pileLettersAt {GenomicAlignments} | R Documentation

## Pile the letters of a set of aligned reads on top of a set of genomic positions

### Description

`pileLettersAt` extracts the letters/nucleotides of a set of reads that align to a set of genomic positions of interest. The extracted letters are returned as "piles of letters" (one per genomic position of interest) stored in an XStringSet (typically DNAStringSet) object.

### Usage

    pileLettersAt(x, seqnames, pos, cigar, at)

### Arguments

`x` | An XStringSet (typically DNAStringSet) object containing N unaligned read sequences (a.k.a. the query sequences) reported with respect to the + strand.
`seqnames` | A factor-Rle parallel to `x`. For each `i`, `seqnames[i]` must be the name of the reference sequence of the i-th alignment.
`pos` | An integer vector parallel to `x`. For each `i`, `pos[i]` must be the 1-based position on the reference sequence of the first aligned letter in `x[[i]]`.
`cigar` | A character vector parallel to `x`. Contains the extended CIGAR strings of the alignments.
`at` | A GPos object containing the genomic positions of interest. `seqlevels(at)` must be identical to `levels(seqnames)`. If `at` is not a GPos object, `pileLettersAt` will first try to turn it into one by calling the `GPos()` constructor function on it. So for example `at` can be a GRanges object (or any other GenomicRanges derivative), and, in that case, each range in it will be interpreted as a run of adjacent genomic positions. See `?GPos` in the GenomicRanges package for more information.

### Details

`x`, `seqnames`, `pos`, `cigar` must be 4 parallel vectors describing N aligned reads.

### Value

An XStringSet (typically DNAStringSet) object parallel to `at` (i.e. with 1 string per genomic position).

### Author(s)

Hervé Pagès

### See Also

- The `pileup` and `applyPileups` functions defined in the Rsamtools package, as well as the SAMtools mpileup command (available at http://samtools.sourceforge.net/ as part of the SAMtools project), for more powerful flexible alternatives.

- The `stackStringsFromGAlignments` function for stacking the read sequences (or their quality strings) stored in a GAlignments object or a BAM file.

- DNAStringSet objects in the Biostrings package.

- GPos objects in the GenomicRanges package.

- GAlignments objects.

- cigar-utils for the CIGAR utility functions used internally by `pileLettersAt`.

### Examples

    ## Input

    ##   - A BAM file:
    bamfile <- BamFile(system.file("extdata", "ex1.bam", package="Rsamtools"))
    seqinfo(bamfile)  # to see the seqlevels and seqlengths
    stackStringsFromBam(bamfile, param="seq1:1-21")  # a quick look at
                                                     # the reads

    ##   - A GPos object containing Genomic Positions Of Interest:
    my_GPOI <- GPos(c("seq1:1-5", "seq1:21-21", "seq1:1575-1575",
                      "seq2:1513-1514"))

    ## Some preliminary massage on 'my_GPOI'

    seqinfo(my_GPOI) <- merge(seqinfo(my_GPOI), seqinfo(bamfile))
    seqlevels(my_GPOI) <- seqlevelsInUse(my_GPOI)

    ## Load the BAM file in a GAlignments object. Note that we load only
    ## the reads aligned to the sequences in 'seqlevels(my_GPOI)'. Also,
    ## in order to be consistent with applyPileups() and SAMtools (m)pileup,
    ## we filter out the following BAM records:
    ##   - secondary alignments (flag bit 0x100);
    ##   - reads not passing quality controls (flag bit 0x200);
    ##   - PCR or optical duplicates (flag bit 0x400).
    ## See ?ScanBamParam and the SAM Spec for more information.

    which <- as(seqinfo(my_GPOI), "GRanges")
    flag <- scanBamFlag(isSecondaryAlignment=FALSE,
                        isNotPassingQualityControls=FALSE,
                        isDuplicate=FALSE)
    what <- c("seq", "qual")
    param <- ScanBamParam(flag=flag, what=c("seq", "qual"), which=which)
    gal <- readGAlignments(bamfile, param=param)
    seqlevels(gal) <- seqlevels(my_GPOI)

    ## Extract the read sequences (a.k.a. query sequences) and quality
    ## strings. Both are reported with respect to the + strand.

    qseq <- mcols(gal)$seq
    qual <- mcols(gal)$qual

    nucl_piles <- pileLettersAt(qseq, seqnames(gal), start(gal), cigar(gal),
                                my_GPOI)
    qual_piles <- pileLettersAt(qual, seqnames(gal), start(gal), cigar(gal),
                                my_GPOI)
    mcols(my_GPOI)$nucl_piles <- nucl_piles
    mcols(my_GPOI)$qual_piles <- qual_piles
    my_GPOI

    ## Finally, to summarize A/C/G/T frequencies at each position:
    alphabetFrequency(nucl_piles, baseOnly=TRUE)

    ## Note that the pileup() function defined in the Rsamtools package
    ## can be used to obtain a similar result:
    scanbam_param <- ScanBamParam(flag=flag, which=my_GPOI)
    pileup_param <- PileupParam(max_depth=5000,
                                min_base_quality=0,
                                distinguish_strands=FALSE)
    pileup(bamfile, scanBamParam=scanbam_param, pileupParam=pileup_param)

[Package GenomicAlignments version 1.23.2 Index]