What is Alternative Splicing Analysis?

What is Alternative Splicing?

Alternative splicing, a pivotal mechanism in eukaryotic gene expression regulation, significantly contributes to the expansion of functional diversity within genes. This process involves the selective splicing of interacting exons and introns in biological gene sequences, allowing a single gene to generate multiple transcripts. Each transcript possesses distinct splice sites in the coding sequence, leading to the production of proteins with diverse functions.

Approximately 95% of genes undergo alternative splicing, resulting in cells expressing around four isoforms per gene. Alternative splicing manifests in five primary types:

  • Exon Skipping (ES)
  • Alternative 5' Splice Site (A5SS)
  • Alternative 3' Splice Site (A3SS)
  • Mutually Exclusive Exon
  • Intron Retention (IR)

Alternative Splicing Analysis in RNA Sequencing

In recent years, massively parallel RNA sequencing (RNA-Seq) has emerged as a powerful tool for conducting comprehensive transcriptomic analyses. The decreasing costs of deep sequencing have facilitated large-scale investigations into gene expression and alternative splicing, leading to a surge in the identification of diseases associated with these splicing variations. The knowledge gained from these studies holds great promise for the development of preventive and therapeutic interventions.

The precise mapping and quantification of alternative splicing events are crucial for downstream analyses, particularly in correlating diseases with specific splicing patterns. Despite the widespread adoption of high-throughput RNA-Seq, accurately discerning homodimer expression and obtaining quantitative results for alternative splicing from the resulting data present ongoing challenges.

In RNA-Seq experiments, mRNA is extracted from tissues, fragmented, and reverse-transcribed into cDNAs. Subsequent amplification and sequencing through high-throughput, short-read sequencing methods generate sequencing data from tissue samples. Ideally, transcriptome read segments can be assembled using comparison software to reconstruct the transcribed genomic region. Alternative splicing events can then be identified and quantified using specialized analysis software.

However, technical limitations persist, primarily due to the constrained read lengths inherent in next-generation sequencing RNA-Seq (typically ranging from 50 bp to 150 bp). The challenge intensifies when attempting to distinguish transcript isoforms of the same gene, as short-read segments rarely cross splice sites. This complexity complicates the inference of full-length transcripts, especially for low-expression transcripts. Additionally, identifying transcription start and termination sites remains a formidable task.

Please read our article PacBio Iso-Seq Enabling In-Depth Exploration of Alternative Splicing, for more information.

Alternative Splicing Analysis in Single-Cell Sequencing

Utilizing alternative splicing analysis software on single-cell data provides valuable insights into different alternative splice sites and associated genes within each subpopulation or sample. Subsequent differential analysis helps identify key alternative splicing sites across different subpopulations or genes linked to specific phenotypes. This exploration allows researchers to unravel the intricate associations between alternative splicing event sites and various cell types or phenotypes.

When assessing alternative splicing, it is crucial to consider the even distribution of cDNA reads across the entire gene. A uniform distribution signifies high randomness in the binding of captured sequences, facilitating full-length coverage of mRNA—an essential prerequisite for accurate alternative splicing analysis.

While single-end single-cell transcriptomes can capture certain alternative splicing site information, the enrichment at the single end restricts the amount of available information. This limitation hinders the comprehensive explanation of alternative splicing isoforms and the subsequent encoded proteins of the gene.

Tools for Accurate Quantification of Alternative Splicing

Over the past decade, computational tools have been developed to address these challenges. Given the author's emphasis on event-based methods and their continued use, the following is a brief overview of six noteworthy alternative splicing analysis software: rMATS, MAJIQ, LeafCutter, SUPPA2, SplAdder, and Whippet.

Algorithm Principle Quantification Differential Analysis Splicing Types Unannotated Transcripts
rMATS Event-based PSI yes 5 Checked
MAJIQ Event-based PSI yes 5 Not Checked
LeafCutter Intron Excision PSI yes Unknown Checked
SUPPA2 Event-based PSI yes 7 Not Checked
SplAdder Event-based PSI yes 5 Checked
Whippet Event-based PSI yes 9 Not Checked
For Research Use Only. Not for use in diagnostic procedures.
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