The identification of differentially spliced isoforms, and their functional effects, is of high importance in the study of both healthy variation and disease, with aberrant splicing implicated in diseases including cancer and neurological disorders. However, traditional short-read RNA-Seq methods typically cannot span full-length isoforms, requiring them to be computationally reassembled; this can lead to incorrect reconstruction. With long nanopore reads, isoforms can be sequenced end-to-end in single reads, enabling their unambiguous characterisation - and simultaneous quantification, in a single dataset.
Accurately resolve and quantify full-length splice variants with long sequencing reads
Identify epigenetic modifications alongside nucleotide sequence through direct RNA sequencing
Scale to your output requirements with a range of sequencing platforms
What is alternative splicing?
Splicing is an important mechanism that regulates isoform expression in a cell-specific or timing-specific (e.g. during development) manner. Different categories of alternative splicing have been described, including exon skipping, the most frequently occurring type, and intron retention (Figure 1). It is estimated that 95% of multiexon genes are alternatively spliced in humans, with an average of 3 transcripts produced per gene Mathur, M. et al. Programmable mutually exclusive alternative splicing for generating RNA and protein diversity. Nat. Commun. 10, 2673 (2019). .
Alternative splicing can have dramatic effects on the protein produced. For example, protein-protein interactions may be altered, enzyme function inhibited, or location of expression may be changed (e.g. from cell-surface to secreted). Such splice variation may significantly impact disease riskScotti, M. and Swanson, M. RNA mis-splicing in disease. Nat. Rev. Genet.17, 19–32 (2016) , disease progression, and even drug responsesGregory, A. et al. TNF receptor 1 genetic risk mirrors outcome of anti-TNF therapy in multiple sclerosis. Nature 488, 508–511 (2012). .
RNA splicing was first discovered by analysis of adenovirus RNA arrangement in the late 1970sBerget, S.M., Moore. C., and Sharp, P.A. Spliced segments at the 5' terminus of adenovirus 2 late mRNA. Proc. Natl. Acad. Sci. USA 74,8: 3171-5 (1977). ,Chow, L.T. et al. An amazing sequence arrangement at the 5′ ends of adenovirus 2 messenger RNA. Cell. 12(1):1-8 (1977). . In contrary to the larger genomes of humans, plants and animals, splicing enables efficient use of the small genomes of viruses. In adenovirus, differential splicing controls gene expression, and therefore protein production, across the different stages of infection.
Accurately characterise splice variation with long nanopore sequencing reads
Comprehensive analysis of splice variation is limited with short-read sequencing; although exon junctions can be observed, resolution of entire isoforms is extremely challenging. With nanopore sequencing, read length is equal to fragment length, meaning entire transcripts can be sequenced in single reads. Full-length transcripts >20 kb in length have been sequenced in single reads. This greatly simplifies the identification and quantification of entire isoforms (Figure 2).
In addition to analysis of cDNA molecules, nanopore technology uniquely enables direct sequencing of native RNA molecules. This allows base modification information to be obtained alongside nucleotide sequence data; no additional sample preparation or sequencing runs are needed to acquire epigenetic data. Investigating the association of methylation with splice variation is therefore greatly simplified with Oxford Nanopore sequencing technology. As an example, splicing regulation by N6-methyladenosine (m6A) modification was recently described in adenovirusPrice, A. M. et al. Direct RNA sequencing reveals m6A modifications on adenovirus RNA are necessary for efficient splicing. bioRxiv 865485 (2019).. With nanopore direct RNA sequencing, m6A modifications could be identified at the nucleotide level, and as the overlapping splice units could be mapped precisely with long reads, methylation could also be accurately resolved at the transcript level.
In-depth splicing analysis of a neuropsychiatric gene
‘Our study highlights the power of long-read sequencing for the annotation and characterisation of alternatively spliced transcripts’
Genetic variation within the gene CACNA1C, encoding the voltage-gated calcium channel CaV1.2, is associated with neuropsychiatric disorders, including schizophrenia and bipolar disorder. However, the basis for the underlying genetic association is unknown. Using long-range PCR and nanopore cDNA sequencing of full-length CACNA1C transcripts, Clark et al. performed an in-depth analysis of its splice variants in post-mortem human brain tissue. This investigation revealed the true complexities of CACNA1C splicing: 38 novel exons were observed, and 241 of 251 total transcripts identified were novel. Many of the novel transcripts were found to be abundantly expressed and encode for aberrant protein products with altered function. The researchers state that such detailed results help to advance our understanding of these neuropsychiatric disorders, and provide potential pharmacological targets.
Revealing mRNA alternative splicing complexity in the human brain
How do I perform alternative splicing analysis using nanopore sequencing?
Oxford Nanopore provides three RNA sequencing kits that can be used for gene expression and downstream splice variation analysis, all of which deliver full-length transcripts. The choice of kit depends on your specific study requirements, including sample amounts, requirement for sample multiplexing, base modification detection, and desired number of reads.
The range of nanopore sequencing platforms enables you to scale according to your throughput and output requirements, from the portable Flongle and MinION devices, which are well suited to targeted splice variation analysis, to the modular GridION and ultra-high-throughput PromethION P24/48 platforms, ideal for transcriptome-wide investigations.
For complete advice on performing research into splice variation with nanopore sequencing technology, from sample prep to data analysis, view our cDNA sequencing Getting Started guide.
Splice variation analysis in the human transcriptome
Library preparation with the Direct cDNA Sequencing Kit, followed by sequencing on the PromethION device, delivers 15-30 million reads per flow cell, ideal for transcriptome-wide analysis of splice variation. Sample multiplexing can be achieved using the Native Barcoding Expansion Packs.