--- name: "Bio::DB::Bam::Alignment" section: "3pm" platform: "linux" package: "libbio-samtools-perl" version: "1.43-5+b1" release: "Debian 13 (Trixie)" platforms: ["linux"] url: "https://new.carta.tech/Bio::DB::Bam::Alignment" --- # Bio::DB::Bam::Alignment(3pm) ## NAME Bio::DB::Bam::Alignment -- The SAM/BAM alignment object ## SYNOPSIS ``` use Bio::DB::Sam; my $sam = Bio::DB::Sam->new(-fasta=>"data/ex1.fa", -bam =>"data/ex1.bam"); my @alignments = $sam->get_features_by_location(-seq_id => 'seq2', -start => 500, -end => 800); for my $a (@alignments) { my $seqid = $a->seq_id; my $start = $a->start; my $end = $a->end; my $strand = $a->strand; my $ref_dna= $a->dna; my $query_start = $a->query->start; my $query_end = $a->query->end; my $query_strand = $a->query->strand; my $query_dna = $a->query->dna; my $cigar = $a->cigar_str; my @scores = $a->qscore; # per-base quality scores my $score = $a->qstring; # TAM-style quality string my $match_qual= $a->qual; # quality of the match my $paired = $a->get_tag_values('PAIRED'); } ``` ## DESCRIPTION The Bio::DB::Bam::Alignment and Bio::DB::Bam::AlignWrapper classes together represent an alignment between a sequence read (the "query") and a reference sequence (the "target"). Bio::DB::Bam::Alignment adheres strictly to the C-level BAM library's definition of a bam1\_t\* and is used in the Bio::DB::Sam low-level API The latter adds convenience methods that make it similar to a BioPerl Bio::SeqFeatureI object. This manual page describes both. ## High-level Bio::DB::Bam::Alignment methods These methods are provided by Bio::DB::Bam::Alignment, and are intended to be compatible with the Bio::SeqFeatureI interfaces. Note that these objects are **not** compatible with Bio::Align::AlignI, as the BAM API is fundamentally incompatible with the BioPerl API for alignments (the first deals with the alignment of a single read against the reference sequence, while the second deals with a multiple alignment). Note that the high-level API return Bio::DB::Bam::AlignWrapper objects **except** in the case of the callback to the **fast\_pileup()** method. In this case only, the object returned by calling `$pileup`-\>b() is a Bio::DB::Bam::Alignment object for performance reasons. - $seq\_id = $align-\>seq\_id Return the seq\_id of the reference (target) sequence. This method is only available in the Bio::DB::Bam::AlignWrapper extension. - $start = $align-\>start Return the start of the alignment in 1-based reference sequence coordinates. - $end = $align-\>end Return the end of the alignment in 1-based reference sequence coordinates. - $len = $align-\>length Return the length of the alignment on the reference sequence. - $mseqid = $align-\>mate\_seq\_id Return the seq\_id of the mate's reference (target) sequence. This method is only available in the Bio::DB::AlignWrapper extension. - $mstart = $align-\>mate\_start For paired reads, return the start of the mate's alignment in 1-based reference sequence coordinates. - $mend = $align-\>mate\_end For paired reads, return the end position of the mate's alignment in 1-based reference sequence coordinates. - $mlen = $align-\>mate\_len For mate-pairs, retrieve the length of the mate's alignment on the reference sequence. - $strand = $align-\>strand Return the strand of the alignment as -1 for reversed, +1 for forward. NOTE: In versions 1.00-1.06, this method always returned +1. As of version 1.07, this behavior is fixed. - $mstrand = $align-\>mstrand If the read has a mate pair, return the strand of the mate in the format -1 or +1. - $ref\_dna = $align-\>dna Returns the **reference** sequence's DNA across the aligned region. If an MD tag is present in the alignment, it will be used preferentially to reconstruct the reference sequence. Otherwise the reference DNA access object passed to Bio::DB::Sam-\>**new()** will be used. - $ref\_dna = $align-\>seq The **reference** sequence's DNA as a Bio::PrimarySeqI object (useful for passing to BioPerl functions and for calculating subsequences and reverse complements). - $query = $align-\>query This method returns a Bio::DB::Alignment::Query object that can be used to retrieve information about the query sequence. The next few entries show how to use this object. - $read\_name = $align-\>query-\>name The name of the read. - $q\_start = $align-\>query-\>start This returns the start position of the query (read) sequence in 1-based coordinates. It acts via a transient Bio::DB::Bam::Query object that is provided for Bio::Graphics compatibility (see Bio::Graphics). - $q\_end = $align-\>query-\>end This returns the end position of the query sequence in 1-based coordinates. - $q\_len = $align-\>query-\>length Return the length of the alignment on the read. - $scores = $align-\>query-\>score Return an array reference containing the unpacked quality scores for each base of the query sequence. The length of this array reference will be equal to the length of the read. - $read\_dna = $align-\>query-\>dna The read's DNA string. - $read\_seq = $align-\>query-\>seq The read's DNA as a Bio::PrimarySeqI object. - $target = $align-\>target; The **target()** method is similar to **query()**, except that it follows Bio::AlignIO conventions for how to represent minus strand alignments. The object returned has **start()**, **end()**, **qscore()**, **dna()** and **seq()** methods, but for minus strand alignments the sequence will be represented as it appears on the reverse strand, rather than on the forward strand. This has the advantage of giving you the read as it came off the machine, before being reverse complemented for use in the SAM file. - $query = $align-\>hit The **hit()** method is identical to **target()** and returns information about the read. It is present for compatibility with some of the Bio::Graphics glyphs, which use **hit()** to represent the non-reference sequence in aligned sequences. - $primary\_id = $align-\>primary\_id This method synthesizes a unique ID for the alignment which can be passed to `$sam`-\>**get\_feature\_by\_id()** to retrieve the alignment at a later date. - @tags = $align-\>get\_all\_tags Return all tag names known to this alignment. This includes SAM flags such as M\_UNMAPPED, as well as auxiliary flags such as H0. The behavior of this method depends on the value of -expand\_flags when the SAM object was created. If false (the default), then the standard SAM flags will be concatenated together into a single string and stored in a tag named 'FLAGS'. The format of this tag value is the list of one or more flag constants separated by the "\|" character, as in: "PAIRED\|MAP\_PAIR\|REVERSED\|SECOND\_MATE". If -expand\_flags was true, then each flag becomes its own named tag, such as "MAP\_PAIR". - @values = $align-\>get\_tag\_values($tag) Given a tag name, such as 'PAIRED' or 'H0', return its value(s). -expand\_flags must be true in order to use the standard SAM flag constants as tags. Otherwise, they can be fetched by asking for the "FLAGS" tag, or by using the low-level methods described below. - $is\_true = $align-\>has\_tag($tag) Return true if the alignment has the indicated tag. - $string = $align-\>cigar\_str Return the CIGAR string for this alignment in conventional human readable format (e.g. "M34D1M1"). - $arrayref = $align-\>cigar\_array Return a reference to an array representing the CIGAR string. This is an array of arrays, in which each subarray consists of a CIGAR operation and a count. Example: ``` [ ['M',34], ['D',1], ['M1',1] ] ``` - ($ref,$matches,$query) = $align-\>padded\_alignment Return three strings that show the alignment between the reference sequence (the target) and the query. It will look like this: ``` $ref AGTGCCTTTGTTCA-----ACCCCCTTGCAACAACC $matches |||||||||||||| ||||||||||||||||| $query AGTGCCTTTGTTCACATAGACCCCCTTGCAACAACC ``` - $str = $align-\>aux Returns the text version of the SAM tags, e.g. "XT:A:M NM:i:2 SM:i:37 AM:i:37 XM:i:1 XO:i:1 XG:i:1 MD:Z:6^C0A47" - $str = $align-\>tam\_line Returns the TAM (text) representation of the alignment (available in the high-level "AlignWrapper" interface only). - $tag = $align-\>primary\_tag This is provided for Bio::SeqFeatureI compatibility. Return the string "match". - $tag = $align-\>source\_tag This is provided for Bio::SeqFeatureI compatibility. Return the string "sam/bam". - @parts = $align-\>get\_SeqFeatures Return subfeatures of this alignment. If you have fetched a "read\_pair" feature, this will be the two mate pair objects (both of type Bio::DB::Bam::AlignWrapper). If you have -split\_splices set to true in the Bio::DB::Sam database, calling **get\_SeqFeatures()** will return the components of split alignments. See "Bio::DB::Sam Constructor and basic accessors" in Bio::DB::Sam for an example of how to use this. ## Low-level Bio::DB::Bam::Alignment methods These methods are available to objects of type Bio::DB::Bam::Alignment as well as Bio::DB::Bam::AlignWrapper and closely mirror the native C API. - $align = Bio::DB::Bam::Alignment-\>new Create a new, empty alignment object. This is usually only needed when iterating through a TAM file using Bio::DB::Tam-\>**read1()**. - $tid = $align-\>tid( \[$new\_tid\] ) Return the target ID of the alignment. Optionally you may change the tid by providing it as an argument (currently this is the only field that you can change; the functionality was implemented as a proof of principle). - $read\_name = $align-\>qname Returns the name of the read. - $pos = $align-\>pos 0-based leftmost coordinate of the aligned sequence on the reference sequence. - $end = $align-\>calend The 0-based rightmost coordinate of the aligned sequence on the reference sequence after taking alignment gaps into account. - $len = $align-\>cigar2qlen The length of the query sequence calculated from the CIGAR string. - $quality = $align-\>qual The quality score for the alignment as a whole. - $flag = $align-\>flag The bitwise flag field (see the SAM documentation). - $mtid = $align-\>mtid For paired reads, the target ID of the mate's alignment. - $mpos = $align-\>mpos For paired reads, the 0-based leftmost coordinate of the mate's alignment on the reference sequence. - $n\_cigar = $align-\>n\_cigar Number of CIGAR operations in this alignment. - $length = $align-\>l\_qseq The length of the query sequence (the read). - $dna = $align-\>qseq The actual DNA sequence of the query. As in the SAM file, reads that are aligned to the minus strand of the reference are returned in reverse complemented form. - $score\_str = $align-\>\_qscore A packed binary string containing the quality scores for each base of the read. It will be the same length as the DNA. You may unpack it using unpack('C\*',$score\_str), or use the high-level **qscore()** method. - $score\_arry = $align-\>qscore - @score\_arry = $align-\>qscore In a scalar context return an array reference containing the unpacked quality scores for each base of the query sequence. In a list context return a list of the scores. This array is in the same orientation as the reference sequence. - $score\_str = $align-\>qstring Returns the quality string in the same format used in the SAM (TAM) file. - $length = $align-\>isize The calculated insert size for mapped paired reads. - $length = $align-\>l\_aux The length of the align "auxiliary" data. - $value = $align-\>aux\_get("tag") Given an auxiliary tag, such as "H0", return its value. - @keys = $align-\>aux\_keys Return the list of auxiliary tags known to this alignment. - $data = $align-\>data Return a packed string containing the alignment data (sequence, quality scores and cigar string). - $length = $align-\>data\_len Return the current length of the alignment data. - $length = $align-\>m\_data Return the maximum length of the alignment data. - $is\_paired = $align-\>paired Return true if the aligned read is part of a mate/read pair (regardless of whether the mate mapped). - $is\_proper = $align-\>proper\_pair Return true if the aligned read is part of a mate/read pair and both partners mapped to the reference sequence. - $is\_unmapped = $align-\>unmapped Return true if the read failed to align. - $mate\_is\_unmapped = $align-\>munmapped Return true if the read's mate failed to align. - $reversed = $align-\>reversed Return true if the aligned read was reverse complemented prior to aligning. - $mate\_reversed = $align-\>mreversed Return true if the aligned read's mate was reverse complemented prior to aligning. - $isize = $align-\>isize For mate-pairs, return the computed insert size. - $arrayref = $align-\>cigar This returns the CIGAR data in its native BAM format. You will receive an arrayref in which each operation and count are packed together into an 8-bit structure. To decode each element you must use the following operations: ``` use Bio::DB::Sam::Constants; my $c = $align->cigar; my $op = $c->[0] & BAM_CIGAR_MASK; my $len = $c->[0] >> BAM_CIGAR_SHIFT; ``` ## SEE ALSO Bio::Perl, Bio::DB::Sam, Bio::DB::Bam::Constants ## AUTHOR Lincoln Stein \. \ Copyright (c) 2009-2015 Ontario Institute for Cancer Research. This package and its accompanying libraries are free software; you can redistribute it and/or modify it under the terms of the Artistic License 2.0, the Apache 2.0 License, or the GNU General Public License (version 1 or higher). Refer to LICENSE for the full license text.