| Bio::DB::HTS(3pm) | User Contributed Perl Documentation | Bio::DB::HTS(3pm) |
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 compliance with the License. You may obtain a copy of the License at
http://www.apache.org/licenses/LICENSE-2.0
Unless required by applicable law or agreed to in writing, software distributed under the License is distributed on an "AS IS" BASIS, WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied. See the License for the specific language governing permissions and limitations under the License.
Bio::DB::HTS -- Read files using HTSlib including BAM/CRAM, Tabix and BCF database files
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);
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 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 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.
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.
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.
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.
The index is not automatically built.
The Bio::DB::HTS object provides the following methods for getting information about the reference sequence(s) contained in the associated Fasta file.
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.
Alternatively, you may call segment() with named -seq_id, -start and -end arguments.
All coordinates are 1-based.
$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.
# 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;
});
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.
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_flag(H0=>1)
Lastly, the high-level API supports two methods for rapidly traversing indexed BAM databases.
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.
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.
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');
}
};
This method can be called as an instance method or a class method.
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.
These methods relate to the indexed Fasta (".fai") files.
For consistency with Bio::DB::HTS->open() this method is also called open().
These methods provide interfaces to alignment files in SAM/BAM/CRAM format.
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');
The Bio::DB::HTS::Index object provides access to index (.bai|.csi, .crai) files.
The index file built will depend on the alignment file type specified. For CRAM this will be a .crai file, for BAM .bai.
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.
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.
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.
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.
To convert from a target ID to a seq_id, simply index into this array:
$seq_id = $header->target_name->[$tid];
$length = $header->target_len->[$tid];
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.
Please see Bio::DB::HTS::Alignment for documentation of the Bio::DB::HTS::Alignment and Bio::DB::HTS::AlignWrapper objects.
Module::Build, Carp, Bio::Perl (>=1.006001), Test::More
None
Rishi Nag <rishi@ebi.ac.uk>, original author.
Alessandro Vullo "<avullo at cpan.org>", the current developer and maintainer.
Andy Yates, Keiran Raine, John Marshall, Zhicheng Liu, Can Wood, Dietmar Rieder, Chris Fields, David Jones, James Gilbert, Alex Hodgkins (Congenica Ltd.), Rob Aganrab
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.
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.
You can find documentation for this module with the perldoc command.
perldoc Bio::DB::HTS
You can also look for information at:
<http://rt.cpan.org/NoAuth/Bugs.html?Dist=Bio-DB-HTS>
<http://annocpan.org/dist/Bio-DB-HTS>
<http://cpanratings.perl.org/d/Bio-DB-HTS>
<http://search.cpan.org/dist/Bio-DB-HTS/>
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";
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
Bio::Perl, Bio::DB::HTS::Alignment, Bio::DB::HTS::Constants
| 2024-03-31 | perl v5.38.2 |