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Splice variation

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

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Introduction

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 three 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 risk

Scotti, M. and Swanson, M. RNA mis-splicing in disease. Nat. Rev. Genet.17, 19–32 (2016)

, disease progression, and even drug responses

Gregory, 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 1970s.

Berget, 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.

Figure 1: Six alternative splicing categories. Exons are represented by a coloured box and introns by a black horizontal line. For each category, the different splicing reactions are symbolised by a red line. For the alternative 5′ splice site (ss) or 3′ ss, the use of the upstream 5′ ss or the downstream 3′ ss, generates a shorter upstream or downstream exon, respectively.

Figure 2: Alternative splicing can produce numerous isoforms per gene. A *Drosophila melanogaster* transcriptome dataset was created, mapped using minimap2, visualised using IGV, and isoforms were reconstructed using the pinfish analysis pipeline (developed by Oxford Nanopore). Compared to the reference isoform set from Ensembl, high exon and transcript-level precision can be seen.

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 adenovirus

Price, 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.

Case study

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’

Clark et al.

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.

Case study

Targeted sequencing of full-length transcripts reveals isoform diversity across human neurodevelopment

At the NCM2022, Rosemary Bamford (University of Exeter, UK) reported on her work characterising alternative splicing in the cerebral cortex of mice and humans. Using targeted nanopore transcript sequencing, her team were able to identify differentially expressed genes and alternative splicing in the brain, and obtain an ultra-deep view of transcripts during neurodevelopment.

Case study

Understanding the mechanisms of RNA processing using direct RNA nanopore sequencing

Newly synthesised messenger RNAs (mRNAs) undergo several processing steps prior to their export to the cytoplasm. At the NCM 2022, Karine Choquet (Harvard Medical School, USA) presented her work exploring the landscape of full-length mRNA isoforms across different subcellular compartments, using direct RNA nanopore sequencing of poly(A)-selected RNA from whole-cell, chromatin, cytoplasm, and polysome fractions in human cells. In her talk, she described the first transcriptome-wide characterisation of splicing and polyadenylation across long mRNA isoforms in distinct subcellular compartments.

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Sequencing workflow

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.

Oxford Nanopore provides analysis tutorials for transcript discovery and annotation; these are available in the Bioinformatics section of the Nanopore Community. Third-party analysis tools can also be found in the Resource Centre of the Oxford Nanopore website.

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Discover more about the advantages of full-length nanopore RNA sequencing for gene expression and alternative splicing analyses.

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Direct cDNA Sequencing Kit (SQK-DCS109)

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Splice variation

Introduction

What is alternative splicing?

Accurately characterise splice variation with long nanopore sequencing reads

Case study

In-depth splicing analysis of a neuropsychiatric gene

Revealing mRNA alternative splicing complexity in the human brain

Case study

Targeted sequencing of full-length transcripts reveals isoform diversity across human neurodevelopment

Case study

Understanding the mechanisms of RNA processing using direct RNA nanopore sequencing

Sequencing workflow

How do I perform alternative splicing analysis using nanopore sequencing?

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