findSpliceOverlaps-methods: Classify ranges (reads) as compatible with existing genomic...

findSpliceOverlaps-methodsR 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

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)

Bioconductor/GenomicAlignments documentation built on July 23, 2022, 4:22 p.m.