NxtIRFcore-package: NxtIRFcore: a command line interface for NxtIRF -...

In alexchwong/NxtIRFcore: Core Engine for NxtIRF: a User-Friendly Intron Retention and Alternative Splicing Analysis using the IRFinder Engine

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

Description

(Important!) NxtIRFcore will be replaced by SpliceWiz from Bioconductor version 3.16 onwards. SpliceWiz replaces the full functionality of NxtIRFcore, plus heaps more! 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:

• Reference generation from user-supplied local and web resources, as well as connectivity to the AnnotationHub repository for Ensembl-based genomes and gene annotations;

• OpenMP and BiocParallel-based multi-threaded support to process short-read BAM files using the IRFinder algorithm written in native C++;

• Stores alignment coverage using the COV format, which is a binary compressed and indexed format for rapid recall of RNA-seq coverage. In contrast to the BigWig format, COV files store coverage of unstranded as well as stranded alignment coverage, and is much more space-efficient, allowing for better portability;

• Memory-efficient collation of hundreds of samples using on-disk memory approaches and H5-based assay storage;

• Streamlined user-friendly functions to construct multi-factor complex experimental designs, and perform differential IR and alternative splicing analysis using well-established statistical methods including limma and DESeq2;

• Advanced RNA-seq coverage visualisation, including the ability to combine RNA-seq coverage of multiple samples using advanced library normalisation methods across samples grouped by conditions;

The main functions are:

• BuildReference - Prepares genome and gene annotation references from FASTA and GTF files, and synthesises the NxtIRF reference for the IRFinder engine and NxtIRF-based downstream analysis.

• STAR-methods - (Optional) Provides wrapper functions to build the STAR genome reference and alignment of short-read FASTQ raw sequencing files. This functionality is only available on systems with STAR installed.

• IRFinder - OpenMP/C++ based IRFinder algorithm to analyse single or multiple BAM files using the NxtIRF/IRFinder reference.

• CollateData - Collates an experiment based on multiple IRFinder outputs for individual samples, into one unified H5-based data structure.

• MakeSE - Constructs a NxtSE (H5-based SummarizedExperiment) object, specialised to house measurements of retained introns and junction counts of alternative splice events.

• apply_filters - Use default or custom filters to remove alternative splicing or IR events pertaining to low-abundance genes and transcripts.

• ASE-methods - one-step method to perform differential alternate splice event (ASE) analysis on a NxtSE object using limma or DESeq2.

• make_plot_data: Functions that compile individual and group-mean percent spliced in (PSI) values of IR and alternative splice events; useful to produce scatter plots or heatmaps.

• Plot_Coverage: Generate RNA-seq coverage plots of individual samples or across samples grouped by user-specified conditions

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

alexchwong/NxtIRFcore documentation built on Oct. 31, 2022, 9:14 a.m.