strandedCounts: Get strand-specific counts

In LTLA/csaw: ChIP-Seq Analysis with Windows

Get strand-specific counts

Description

Obtain strand-specific counts for each genomic window or region.

Usage

strandedCounts(bam.files, param=readParam(forward=NULL), regions=NULL, ...)

Arguments

bam.files	a character vector containing paths to sorted and indexed BAM files. Alternatively, a list of BamFile objects.
param	a readParam object containing read extraction parameters, where the forward slot must be set to NULL
regions	a GRanges object specifying the regions over which reads are to be counted
...	other arguments to be passed to windowCounts or regionCounts

Details

Some applications require strand-specific counts for each genomic region. This function calls windowCounts after setting param$forward to TRUE and FALSE. Any existing value of param$forward is ignored. If regions is specified, regionCounts is used instead of windowCounts.

The function then concatenates the two RangedSummarizedExperiment objects (one from each strand). The total numbers of reads are added together to form the new totals. However, the total numbers of reads for each strand are also stored for future reference. Count loading parameters are also stored in the metadata.

Each row in the concatenated object corresponds to a stranded genomic region, where the strand of the region indicates the strand of the reads that were counted in that row. Note that there may not be two rows for each genomic region. This is because any empty rows, or those with counts below filter, will be removed within each call to windowCounts.

Value

A RangedSummarizedExperiment object containing strand-specific counts for genomic regions.

Warnings

Users should be aware that many of the downstream range-processing functions are not strand-aware by default, e.g., mergeWindows. Any strandedness of the ranges will be ignored in these functions. If strand-specific processing is desired, users must manually set ignore.strand=FALSE.

The input param$forward should be set to NULL, as a safety measure. This is because the returned object is a composite of two separate calls to the relevant counting function. If the same param object is supplied to other functions, an error will be thrown if param$forward is NULL. This serves to remind users that such functions should instead be called twice, i.e., separately for each strand after setting param$forward to TRUE or FALSE.

Author(s)

Aaron Lun

See Also

windowCounts, regionCounts

Examples

bamFiles <- system.file("exdata", c("rep1.bam", "rep2.bam"), package="csaw")
xparam <- readParam(forward=NULL)
out <- strandedCounts(bamFiles, filter=1, param=xparam)
out

strandedCounts(bamFiles, filter=1, width=100, param=xparam)
strandedCounts(bamFiles, filter=1, param=reform(xparam, minq=20))

incoming <- GRanges(c('chrA', 'chrA', 'chrB', 'chrC'), 
    IRanges(c(1, 500, 100, 1000), c(200, 1000, 700, 1500)))
strandedCounts(bamFiles, regions=incoming, param=xparam)
strandedCounts(bamFiles, regions=incoming, param=reform(xparam, dedup=TRUE))

# Throws an error, as the same reads are not involved.
try(windowCounts(bamFiles, filter=1, width=100, param=xparam))

# Library sizes should be the same as that without strand-specificity.
colData(out)
out.ref <- windowCounts(bamFiles, param=reform(xparam, forward=NA))
stopifnot(identical(out.ref$totals, out$totals))

# Running assorted functions on strandedCounts output.
mergeWindows(rowRanges(out), tol=100)
mergeWindows(rowRanges(out), tol=100, ignore.strand=FALSE)

rwsms <- rowSums(assay(out))
summary(findMaxima(rowRanges(out), range=100, metric=rwsms))
summary(findMaxima(rowRanges(out), range=100, metric=rwsms, ignore.strand=FALSE))

LTLA/csaw documentation built on Dec. 11, 2023, 5:11 a.m.