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.
What is alternative splicing?
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-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.
Revealing mRNA alternative splicing complexity in the human brain
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.
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.
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.
Discover more about the advantages of full-length nanopore RNA sequencing for gene expression and alternative splicing analyses.
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.
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