predictTxFeaturesPerSample: Identification of splice junctions and exons from BAM file
In SGSeq: Splice event prediction and quantification from RNA-seq data

Description Usage Arguments Details Value Author(s)

Splice junctions and exons are predicted from genomic RNA-seq read alignments in BAM format.

predictTxFeaturesPerSample(file_bam, which, paired_end, read_length,
  frag_length, lib_size, min_junction_count, alpha, psi, beta, gamma,
  min_anchor, include_counts, retain_coverage, junctions_only, max_complexity,
  sample_name, verbose, cores)

`file_bam`	BAM file with genomic RNA-seq read alignments
`which`	`GRanges` of genomic regions to be considered for feature prediction, passed to `ScanBamParam`
`paired_end`	Logical, `TRUE` for paired-end data, `FALSE` for single-end data
`read_length`	Read length required for use with `alpha`
`frag_length`	Fragment length for paired-end data required for use with `alpha`
`lib_size`	Number of aligned fragments required for use with `alpha`
`min_junction_count`	Minimum fragment count required for a splice junction to be included. If specified, argument `alpha` is ignored.
`alpha`	Minimum FPKM required for a splice junction to be included. Internally, FPKMs are converted to counts, requiring arguments `read_length`, `frag_length` and `lib_size`. `alpha` is ignored if argument `min_junction_count` is specified.
`psi`	Minimum splice frequency required for a splice junction to be included
`beta`	Minimum relative coverage required for an internal exon to be included
`gamma`	Minimum relative coverage required for a terminal exon to be included
`min_anchor`	Integer specifiying minimum anchor length
`include_counts`	Logical indicating whether counts of compatible fragments should be included in metadata column “N”
`retain_coverage`	Logical indicating whether coverage for each exon should be retained as an `RleList` in metadata column “coverage”. This allows filtering of features using more stringent criteria after the initial prediction.
`junctions_only`	Logical indicating whether predictions should be limited to identification of splice junctions only
`max_complexity`	Maximum allowed complexity. If a locus exceeds this threshold, it is skipped, resulting in a warning. Complexity is defined as the maximum number of unique predicted splice junctions overlapping a given position. High complexity regions are often due to spurious read alignments and can slow down processing. To disable this filter, set to `NA`.
`sample_name`	Sample name used in messages
`verbose`	If `TRUE`, generate messages indicating progress
`cores`	Number of cores available for parallel processing

For spliced alignments, the direction of transcription is inferred from the XS tag in the BAM file and used to assign strand information to the read, or fragment for paired-end data.

Feature prediction is performed in two steps. First, splice junctions are identified from spliced alignments. Second, exons are identified based on regions that are flanked by splice junctions and show sufficient coverage with compatible reads.

Splice junctions implied by read alignments are filtered based on fragment count and splice frequency. The splice frequency at the splice donor (acceptor) is defined as x_J/x_D (x_J/x_A), where x_J is the number of fragments containing the splice junction, and x_D (x_A) is the number of fragments overlapping the exon/intron (intron/exon) boundary. Fragments overlapping the spliced boundary can be either spliced or extend into the intron. To be included in predicted features, splice junctions must have fragment count at least min_junction_count or FPKM at least alpha, and splice frequency at both donor and acceptor at least psi.

Regions between any pair of identified splice junctions with sufficient compatible read coverage are considered candidate internal exons. Read coverage for a candidate exon is computed based on compatible fragments, i.e. fragments with matching (or missing) strand information and introns consistent with the exon under consideration. Candidate exons are included in predicted features if the minimum coverage is at least beta * number of junction-containing fragments for either flanking junctions.

Terminal exons are regions downstream or upstream of splice junctions with compatible fragment coverage at least gamma * number of junction-containing fragments.

TxFeatures object

Leonard Goldstein

SGSeq documentation built on Nov. 8, 2020, 8:31 p.m.