() method. Given that ID, this method returns
the feature.
$iterator = $hts->get_seq_stream(%options)
Convenience method. This is the same as calling $hts->features(%options,-iterator=>1).
$fh = $hts->get_seq_fh(%options)
Convenience method. This is the same as calling $hts->features(%options,-fh=>1).
$fh = $hts->tam_fh
Convenience method. It is the same as calling $hts->features(-fh=>1).
@types = $hts->types
This method returns the list of feature types (e.g. "read_pair") returned by the current version of
the interface.
The generic fetch() and pileup() methods
Lastly, the high-level API supports two methods for rapidly traversing indexed BAM databases.
$hts->fetch($region,$callback)
This method traverses the indicated region and invokes a callback code reference on each match.
Specify a region using the syntax "seqid:start-end", or either of the alternative syntaxes
"seqid:start..end" and "seqid:start,end". If start and end are absent, then the entire reference
sequence is traversed. If end is absent, then the end of the reference sequence is assumed.
The callback will be called repeatedly with a Bio::DB::HTS::AlignWrapper on the argument list.
Example:
$hts->fetch('seq1:600-700',
sub {
my $a = shift;
print $a->display_name,' ',$a->cigar_str,"\n";
});
Note that the fetch() operation works on reads that overlap the indicated region. Therefore the
callback may be called for reads that align to the reference at positions that start before or end
after the indicated region.
$hts->pileup($region,$callback [,$keep_level])
This method, which is named after the native bam_lpileupfile() function in the C interfaces,
traverses the indicated region and generates a "pileup" of all the mapped reads that cover it. The
user-provided callback function is then invoked on each position of the alignment along with a data
structure that provides access to the individual aligned reads.
As with fetch(), the region is specified as a string in the format "seqid:start-end",
"seqid:start..end" or "seqid:start,end".
The callback is a coderef that will be invoked with three arguments: the seq_id of the reference
sequence, the current position on the reference (in 1-based coordinates!), and a reference to an
array of Bio::DB::HTS::Pileup objects. Here is the typical call signature:
sub {
my ($seqid,$pos,$pileup) = @_;
# do something
}
For example, if you call pileup on the region "seq1:501-600", then the callback will be invoked for
all reads that overlap the indicated region. The first invocation of the callback will typically have
a $pos argument somewhat to the left of the desired region and the last call will be somewhat to the
right. You may wish to ignore positions that are outside of the requested region. Also be aware that
the reference sequence position uses 1-based coordinates, which is different from the low-level
interface, which use 0-based coordinates.
The size of the $pileup array reference indicates the read coverage at that position. Here is a
simple average coverage calculator:
my $depth = 0;
my $positions = 0;
my $callback = sub {
my ($seqid,$pos,$pileup) = @_;
next unless $pos >= 501 && $pos <= 600;
$positions++;
$depth += @$pileup;
}
$hts->pileup('seq1:501-600',$callback);
print "coverage = ",$depth/$positions;
Each Bio::DB::HTS::Pileup object describes the position of a read in the alignment. Briefly,
Bio::DB::HTS::Pileup has the following methods:
$pileup->alignment The alignment at this level (a
Bio::DB::HTS::AlignWrapper object).
$pileup->qpos The position of the read base at the pileup site,
in 0-based coordinates.
$pileup->pos The position of the read base at the pileup site,
in 1-based coordinates;
$pileup->level The level of the read in the multiple alignment
view. Note that this field is only valid when
$keep_level is true, so it may not be relevant post
htslib move.
$pileup->indel Length of the indel at this position: 0 for no indel, positive
for an insertion (relative to the reference), negative for a
deletion (relative to the reference.)
$pileup->is_del True if the base on the padded read is a deletion.
$pileup->is_refskip True if the base on the padded read is a gap relative to the reference (denoted as < or > in the pileup)
$pileup->is_head True if this is the first base in the query sequence.
$pileup->is_tail True if this is the last base in the query sequence.
See "Examples" for a very simple SNP caller.
$hts->fast_pileup($region,$callback [,$keep_level])
This is identical to pileup() except that the pileup object returns low-level Bio::DB::HTS::Alignment
objects rather than the higher-level Bio::DB::HTS::AlignWrapper objects. This makes it roughly 50%
faster, but you lose the align objects' seq_id() and get_tag_values() methods. As a compensation, the
callback receives an additional argument corresponding to the Bio::DB::HTS object. You can use this
to create AlignWrapper objects on an as needed basis:
my $callback = sub {
my($seqid,$pos,$pileup,$hts) = @_;
for my $p (@$pileup) {
my $alignment = $p->alignment;
my $wrapper = Bio::DB::HTS::AlignWrapper->new($alignment,$hts);
my $has_mate = $wrapper->get_tag_values('PAIRED');
}
};
Bio::DB::HTS->max_pileup_cnt([$new_cnt])
$hts->max_pileup_cnt([$new_cnt])
The HTSlib library caps pileups at a set level, defaulting to 8000. The callback will not be invoked
on a single position more than the level set by the cap, even if there are more reads. Called with no
arguments, this method returns the current cap value. Called with a numeric argument, it changes the
cap. There is currently no way to specify an unlimited cap.
This method can be called as an instance method or a class method.
$hts->coverage2BedGraph([$fh])
This special-purpose method will compute a four-column BED graph of the coverage across the entire
alignment file and print it to STDOUT. You may provide a filehandle to redirect output to a file or
pipe.
The next sections correspond to the low-level API, which let you create and manipulate Perl objects that
correspond directly to data structures in the C interface. A major difference between the high and low
level APIs is that in the high-level API, the reference sequence is identified using a human-readable
seq_id. However, in the low-level API, the reference is identified using a numeric target ID ("tid"). The
target ID is established during the creation of the alignment file and is a small 0-based integer index.
The Bio::DB::HTS::Header object provides methods for converting from seq_ids to tids.
Indexed Fasta Files
These methods relate to the indexed Fasta (".fai") files.
$fai = Bio::DB::HTS::Fai->load('/path/to/file.fa')
Load an indexed Fasta file and return the object corresponding to it. If the index does not exist, it
will be created automatically. Note that you pass the path to the Fasta file, not the index.
For consistency with Bio::DB::HTS->open() this method is also called open().
$dna_string = $fai->fetch("seqid:start-end")
Given a sequence ID contained in the Fasta file and optionally a subrange in the form "start-end",
finds the indicated subsequence and returns it as a string.
Alignment Files
These methods provide interfaces to alignment files in SAM/BAM/CRAM format.
$hts_file = Bio::DB::HTSfile->open('/path/to/file.bam' [,$mode])
Open the alignment file at the indicated path. Mode, if present, must be one of the file stream open
flags ("r", "w", "wb", "wc", "a", "r+", etc.). If absent, mode defaults to "r". [write formats: w =
SAM, wb = BAM, wc = CRAM]
Note that Bio::DB::HTS objects are not stable across fork() operations. If you fork, and intend to
use the object in both parent and child, you must reopen the Bio::DB::HTS in either the child or the
parent (but not both) before attempting to call any of the object's methods.
The path may be an http: or ftp: URL, in which case a copy of the index file will be downloaded to
the current working directory (see below) and all accesses will be performed on the remote BAM file.
Example:
$hfile = Bio::DB::HTSfile->open('http://some.site.com/nextgen/chr1_bowtie.bam');
$header = $hfile->header_read()
Given an open alignment file, return a Bio::DB::HTS::Header object containing information about the
reference sequence(s). Note that you must invoke header_read() at least once before calling read1().
$status_code = $hfile->header_write($header, [$reference])
Given a Bio::DB::HTSfile::Header object and a BAM file opened in write mode, write the header to the
file. If the write fails the process will be terminated at the C layer. If $hfile is CRAM formated a
second argument $reference, which is the path to the reference Fasta file, must be passed. The
result code is (currently) always zero.
$alignment = $hfile->read1($header)
Read one alignment from the alignment file and return it as a Bio::DB::HTS::Alignment object. The
$header parameter is returned by invoking header().
$bytes = $hfile->write1($header, $alignment)
Given a BAM file that has been opened in write mode and a Bio::DB::HTS::Alignment object, write the
alignment to the BAM file and return the number of bytes successfully written.
Index methods
The Bio::DB::HTS::Index object provides access to index (.bai|.csi, .crai) files.
$status_code = Bio::DB::HTS->index_build('/path/to/file.?am')
Given the path to an alignment file, this function attempts to build an index. The process in which
the alignment file exists must be writable by the current process and there must be sufficient disk
space for the operation or the process will be terminated in the C library layer. The result code is
currently always zero, but in the future may return a negative value to indicate failure.
The index file built will depend on the alignment file type specified. For CRAM this will be a .crai
file, for BAM .bai.
$index = Bio::DB::HTS->index('/path/to/file.?am',$reindex)
Attempt to open the index for the indicated alignment file. If $reindex is true, and the index either
does not exist or is out of date with respect to the alignment file (by checking modification dates),
then attempt to rebuild the index. Will throw an exception if the index does not exist or if
attempting to rebuild the index was unsuccessful.
$index = Bio::DB::HTS->index_load('/path/to/file.?am')
Attempt to open the index file for an alignment file, returning a Bio::DB::HTS::Index object. The
filename path to use is the alignment file, not the index file (i.e. .bam or .cram, not .bai|.csi or
.crai)
$index = Bio::DB::HTS->index_open_in_safewd('/path/to/file.?am' [,$mode])
When opening a remote alignmentfile, you may not wish for the index to be downloaded to the current
working directory. This version of index_open copies the index into the directory indicated by the
TMPDIR environment variable or the system-defined /tmp directory if not present. You may change the
environment variable just before the call to change its behavior.
$code = $index->fetch($hfile,$tid,$start,$end,$callback [,$callback_data])
This is the low-level equivalent of the $hts->fetch() function described for the high-level API.
Given a open BAM file object, the numeric ID of the reference sequence, start and end ranges on the
reference, and a coderef, this function will traverse the region and repeatedly invoke the coderef
with each Bio::DB::HTS::Alignment object that overlaps the region.
Arguments:
Argument Description
-------- -----------
$hts_file The Bio::DB::HTSfile object that corresponds to the
index object.
$tid The target ID of the reference sequence. This can
be obtained by calling $header->parse_region() with
an appropriate opened Bio::DB::HTS::Header object.
$start The start and end positions of the desired range on
the reference sequence given by $tid, in 0-based
$end coordinates. Like the $tid, these can be obtained from
$header->parse_region().
$callback A coderef that will be called for each read overlapping
the designated region.
$callback_data Any arbitrary Perl data that you wish to pass to the
$callback (optional).
The coderef's call signature should look like this:
my $callback = sub {
my ($alignment,$data) = @_;
...
}
The first argument is a Bio::DB::HTS::Alignment object. The second is the callback data (if any)
passed to fetch().
Fetch() returns an integer code, but its meaning is not described in the SAM/BAM C library
documentation.
$index->pileup($htsfile,$tid,$start,$end,$callback [,$callback_data])
This is the low-level version of the pileup() method, which allows you to invoke a coderef for every
position in a BAM alignment. Arguments are:
Argument Description
-------- -----------
$hts_file The Bio::DB::HTSfile object that corresponds to the
index object.
$tid The target ID of the reference sequence. This can
be obtained by calling $header->parse_region() with
an appropriate opened Bio::DB::HTS::Header object.
$start The start and end positions of the desired range on
the reference sequence given by $tid, in 0-based
$end coordinates. Like the $tid, these can be obtained from
$header->parse_region().
$callback A coderef that will be called for each position of the
alignment across the designated region.
$callback_data Any arbitrary Perl data that you wish to pass to the
$callback (optional).
The callback will be invoked with four arguments corresponding to the numeric sequence ID of the
reference sequence, the zero-based position on the alignment, an arrayref of Bio::DB::HTS::Pileup
objects, and the callback data, if any. A typical call signature will be this:
$callback = sub {
my ($tid,$pos,$pileups,$callback_data) = @_;
for my $pileup (@$pileups) {
# do something
};
Note that the position argument is zero-based rather than 1-based, as it is in the high-level API.
The Bio::DB::HTS::Pileup object was described earlier in the description of the high-level pileup()
method.
$coverage = $index->coverage($hfile,$tid,$start,$end [,$bins [,maxcnt]])
Calculate coverage for the region on the target sequence given by $tid between positions $start and
$end (zero-based coordinates). This method will return an array reference equal to the size of the
region (by default). Each element of the array will be an integer indicating the number of reads
aligning over that position. If you provide an option binsize in $bins, the array will be $bins
elements in length, and each element will contain the average coverage over that region as a floating
point number.
By default, the underlying Samtools library caps coverage counting at a fixed value of 8000. You may
change this default by providing an optional numeric sixth value, which changes the cap for the
duration of the call, or by invoking Bio::DB::HTS->max_pileup_cnt($new_value), which changes the cap
permanently. Unfortunately there is no way of specifying that you want an unlimited cap.
HTS header methods
The Bio::DB::HTS::Header object contains information regarding the reference sequence(s) used to
construct the corresponding alignment file. It is most frequently used to translate between numeric
target IDs and human-readable seq_ids. Headers can be created by reading from a BAM file using
Bio::DB::HTS->header(). You can also create header objects from scratch, although there is not much that
you can do with such objects at this point.
$header = Bio::DB::HTS::Header->new()
Return a new, empty, header object.
$n_targets = $header->n_targets
Return the number of reference sequences in the database.
$name_arrayref = $header->target_name
Return a reference to an array of reference sequence names, corresponding to the high-level API's
seq_ids.
To convert from a target ID to a seq_id, simply index into this array:
$seq_id = $header->target_name->[$tid];
$length_arrayref = $header->target_len
Return a reference to an array of reference sequence lengths. To get the length of the sequence
corresponding to $tid, just index into the array returned by target_len():
$length = $header->target_len->[$tid];
$text = $header->text
$header->text("new value")
Read the text portion of the header. The text can be replaced by providing the replacement string as
an argument. Note that you should follow the header conventions when replacing the header text. No
parsing or other error-checking is performed.
($tid,$start,$end) = $header->parse_region("seq_id:start-end")
Given a string in the format "seqid:start-end" (using a human-readable seq_id and 1-based start and
end coordinates), parse the string and return the target ID and start and end positions in 0-based
coordinates. If the range is omitted, then the start and end coordinates of the entire sequence is
returned. If only the end position is omitted, then the end of the sequence is assumed.
$header->view1($alignment)
This method will accept a Bio::DB::HTS::Alignment object, convert it to a line of TAM output, and
write the output to STDOUT. In the low-level API there is currently no way to send the output to a
different filehandle or capture it as a string.
Bio::DB::HTS::Pileup methods
An array of Bio::DB::HTS::Pileup object is passed to the pileup() callback for each position of a multi-
read alignment. Each pileup object contains information about the alignment of a single read at a single
position.
$alignment = $pileup->alignment
Return the Bio::DB::HTS::Alignment object at this level. This provides you with access to the
aligning read.
$alignment = $pileup->b
An alias for alignment(), provided for compatibility with the C API.
$pos = $pileup->qpos
The position of the aligning base in the read in zero-based coordinates.
$pos = $pileup->pos
The position of the aligning base in 1-based coordinates.
$level = $pileup->level
The "level" of the read in the BAM-generated text display of the alignment.
$indel = $pileup->indel
Length of the indel at this position: 0 for no indel, positive for an insertion (relative to the
reference), negative for a deletion (relative to the reference sequence.)
$flag = $pileup->is_del
True if the base on the padded read is a deletion.
$flag = $pileup->is_refskip
True if the base on the padded read is a gap relative to the reference (denoted as < or > in the
pileup)
$flag = $pileup->is_head
True if this is the first base in the query sequence.
$flag = $pileup->is_tail
True if this is the last base in the query sequence.
The alignment objects
Please see Bio::DB::HTS::Alignment for documentation of the Bio::DB::HTS::Alignment and
Bio::DB::HTS::AlignWrapper objects.
DEPENDENCIES
Module::Build, Carp, Bio::Perl (>=1.006001), Test::More
EXPORT
None
AUTHORS
Rishi Nag <rishi@ebi.ac.uk>, original author. Alessandro Vullo "<avullo at cpan.org>", the current developer and maintainer.
CONTRIBUTORS
Andy Yates, Keiran Raine, John Marshall, Zhicheng Liu, Can Wood, Dietmar Rieder, Chris Fields, David
Jones, James Gilbert, Alex Hodgkins (Congenica Ltd.), Rob Aganrab
KNOWN BUGS
• SAM file reading and iterating over alignments does not work with older htslib versions (<1.5)
• The padded_alignment() function with CRAM files may produce invalid output: unequal lenght of the
strings that specify the pairwise alignment
Please report any bugs or feature requests to "bug-bio-db-hts at rt.cpan.org", or through the web
interface at <http://rt.cpan.org/NoAuth/ReportBug.html?Queue=Bio-DB-HTS>. I will be notified, and then
you'll automatically be notified of progress on your bug as I make changes.
TESTING AND CONTRIBUTING
You can obtain the most recent development version of this module via the GitHub repository at
https://github.com/Ensembl/Bio-DB-HTS. Please feel free to submit bug reports, patches etc.
SUPPORT
You can find documentation for this module with the perldoc command.
perldoc Bio::DB::HTS
You can also look for information at:
• RT: CPAN's request tracker (report bugs here)
<http://rt.cpan.org/NoAuth/Bugs.html?Dist=Bio-DB-HTS>
• AnnoCPAN: Annotated CPAN documentation
<http://annocpan.org/dist/Bio-DB-HTS>
• CPAN Ratings
<http://cpanratings.perl.org/d/Bio-DB-HTS>
• Search CPAN
<http://search.cpan.org/dist/Bio-DB-HTS/>
EXAMPLES
For illustrative purposes only, here is an extremely stupid SNP caller that tallies up bases that are
q>20 and calls a SNP if there are at least 4 non-N/non-indel bases at the position and at least 25% of
them are a non-reference base.
my @SNPs; # this will be list of SNPs
my $snp_caller = sub {
my ($seqid,$pos,$p) = @_;
my $refbase = $hts->segment($seqid,$pos,$pos)->dna;
my ($total,$different);
for my $pileup (@$p) {
my $b = $pileup->alignment;
next if $pileup->indel or $pileup->is_refskip; # don't deal with these ;-)
my $qbase = substr($b->qseq,$pileup->qpos,1);
next if $qbase =~ /[nN]/;
my $qscore = $b->qscore->[$pileup->qpos];
next unless $qscore > 25;
$total++;
$different++ if $refbase ne $qbase;
}
if ($total >= 4 && $different/$total >= 0.25) {
push @SNPs,"$seqid:$pos";
}
};
$hts->pileup('seq1',$snp_caller);
print "Found SNPs: @SNPs\n";
GBrowse Compatibility
The Bio::DB::HTS interface can be used as a backend to GBrowse (gmod.sourceforge.net/gbrowse). GBrowse
can calculate and display coverage graphs across large regions, alignment cartoons across intermediate
size regions, and detailed base-pair level alignments across small regions.
Here is a typical configuration for a BAM database that contains information from a shotgun genomic
sequencing project. Some notes:
* It is important to set "search options = none" in order to avoid
GBrowse trying to scan through the BAM database to match read
names. This is a time-consuming operation.
* The callback to "bgcolor" renders pairs whose mates are unmapped in
red.
* The callback to "balloon hover" causes a balloon to pop up with the
read name when the user hovers over each paired read. Otherwise the
default behavior would be to provide information about the pair as
a whole.
* When the user zooms out to 1001 bp or greaterp, the track switches
to a coverage graph.
[bamtest:database]
db_adaptor = Bio::DB::HTSfile
db_args = -bam /var/www/gbrowse2/databases/bamtest/ex1.bam
search options= default
[Pair]
feature = read_pair
glyph = segments
database = bamtest
draw_target = 1
show_mismatch = 1
bgcolor = sub {
my $f = shift;
return $f->get_tag_values('M_UNMAPPED') ? 'red' : 'green';
}
fgcolor = green
height = 3
label = sub {shift->display_name}
label density = 50
bump = fast
connector = dashed
balloon hover = sub {
my $f = shift;
return '' unless $f->type eq 'match';
return 'Read: '.$f->display_name.' : '.$f->flag_str;
}
key = Read Pairs
[Pair:1000]
feature = coverage:1001
glyph = wiggle_xyplot
height = 50
min_score = 0
autoscale = local
To show alignment data correctly when the user is zoomed in, you should also provide a pointer to the
FASTA file containing the reference genome. In this case, modify the db_args line to read:
db_args = -bam /var/www/gbrowse2/databases/bamtest/ex1.bam
-fasta /var/www/gbrowse2/databases/bamtest/ex1.fa
SEE ALSO
Bio::Perl, Bio::DB::HTS::Alignment, Bio::DB::HTS::Constants