findSpliceOverlaps-methods {GenomicAlignments} | R Documentation

## Classify ranges (reads) as compatible with existing genomic annotations or as having novel splice events

### Description

The `findSpliceOverlaps` function identifies ranges (reads) that are compatible with a specific transcript isoform. The non-compatible ranges are analyzed for the presence of novel splice events.

### Usage

    findSpliceOverlaps(query, subject, ignore.strand=FALSE, ...)

    ## S4 method for signature 'GRangesList,GRangesList'
    findSpliceOverlaps(query, subject, ignore.strand=FALSE, ..., cds=NULL)

    ## S4 method for signature 'GAlignments,GRangesList'
    findSpliceOverlaps(query, subject, ignore.strand=FALSE, ..., cds=NULL)

    ## S4 method for signature 'GAlignmentPairs,GRangesList'
    findSpliceOverlaps(query, subject, ignore.strand=FALSE, ..., cds=NULL)

    ## S4 method for signature 'BamFile,ANY'
    findSpliceOverlaps(query, subject, ignore.strand=FALSE, ...,
                       param=ScanBamParam(), singleEnd=TRUE)

### Arguments

`query` | A GRangesList, GAlignments, GAlignmentPairs, or BamFile object containing the reads. Can also be a single string containing the path to a BAM file. Single or paired-end reads are specified with the `singleEnd` argument (default FALSE). Paired-end reads can be supplied in a BAM file or GAlignmentPairs object. Single-end are expected to be in a BAM file, GAlignments or GRanges object.
`subject` | A GRangesList containing the annotations. This list is expected to contain exons grouped by transcripts.
`ignore.strand` | When set to `TRUE`, strand information is ignored in the overlap calculations.
`...` | Additional arguments such as `param` and `singleEnd` used in the method for BamFile objects. See below.
`cds` | Optional GRangesList of coding regions for each transcript in the `subject`. If provided, the "coding" output column will be a `logical` vector indicating if the read falls in a coding region. When not provided, the "coding" output is `NA`.
`param` | An optional `ScanBamParam` instance to further influence scanning, counting, or filtering.
`singleEnd` | A logical value indicating if reads are single or paired-end. See `summarizeOverlaps` for more information.

### Details

When a read maps compatibly and uniquely to a transcript isoform we can quantify the expression and look for shifts in the balance of isoform expression. If a read does not map in compatible way, novel splice events such as splice junctions, novel exons or retentions can be quantified and compared across samples.

`findSpliceOverlaps` detects which reads (query) match to transcripts (subject) in a compatible fashion. Compatibility is based on both the transcript bounds and splicing pattern. Assessing the splicing pattern involves comparision of the read splices (i.e., the N operations in the CIGAR) with the transcript introns. For paired-end reads, the inter-read gap is not considered a splice junction. The analysis of non-compatible reads for novel splice events is under construction.

### Value

The output is a Hits object with the metadata columns defined below. Each column is a `logical` indicating if the read (query) met the criteria.

- compatible: Every splice (N) in a read alignment matches an intron in an annotated transcript. The read does not extend into an intron or outside the transcript bounds.

- unique: The read is compatible with only one annotated transcript.

- strandSpecific: The query (read) was stranded.

### Note

WARNING: The current implementation of `findSpliceOverlaps` doesn't work properly on paired-end reads where the 2 ends overlap!

### Author(s)

Michael Lawrence and Valerie Obenchain <Valerie.Obenchain@RoswellPark.org>

### See Also

- GRangesList objects in the GenomicRanges package.

- GAlignments and GAlignmentPairs objects.

- BamFile objects in the Rsamtools package.

### Examples

    ## -----------------------------------------------------------------------
    ## Isoform expression :
    ## -----------------------------------------------------------------------
    ## findSpliceOverlaps() can assist in quantifying isoform expression
    ## by identifying reads that map compatibly and uniquely to a
    ## transcript isoform.
    library(TxDb.Dmelanogaster.UCSC.dm3.ensGene)
    library(pasillaBamSubset)
    se <- untreated1_chr4() ## single-end reads
    txdb <- TxDb.Dmelanogaster.UCSC.dm3.ensGene
    exbytx <- exonsBy(txdb, "tx")
    cdsbytx <- cdsBy(txdb, "tx")
    param <- ScanBamParam(which=GRanges("chr4", IRanges(1e5,3e5)))
    sehits <- findSpliceOverlaps(se, exbytx, cds=cdsbytx, param=param)

    ## Tally the reads by category to get an idea of read distribution.
    lst <- lapply(mcols(sehits), table)
    nms <- names(lst)
    tbl <- do.call(rbind, lst[nms])
    tbl

    ## Reads compatible with one or more transcript isoforms.
    rnms <- rownames(tbl)
    tbl[rnms == "compatible","TRUE"]/sum(tbl[rnms == "compatible",])

    ## Reads compatible with a single isoform.
    tbl[rnms == "unique","TRUE"]/sum(tbl[rnms == "unique",])

    ## All reads fall in a coding region as defined by
    ## the txdb annotation.
    lst[["coding"]]

    ## Check : Total number of reads should be the same across categories.
    lapply(lst, sum)

    ## -----------------------------------------------------------------------
    ## Paired-end reads :
    ## -----------------------------------------------------------------------
    ## 'singleEnd' is set to FALSE for a BAM file with paired-end reads.
    pe <- untreated3_chr4()
    hits2 <- findSpliceOverlaps(pe, exbytx, singleEnd=FALSE, param=param)

    ## In addition to BAM files, paired-end reads can be supplied in a
    ## GAlignmentPairs object.
    genes <- GRangesList(
        GRanges("chr1", IRanges(c(5, 20), c(10, 25)), "+"),
        GRanges("chr1", IRanges(c(5, 22), c(15, 25)), "+"))
    galp <- GAlignmentPairs(
        GAlignments("chr1", 5L, "11M4N6M", strand("+")),
        GAlignments("chr1", 50L, "6M", strand("-")))
    findSpliceOverlaps(galp, genes)

[Package GenomicAlignments version 1.24.0 Index]