readGAlignments: Reading genomic alignments from a file

Description

Read genomic alignments from a file (typically a BAM file) into a GAlignments, GAlignmentPairs, GAlignmentsList, or GappedReads object.

Usage

## Front-ends
readGAlignments(file, format="BAM", use.names=FALSE, ...)
readGAlignmentPairs(file, format="BAM", use.names=FALSE, ...)
readGAlignmentsList(file, format="BAM", use.names=FALSE, ...)
readGappedReads(file, format="BAM", use.names=FALSE, ...)
## BAM specific back-ends
readGAlignmentsFromBam(file, index=file, ..., use.names=FALSE, param=NULL, with.which_label=FALSE)
readGAlignmentPairsFromBam(file, index=file, use.names=FALSE, param=NULL, with.which_label=FALSE)
readGAlignmentsListFromBam(file, index=file, ..., use.names=FALSE, param=ScanBamParam(), with.which_label=FALSE)
readGappedReadsFromBam(file, index=file, use.names=FALSE, param=NULL, with.which_label=FALSE)

Arguments

file

The path to the file to read or a BamFile object. Can also be a BamViews object for readGAlignmentsFromBam.

format

Only "BAM" (the default) is supported for now.

use.names

Use the query template names (QNAME field) as the names of the returned object? If not (the default), then the returned object has no names.

...

Arguments passed to other methods.

index

The path to the index file of the BAM file to read. Must be given without the '.bai' extension. See scanBam in the Rsamtools packages for more information.

param

NULL or a ScanBamParam object. Like for scanBam, this influences what fields and which records are imported. However, note that the fields specified thru this ScanBamParam object will be loaded in addition to any field required for generating the returned object (GAlignments, GAlignmentPairs, or GappedReads object), but only the fields requested by the user will actually be kept as metadata columns of the object.

By default (i.e. param=NULL or param=ScanBamParam()), no additional field is loaded. The flag used is scanBamFlag(isUnmappedQuery=FALSE) for readGAlignmentsFromBam, readGappedReadsFromBam and readGAlignmentsListFromBam (i.e. only records corresponding to mapped reads are loaded), and scanBamFlag(isUnmappedQuery=FALSE, isPaired=TRUE, hasUnmappedMate=FALSE) for readGAlignmentPairsFromBam (i.e. only records corresponding to paired-end reads with both ends mapped are loaded).

with.which_label

TRUE or FALSE (the default). If TRUE and if param has a which component, a "which_label" metadata column is added to the returned GAlignments or GappedReads object, or to the first and last components of the returned GAlignmentPairs object. In the case of readGAlignmentsListFromBam, it's added as an inner metadata column, that is, the metadata column is placed on the GAlignments object obtained by unlisting the returned GAlignmentsList object.

The purpose of this metadata column is to unambiguously identify the range in which where each element in the returned object originates from. The labels used to identify the ranges are normally of the form "seq1:12250-246500", that is, they're the same as the names found on the outer list that scanBam would return if called with the same param argument. If some ranges are duplicated, then the labels are made unique by appending a unique suffix to all of them. The "which_label" metadata column is represented as a factor-Rle.

Value

A GAlignments object for readGAlignmentsFromBam.A GAlignmentPairs object for readGAlignmentPairsFromBam. Note that a BAM (or SAM) file can in theory contain a mix of single-end and paired-end reads, but in practise it seems that single-end and paired-end are not mixed. In other words, the value of flag bit 0x1 (isPaired) is the same for all the records in a file. So if readGAlignmentPairsFromBam returns a GAlignmentPairs object of length zero, this almost certainly means that the BAM (or SAM) file contains alignments for single-end reads (although it could also mean that the user-supplied ScanBamParam is filtering out everything, or that the file is empty, or that all the records in the file correspond to unmapped reads).A GAlignmentsList object for readGAlignmentsListFromBam. When the list contains paired-end reads a metadata data column of mate_status is added to the object. See details in the `Bam specific back-ends' section on this man page.A GappedReads object for readGappedReadsFromBam.

Details

See ?GAlignments for a description of GAlignments objects.

See ?GappedReads for a description of GappedReads objects.

Front-ends readGAlignments reads a file containing aligned reads as a GAlignments object.

readGAlignmentPairs reads a file containing aligned paired-end reads as a GAlignmentPairs object.

readGAlignmentsList reads a file containing aligned reads as a GAlignmentsList object.

readGappedReads reads a file containing aligned reads as a GappedReads object.

By default (i.e. use.names=FALSE), the resulting object has no names. If use.names is TRUE, then the names are constructed from the query template names (QNAME field in a SAM/BAM file). Note that the 2 records in a pair (when using readGAlignmentPairs or the records in a group (when using readGAlignmentsList) have the same QNAME.

These functions are just front-ends that delegate to a format-specific back-end function depending on the supplied format argument. The use.names argument and any extra argument are passed to the back-end function. Only the BAM format is supported for now via the read*FromBam back-end functions.

BAM specific back-ends When file is BamViews object readGAlignmentsFromBam visits each path in bamPaths(file), returning the result of readGAlignmentsFromBam applied to the specified path. When index is missing, it is set equal to bamIndicies(file). Only reads in bamRanges(file) are returned (if param is supplied, bamRanges(file) takes precedence over bamWhich(param)). The return value is a SimpleList object, with elements of the list corresponding to each path. bamSamples(file) is available as metadata columns (accessed with mcols) of the returned SimpleList object.

readGAlignmentPairsFromBam proceeds in 2 steps:

Load the BAM file into a GAlignmentsList object with readGAlignmentsListFromBam (see below);
Turn this GAlignmentsList object into a GAlignmentPairs object. Only list elements marked with mate status "mated" go into the returned GAlignmentPairs object.

See ?GAlignmentPairs for a description of GAlignmentPairs objects.

readGAlignmentsListFromBam pairs records into mates according to the pairing criteria described below. The 1st mate will always be 1st in the GAlignmentsList list elements that have mate_status set to "mated", and the 2nd mate will always be 2nd.

A GAlignmentsList is returned with a ‘mate_status’ metadata column on the outer list elements. mate_status is a factor with 3 levels indicating mate status, ‘mated’, ‘ambiguous’ or unmated.

Mate status:

mated: primary or non-primary pairs
ambiguous: multiple segments matching to the same location (indistinguishable)
unmated: mate does not exist or is unmapped

When the ‘file’ argument is a BamFile, ‘asMates=TRUE’ must be set, otherwise the data are treated as single-end reads. See the ‘asMates’ section of ?BamFile for details.

Flags, tags and ranges may be specified in the ScanBamParam for fine tuning of results.

See ?GAlignmentsList-class for a description of GAlignmentsList objects.

Pairing criteria This section describes the pairing criteria used by readGAlignmentsListFromBam and readGAlignmentPairsFromBam.

First, only records with flag bit 0x1 (multiple segments) set to 1, flag bit 0x4 (segment unmapped) set to 0, and flag bit 0x8 (next segment in the template unmapped) set to 0, are candidates for pairing (see the SAM Spec for a description of flag bits and fields). Records that correspond to single-end reads, or records that correspond to paired-end reads where one or both ends are unmapped, will remain unmated.

Then the following fields and flag bits are considered:

(A) QNAME
(B) RNAME, RNEXT
(C) POS, PNEXT
(D) Flag bits Ox10 (segment aligned to minus strand) and 0x20 (next segment aligned to minus strand)
(E) Flag bits 0x40 (first segment in template) and 0x80 (last segment in template)
(F) Flag bit 0x2 (proper pair)
(G) Flag bit 0x100 (secondary alignment)

2 records rec1 and rec2 are considered mates iff all the following conditions are satisfied:

(A) QNAME(rec1) == QNAME(rec2)
(B) RNEXT(rec1) == RNAME(rec2) and RNEXT(rec2) == RNAME(rec1)
(C) PNEXT(rec1) == POS(rec2) and PNEXT(rec2) == POS(rec1)
(D) Flag bit 0x20 of rec1 == Flag bit 0x10 of rec2 and Flag bit 0x20 of rec2 == Flag bit 0x10 of rec1
(E) rec1 corresponds to the first segment in the template and rec2 corresponds to the last segment in the template, OR, rec2 corresponds to the first segment in the template and rec1 corresponds to the last segment in the template
(F) rec1 and rec2 have same flag bit 0x2
(G) rec1 and rec2 have same flag bit 0x100

Note that this is actually the pairing criteria used by scanBam (when the BamFile passed to it has the asMates toggle set to TRUE), which readGAlignmentsListFromBam and readGAlignmentPairsFromBam call behind the scene. It is also the pairing criteria used by findMateAlignment.

Examples

Run this code

## ---------------------------------------------------------------------
## A. readGAlignmentsFromBam()
## ---------------------------------------------------------------------

## Simple use:
bamfile <- system.file("extdata", "ex1.bam", package="Rsamtools",
                       mustWork=TRUE)
gal1 <- readGAlignmentsFromBam(bamfile)
gal1
names(gal1)

## Using the 'use.names' arg:
gal2 <- readGAlignmentsFromBam(bamfile, use.names=TRUE)
gal2
head(names(gal2))

## Using the 'param' arg to drop PCR or optical duplicates as well as
## secondary alignments, and to load additional BAM fields:
param <- ScanBamParam(flag=scanBamFlag(isDuplicate=FALSE,
                                       isNotPrimaryRead=FALSE),
                      what=c("qual", "flag"))
gal3 <- readGAlignmentsFromBam(bamfile, param=param)
gal3
mcols(gal3)

## Using the 'param' arg to load reads from particular regions.
## Note that if we weren't providing a 'what' argument here, all the
## BAM fields would be loaded:
which <- RangesList(seq1=IRanges(1000, 2000),
                    seq2=IRanges(c(100, 1000), c(1000, 2000)))
param <- ScanBamParam(which=which)
gal4 <- readGAlignmentsFromBam(bamfile, param=param)
gal4

## Note that a given record is loaded one time for each region it
## belongs to (this is a scanBam() feature, readGAlignmentsFromBam()
## is based on scanBam()):
which <- IRangesList(seq2=IRanges(c(1563, 1567), width=1))
param <- ScanBamParam(which=which)
gal5 <- readGAlignmentsFromBam(bamfile, param=param)
gal5

## Use 'with.which_label=TRUE' to identify the range in 'which'
## where each element in 'gal5' originates from.
gal5 <- readGAlignmentsFromBam(bamfile, param=param,
                               with.which_label=TRUE)
gal5

## Using the 'param' arg to load tags. Except for MF and Aq, the tags
## specified below are predefined tags (see the SAM Spec for the list
## of predefined tags and their meaning).
param <- ScanBamParam(tag=c("MF", "Aq", "NM", "UQ", "H0", "H1"),
                      what="isize")
gal6 <- readGAlignmentsFromBam(bamfile, param=param)
mcols(gal6)  # "tag" cols always after "what" cols

## With a BamViews object:
fls <- system.file("extdata", "ex1.bam", package="Rsamtools",
                   mustWork=TRUE)
bv <- BamViews(fls,
               bamSamples=DataFrame(info="test", row.names="ex1"),
               auto.range=TRUE)
aln <- readGAlignmentsFromBam(bv)
aln
aln[[1]]
aln[colnames(bv)]
mcols(aln)

## ---------------------------------------------------------------------
## B. readGAlignmentPairsFromBam()
## ---------------------------------------------------------------------
galp1 <- readGAlignmentPairsFromBam(bamfile)
head(galp1)
names(galp1)
## Using the 'param' arg to drop PCR or optical duplicates as well as
## secondary alignments (dropping secondary alignments can help make the
## pairing algorithm run significantly faster, see ?findMateAlignment):
param <- ScanBamParam(flag=scanBamFlag(isDuplicate=FALSE,
                                       isNotPrimaryRead=FALSE))
galp2 <- readGAlignmentPairsFromBam(bamfile, use.names=TRUE, param=param)
galp2
head(galp2)
head(names(galp2))

## ---------------------------------------------------------------------
## C. readGAlignmentsListFromBam()
## ---------------------------------------------------------------------
library(pasillaBamSubset)

## 'file' as character.
fl <- untreated3_chr4() 
galist1 <- readGAlignmentsListFromBam(fl)
galist1[1:3]
length(galist1)
table(elementLengths(galist1))

## When 'file' is a BamFile, 'asMates' must be TRUE. If FALSE,
## the data are treated as single-end and each list element of the
## GAlignmentsList will be of length 1. For single-end data 
## use readGAlignments().
bf <- BamFile(fl, yieldSize=3, asMates=TRUE)
readGAlignmentsList(bf)

## Use a 'param' to fine tune the results.
param <- ScanBamParam(flag=scanBamFlag(isProperPair=TRUE))
galist2 <- readGAlignmentsListFromBam(fl, param=param)
length(galist2)

## ---------------------------------------------------------------------
## D. readGappedReadsFromBam()
## ---------------------------------------------------------------------
greads1 <- readGappedReadsFromBam(bamfile)
greads1
names(greads1)
qseq(greads1)
greads2 <- readGappedReadsFromBam(bamfile, use.names=TRUE)
head(greads2)
head(names(greads2))

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