AlignedData-class: Class 'AlignedData'

In Pasha: Preprocessing of Aligned Sequences from HTS Analyses

Description

This class gives tools to read, store and manage simple data from HTS experiments. This class is specially optimized for chromatin-oriented sequencing experiments (ChIP-Seq, MNase-Seq, Faire-Seq) since it is mainly focused on reads position rather than sequences, ignores alignments with non-simple CIGAR strings (non canonical alignments, ie. insertions/deletions) and secondary alignments, and is not designed to represent features with undefined strands. It directly uses Rsamtools to read data from BAM files. It has a similar global structure to ShortRead and GAlignments classes but have some specific features.

Details

The specific features of this class are that (i) read sequences are not read from aligned files, (ii) only R native types are used to store data in memory, and (iii) it can represent both paired-end or single-end data by using a similar internal structure as what has been defined for SAM/BAM formats. For reading data from 'non-BAM' formats (aligner-specific), the constructor relies on ShortRead library (see ShortRead for details).

This class is a simplistic representation of aligned reads for users aiming at using piling options offered by Pasha package. Secondary alignments, reads aligned with insertions/deletions (non simple CIGAR), and not aligned reads are discarded by the constructor, and reads with a strand value other than -/+ will raise a warning (strand representation keeps levels c('+', '-', '*') for consistency with bioconductor packages). For other applications requiring exhaustive handling of CIGAR strings (insertions/deletions), undefined strand values, or to keep reads that could not be aligned, the use of GAlignments class family is recommended.

Specific functions are provided for a seamless conversion to/from GAlignments class. It is however important to note that eventual reads with non-simple CIGAR string will be dropped (with a warning) during conversions from GAlignments objects.

Internally, this class defines different kinds of slots. First, slots where a value is mandatory for each read. Second, slots with atomic values (one for the whole object : "notAligned", "pairedEnds"), and finally optionnal slots which can be empty (no information) or should provide information for each read ("mapq", "isize", "flag", "mrnm", "weight", "qwidth").

readID:

integer - Read identifier. This value is inferred from 'groupid' field when reading paired-end BAM files (see BamFile and 'asMates'). A simple integer if inferred from 'qname' field otherwise.)

seqnames:

factor - Chromosome sequence name

strand:

factor - Strand value (accessors and subsetters methods will try to maintain consistent values for factor levels ('+','-'))

position:

integer - The 1-based leftmost position/coordinate (POS field in a SAM/BAM record)

mapq:

integer - Mapping quality

isize:

integer - Insert size (paired-ends only)

qwidth:

integer - Query width (read sequence length)

flag:

integer - Flag (see SAM format specification)

mrnm:

factor - Mate-pair chromosome sequence name

notAligned:

integer (single value) - Number of reads that wouldn't be aligned during mapping (if information availabe)

pairedEnds:

logical (single value) - Object declared as paired-ends

weight:

numeric - Weight attributed to read for piling (multireads only)

Paired-end specificities

The limitations imposed by the constructor when reading paired-end datasets (no unaligned reads, and no secondary alignments) allow to represent the mate reads association by their shared 'qname' (reported as 'readID'). Altering such IDs can thus break the pairs relation as seen by methods of the class.

Several methods are dedicated for paired-ends datasets (see 'checkPairsOK', 'filterInsertSize', 'getOrphansIndexes', 'cleanNonSimplePairs', 'sortByPairs'). These methods will emit a warning and not process data when called for a single-end object.

Objects from the Class

Objects can be created by a call of the form new("AlignedData", readID, seqnames, strand, position, mapq, isize, qwidth, flag, mrnm, notAligned, pairedEnds, weight), or by using the dedicated constructor that builds an object from a file.

Constructors

readAlignedData(folderName, fileName, fileType="BAM", pairedEnds=FALSE)

This function reads data from a file and creates an AlignedData object from it. It handles BAM files using the Rsamtools library (ignores reads with non-simple CIGAR string) and uses ShortRead library to handle several other proprietary formats (see ShortRead readAligned function).

Subsetting

[

Allow to select a subpopulation of reads, all slots are affected except the atomic ones for which the value is copied in resulting object. This function can also drop the unused levels for chromosome (seqnames) and mrnm slots only if the argument drop=TRUE is used.

Coercion

as(object, 'GAlignments')

Converts an AlignedData object to a GAlignment object. This conversion drops the eventual pairs related information, as well as mapping quality, number of unaligned read in the dataset, and flags.

as(object, 'AlignedData')

Converts a GAlignments object to a AlignedData object. This conversion will drop eventual reads that aligned with a non-simple CIGAR string.

Accessors

readID(object), readID(object) <- value

Get or set the numeric ID of reads (typically inferred from read name by constructor, see details). Altering these values can break the reads mating for paired-end data.

seqnames(object)), seqnames(object) <- value

Get or set a factor describing the seqnames (chromosome name) to which each read has been aligned.

strand(object), strand(object) <- value

Get or set the factor describing the strand to which reads are aligned.

position(object), position(object) <- value

Get or set the 1-base leftmost genomic coordinate of the reads.

mapq(object), mapq(object) <- value

Get or set the numeric quality of alignment (if available). See Rsamtools library for details.

isize(object), isize(object) <- value

Get or set the insert size for paired reads (if available).

qwidth(object), qwidth(object) <- value

Get or set the length of each read sequence (if available).

flag(object), flag(object) <- value

Get or set the numeric (integer) flag values of reads (if available). See BAM/SAM documentation for flag interpretation. Modification of this slot can lead to inconsistencies in further processing (specially for paired-end experiments).

mrnm(object), mrnm(object) <- value

Get or set the mate read chromosome name (if available).

notAligned(object), notAligned(object) <- value

Get or set the number of reads reported as not aligned by aligner (only available from BAM format when object is created via the constructor).

pairedEnds(object), pairedEnds(object) <- value

Get or set the declared status of the object concerning paired/single ends reads.

weight(object), weight(object) <- value

Get or set the weight that has been attributed to each read for piling (if available).

Methods

show(object)

Writes a quick summary of the object content to the standard output (uses cat).

checkPairsOK(object)

Perform a validity test on paired objects. Checks that the object only contain 'full-pairs' (no orhpans) and that all reads are correctly sorted (eg. interlaced reads and mates).

length(object)

Get the number of elements in the object.

dropChromosomePattern(object, pattern, quiet=TRUE)

Filter out reads for which the chromosome name (seqnames) match a certain pattern. Helps to get rid of undesired reads (align on mitochondrial chromosomes for instance).

filterInsertSize(object, rangeMin, rangeMax, includeLower=FALSE, quiet=TRUE)

Select pairs of reads for which insert size is defined in a specific range. Returns a new object with the selected reads. By default (includeLower=FALSE), the lower range value ('rangeMin') is EXCLUDED from selection whereas the upper one is INCLUDED in the selection. Insert size is defined as a symmetric (positive and negative numeric) value for reads of a pair. This function is designed for selecting both reads of pairs and therefore ignores the numeric sign for filtering.

filterReadSize(object, rangeMin, rangeMax, includeLower=FALSE, quiet=TRUE)

Select reads for which the sequence length is in a specified range. Returns a new object with the selected reads. By default (includeLower=FALSE), the lower range value ('rangeMin') is EXCLUDED from selection whereas the upper one is INCLUDED in the selection.

getOrphansIndexes(object, quiet=TRUE)

Get the indexes of reads for which a mate can't be identified (based on their ID).

cleanNonSimplePairs(object, quiet=TRUE)

For paired-ends objects, return a filtered object from which the pairs with flag 'unmapped mate' and/or the pairs with both reads on the same strand are "orphanized"

normalizeChrNames(object, chrPrefix, chrSuffix)

The main functions of this library (see processPipeline) assumes that chromosomes are always represented as chr*, * being the actual chromosome name. This functions helps to fit this requirement by removing eventual prefix/suffix around the actual chromosome name and then adding 'chr' as prefix. This function returns an object with modified seqnames (chromosome names).

sortByPairs(object, quiet=TRUE)

In case of pairedEnds experiments, this function sorts the reads. It tries to group the index of reads from pairs and uses the flag to order the reads by 'first in pair' and 'second in pair' bits as firsts and seconds respectively.

dropUndefinedStrand, quiet=TRUE

Return a AlignedData object after removing eventual reads with unsupported strand values (supported strand values are '+' and '-').

Arguments

folderName

Path to the directory containing the file to read.

fileName

File containing aligned reads. Several formats are handled.

fileType

File format for reads description. This format can vary depending on the aligner and/or the parameters used. Typical format will be BAM files from which the maximum of information can be read from. Other proprietary formats are handled thanks to the ShortRead library (see corresponding documentation for details)

pairedEnds

A logical defining if the data read from file should be considered as paired-end or not.

object

Object of the class 'AlignedData' to which the function call is applied.

pattern

A regular expression describing the pattern to be recognized and replaced

quiet

An atomic logical. If TRUE, the function will not write messages to stdout

rangeMin

An atomic integer, defining the minimal size to be selected

rangeMax

An atomic integer, defining the maximal size to be selected

includeLower

An tomic logical defining if the lower value should be included when filtering reads/inserts size based on a range of values (default FALSE)

chrPrefix

An atomic regular expression describing the pattern to be recognized and replaced

chrSuffix

An atomic regular expression describing the pattern to be recognized and replaced

breaks

An integer, or a vector of integer. This parameters behaves like the 'breaks' parameter in function 'cut'

Author(s)

Romain Fenouil

See Also

readAligned GAlignments scanBam BamFile

Examples

# Get the BAM file used in RSamTools as example
BAMfile <- system.file("extdata","ex1.bam",package="Rsamtools")
# Testing the public constructor of AlignedData class (from Pasha)
alignedDataObject <- readAlignedData(folderName="" ,
                                     fileName=BAMfile, 
                                     fileType="BAM", 
                                     pairedEnds=TRUE)
print(alignedDataObject)
                             
# Rename the chromosomes from seqX to chrX
alignedDataObject <- normalizeChrNames(alignedDataObject,
                                       chrPrefix="seq",
                                       chrSuffix="")
print(alignedDataObject)
                               
                               
#### Splitting object (indirect use of '[') 
# Split the object and the associated data by chromosomes 
splitListalignedDataObject <- split(alignedDataObject, 
                                    seqnames(alignedDataObject))
# check that the object has correctly been split
print(names(splitListalignedDataObject))
print(sapply(lapply(splitListalignedDataObject, seqnames), unique))


#### Orphans and pairs consistency
print(length(alignedDataObject))
# Identify the reads with no mate and remove them
orphansReadsIndex <- getOrphansIndexes(alignedDataObject)
print(paste("Nb of orphan reads :", sum(orphansReadsIndex)))
alignedDataObject <- alignedDataObject[!orphansReadsIndex]
print(length(alignedDataObject))

# Check that pairs are correctly represented in the object
# ie. mate pairs are consecutive
print(checkPairsOK(alignedDataObject))
# Sort by pairs
alignedDataObject <- sortByPairs(alignedDataObject)
# Check pairs consistency again
print(checkPairsOK(alignedDataObject))


#### Filtering

## Chromosome patterns
# (drop reads on chromosomes containing the character '1')
print(alignedDataObject)
resultFiltering <- dropChromosomePattern(alignedDataObject, 
                                           pattern="1")
print(resultFiltering)

## Range of insert size
print(table(isize(alignedDataObject)))
resultFiltering <- filterInsertSize(alignedDataObject, 
                                    rangeMin=190, 
                                    rangeMax=195)
print(table(isize(resultFiltering)))
print(resultFiltering)

## Reads sequence size
print(table(qwidth(alignedDataObject)))
resultFiltering <- filterReadSize(alignedDataObject, 
                                  rangeMin=35, 
                                  rangeMax=50)
print(table(qwidth(resultFiltering)))
print(resultFiltering)
                          
## Reads with atypic characteristics for chromatin alignments
# Mate unmapped flag set, or mates aligned on same strand
resultFiltering <- cleanNonSimplePairs(alignedDataObject, quiet=FALSE)
# Reads with a strand value different from expected '+' or '-'
resultFiltering <- dropUndefinedStrand(resultFiltering, quiet=FALSE)

Pasha documentation built on Jan. 15, 2017, 6:21 p.m.