NxtIRFcore-package {NxtIRFcore}R Documentation

NxtIRFcore: a command line interface for NxtIRF - IRFinder-based differential Alternative Splicing and Intron Retention analysis

Description

NxtIRF is a computationally efficient and user friendly workflow that analyses aligned short-read RNA sequencing for differential intron retention and alternative splicing. It utilises an improved IRFinder-based OpenMP/C++ algorithm. A streamlined downstream analysis pipeline allows for GLM-based differential IR and splicing analysis, suited for large datasets of up to hundreds of samples. Additionally NxtIRF provides a novel visualisation of per-nucleotide mean and variations of alignment coverage across splice and IR events, grouped by user-defined experimental conditions.

Details

IRFinder is a well-established bioinformatic tool that measures intron retention (IR) in annotated and novel retained introns in short-read RNA sequencing samples. It is a computationally-efficient algorithm that measures alignment coverage across introns, accounting for regions of low-mappable intronic regions. Unlike other algorithms that measure exon-intron spanning reads, IRFinder considers the alignment coverage across the whole intron, allowing it to distinguish between full-length and partial IR. This distinction is important as partial IR is often confounded with novel alternate splice site usage, alternate transcription start site and intronic polyadenylation events.

NxtIRF is a R/Bioconductor package that provides a user-friendly workflow using the IRFinder algorithm to perform both IR and alternative splicing analysis in large datasets. By incorporating the core C++ based IRFinder algorithm using Rcpp, NxtIRF is multi-platform and further improves computational efficiency using OpenMP-based multi-threading. Besides analysing IR, NxtIRF analyses other forms of alternative splicing events that depend on alternate splice site selection, including skipped exons, mutually exclusive exons, alternate 5'- and 3'- splice sites, alternate first exons and alternate last exons.

Downstream, NxtIRF provides functions to collate individual NxtIRF/IRFinder outputs of multiple samples in an experiment / dataset, and assembles these into a specialised NxtSE object that inherits the SummarizedExperiment class. Users can easily define experimental conditions, perform differential analysis and filter out lowly-expressed splice events.

Finally, NxtIRF provides visualisation tools to illustrate alternative splicing using coverage plots, including a novel method to normalise RNA-seq coverage grouped by experimental condition. This approach accounts for variations introduced by sequenced library size and gene expression. NxtIRF efficiently computes and visualises means and variations in per-nucleotide coverage depth across alternate exons in genomic loci.

NxtIRFcore is the command line interface for R/Bioconductor. NxtIRF (coming soon) will feature an interactive graphical user interface with additional functions.

Features include:

The main functions are:

See the NxtIRF vignette for worked examples on how to use NxtIRF

Author(s)

Alex Wong

References

Middleton R, Gao D, Thomas A, Singh B, Au A, Wong JJ, Bomane A, Cosson B, Eyras E, Rasko JE, Ritchie W. IRFinder: assessing the impact of intron retention on mammalian gene expression. Genome Biol. 2017 Mar 15;18(1):51. https://doi.org/10.1186/s13059-017-1184-4


[Package NxtIRFcore version 1.0.0 Index]