Description

The GAlignmentPairs class is a container for storing pairs of genomic alignments. These pairs are typically obtained by aligning paired-end reads to a reference genome or transcriptome.

Details

A GAlignmentPairs object is a list-like object where each list element represents a pair of genomic alignment.

An alignment pair is made of a "first" and a "last"/"second" alignment, and is formally represented by a GAlignments object of length 2. In most applications, an alignment pair will represent an aligned paired-end read. In that case, the "first" member of the pair represents the alignment of the first end of the read (aka "first segment in the template", using SAM Spec terminology), and the "last" member of the pair represents the alignment of the second end of the read (aka "last segment in the template", using SAM Spec terminology).

In general, a GAlignmentPairs object will be created by loading records from a BAM (or SAM) file containing aligned paired-end reads, using the readGAlignmentPairs function (see below). Each element in the returned object will be obtained by pairing 2 records.

Constructor

GAlignmentPairs(first, last, strandMode=1, isProperPair=TRUE, names=NULL): Low-level GAlignmentPairs constructor. Generally not used directly.

Accessors

In the code snippets below, x is a GAlignmentPairs object.

strandMode(x), strandMode(x) <- value: The strand mode is a per-object switch on GAlignmentPairs objects that controls the behavior of the strand getter. More precisely, it indicates how the strand of a pair should be inferred from the strand of the first and last alignments in the pair:

• 0: strand of the pair is always *.

• 1: strand of the pair is strand of its first alignment. This mode should be used when the paired-end data was generated using one of the following stranded protocols: Directional Illumina (Ligation), Standard SOLiD.

• 2: strand of the pair is strand of its last alignment. This mode should be used when the paired-end data was generated using one of the following stranded protocols: dUTP, NSR, NNSR, Illumina stranded TruSeq PE protocol.

These modes are equivalent to strandSpecific equal 0, 1, and 2, respectively, for the featureCounts function defined in the Rsubread package.

Note that, by default, the readGAlignmentPairs function sets the strand mode to 1 on the returned GAlignmentPairs object. The function has a strandMode argument to let the user set a different strand mode. The strand mode can also be changed any time with the strandMode setter or with invertStrand.

Also note that 3rd party programs TopHat2 and Cufflinks have a --library-type option to let the user specify which protocol was used. Please refer to the documentation of these programs for more information.

length(x): Return the number of alignment pairs in x.

names(x), names(x) <- value: Get or set the names on x. See readGAlignmentPairs for how to automatically extract and set the names when reading the alignments from a file.

first(x, real.strand=FALSE), last(x, real.strand=FALSE), second(x, real.strand=FALSE): Get the "first" or "last"/"second" alignment for each alignment pair in x. The result is a GAlignments object of the same length as x.

If real.strand=TRUE, then the strand is inverted on-the-fly according to the strand mode currently set on the object (see strandMode(x) above). More precisely, if strandMode(x) is 0, then the strand is set to * for the GAlignments object returned by both, first() and last(). If strandMode(x) is 1, then the strand of the object returned by last() is inverted. If strandMode(x) is 2, then the strand of the object returned by first() is inverted.

seqnames(x): Get the sequence names of the pairs in x i.e. the name of the reference sequence for each alignment pair in x. The sequence name of a pair is the sequence name of the 2 alignments in the pair if they are the same (concordant seqnames), or NA if they differ (discordant seqnames).

The sequence names are returned in a factor-Rle object that is parallel to x, i.e. the i-th element in the returned object is the sequence name of the i-th pair in x.

strand(x): Get the strand for each alignment pair in x. Obey strandMode(x) above to infer the strand of a pair. Return * for pairs with discordant strand, or for all pairs if strandMode(x) is 0.

njunc(x): Equivalent to njunc(first(x)) + njunc(last(x)).

isProperPair(x): Get the "isProperPair" flag bit (bit 0x2 in SAM Spec) set by the aligner for each alignment pair in x.

seqinfo(x), seqinfo(x) <- value: Get or set the information about the underlying sequences. value must be a Seqinfo object.

seqlevels(x), seqlevels(x) <- value: Get or set the sequence levels. seqlevels(x) is equivalent to seqlevels(seqinfo(x)) or to levels(seqnames(x)), those 2 expressions being guaranteed to return identical character vectors on a GAlignmentPairs object. value must be a character vector with no NAs. See ?seqlevels for more information.

seqlengths(x), seqlengths(x) <- value: Get or set the sequence lengths. seqlengths(x) is equivalent to seqlengths(seqinfo(x)). value can be a named non-negative integer or numeric vector eventually with NAs.

isCircular(x), isCircular(x) <- value: Get or set the circularity flags. isCircular(x) is equivalent to isCircular(seqinfo(x)). value must be a named logical vector eventually with NAs.

genome(x), genome(x) <- value: Get or set the genome identifier or assembly name for each sequence. genome(x) is equivalent to genome(seqinfo(x)). value must be a named character vector eventually with NAs.

seqnameStyle(x): Get or set the seqname style for x. Note that this information is not stored in x but inferred by looking up seqnames(x) against a seqname style database stored in the seqnames.db metadata package (required). seqnameStyle(x) is equivalent to seqnameStyle(seqinfo(x)) and can return more than 1 seqname style (with a warning) in case the style cannot be determined unambiguously.

Vector methods

In the code snippets below, x is a GAlignmentPairs object.

x[i]: Return a new GAlignmentPairs object made of the selected alignment pairs.

List methods

In the code snippets below, x is a GAlignmentPairs object.

x[[i]]: Extract the i-th alignment pair as a GAlignments object of length 2. As expected x[[i]][1] and x[[i]][2] are respectively the "first" and "last" alignments in the pair.

unlist(x, use.names=TRUE): Return the GAlignments object conceptually defined by c(x[[1]], x[[2]], ..., x[[length(x)]]). use.names determines whether x names should be propagated to the result or not.

Coercion

In the code snippets below, x is a GAlignmentPairs object.

granges(x, use.names=TRUE, use.mcols=FALSE, on.discordant.seqnames=c("error", "drop", "split")), ranges(x, use.names=TRUE, use.mcols=FALSE, on.discordant.seqnames=c("error", "drop", "split")):

Return a GRanges object (for granges()) or IRanges) object (for ranges()).

If x contains no pairs with discordant seqnames, the operation is guaranteed to be successful and to return an object parallel to x, that is, an object where the i-th element is the range of the genomic region spanned by the i-th alignment in x (all gaps in the region are ignored).

If x contains pairs with discordant seqnames, then an error is raised, unless the on.discordant.seqnames argument is set to "drop" or "split", in which case the pairs with discordant seqnames are either dropped or represented with 2 genomic ranges (or 2 ranges for ranges()) in the returned object. In that case, the returned object is NOT parallel to x.

If use.names is TRUE, then the names on x (if any) are propagated to the returned object. If use.mcols is TRUE, then the metadata columns on x (if any) are propagated to the returned object.

grglist(x, use.mcols=FALSE, drop.D.ranges=FALSE):

Return a GRangesList object of length length(x) where the i-th element represents the ranges (with respect to the reference) of the i-th alignment pair in x. The strand of the returned ranges obeys the strand mode currently set on the object (see strandMode(x) above).

More precisely, if grl1 and grl2 are grglist(first(x, real.strand=TRUE), order.as.in.query=TRUE) and grglist(last(x, real.strand=TRUE), order.as.in.query=TRUE), respectively, then the i-th element in the returned GRangesList object is c(grl1[[i]], grl2[[i]]) if strandMode(x) is 0 or 1, or c(grl2[[i]], grl1[[i]]) if strandMode(x) is 2.

Note that, if strandMode(x) is 1 or 2, this results in the ranges being in consistent order with the original "query template", that is, being in the order defined by walking the "query template" from the beginning to the end.

If use.names is TRUE, then the names on x (if any) are propagated to the returned object. If use.mcols is TRUE, then the metadata columns on x (if any) are propagated to the returned object.

If drop.D.ranges is TRUE, then deletions (Ds in the CIGAR) are treated like junctions (Ns in the CIGAR), that is, the ranges corresponding to deletions are dropped.

as(x, "GRanges"), as(x, "IntegerRanges"), as(x, "GRangesList"): Alternate ways of doing granges(x, use.names=TRUE, use.mcols=TRUE), ranges(x, use.names=TRUE, use.mcols=TRUE), and grglist(x, use.names=TRUE, use.mcols=TRUE), respectively.

as(x, "GAlignments"): Equivalent of unlist(x, use.names=TRUE).

Other methods

In the code snippets below, x is a GAlignmentPairs object.

show(x): By default the show method displays 5 head and 5 tail elements. This can be changed by setting the global options showHeadLines and showTailLines. If the object length is less than (or equal to) the sum of these 2 options plus 1, then the full object is displayed. Note that these options also affect the display of GRanges and GAlignments objects, as well as other Vector derivatives defined in the IRanges and Biostrings packages (e.g. IRanges and XStringSet objects).

Author(s)

Hervé Pagès

See Also

• readGAlignmentPairs for reading aligned paired-end reads from a file (typically a BAM file) into a GAlignmentPairs object.

• GAlignments objects for handling aligned single-end reads.

• makeGAlignmentPairs for pairing the elements of a GAlignments object into a GAlignmentPairs object.

• junctions-methods for extracting and summarizing junctions from a GAlignmentPairs object.

• coverage-methods for computing the coverage of a GAlignmentPairs object.

• findOverlaps-methods for finding range overlaps between a GAlignmentPairs object and another range-based object.

• seqinfo in the GenomeInfoDb package for getting/setting/modifying the sequence information stored in an object.

• The GRanges and GRangesList classes defined and documented in the GenomicRanges package.

Examples