Fusion transcripts

The accurate characterisation of fusion transcripts is of high importance for clinical research into diseases, including some forms of cancer. However, their identification via traditional short-read sequencing approaches requires transcripts to be sequenced in small fragments before being reassembled computationally, which can lead to multimapping and misassembly. With long nanopore sequencing reads, fusion transcripts can be sequenced end-to-end in single reads, enabling comprehensive characterisation of fusions and their precise splice junctions.

  • Sequence full-length fusion transcripts with long nanopore reads
  • Target fusion transcripts with or without PCR with simple, flexible workflows
  • Rapidly identify fusions with real-time sequencing and analysis

Full-length sequencing of fusion transcripts

Fusion transcripts, representing the product of a fusion gene or the splicing together of transcripts encoded by different genes, are significant in clinical research due to their association with many diseases, including some cancers. Sequencing of transcripts using traditional RNA-Seq approaches requires cDNA sequencing libraries to be fragmented and sequenced in reads of ~50–100 bases; the full transcript sequences are then deduced via computational assembly of the reads. However, such short reads may make precise breakpoint mapping of fusions difficult, and can result in incorrectly assembled transcripts and high levels of multimapping (Figure 1), whereby reads cannot be assigned to a single transcript. Furthermore, the requirement for PCR may mean that transcripts which are difficult to amplify may be poorly represented or missing from sequencing data. With nanopore sequencing, there is no upper read length limit, and fragmentation is not required: transcripts can be sequenced end-to-end in single long reads, enabling unambiguous identification of fusion transcripts.

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Figure 1. Long-read nanopore cDNA sequencing greatly reduces rates of multimapping compared with short-read sequencing techniques. Fusion transcripts can be sequenced end-to-end, enabling their accurate, unambiguous detection.

Figure 2. Both qualitative and accurate quantitative data can be generated with PCR-cDNA and direct RNA nanopore sequencing: here, good concordance was seen between observed and expected counts when sequencing an RNA spike-in via these methods. This enables both isoform identification and counting in one experiment.

Tailor your workflow to your experimental goals with versatile RNA and cDNA sequencing methods

With versatile options for library prep and sequencing available, nanopore workflows can be tailored to suit your needs. Oxford Nanopore has developed the only method of sequencing RNA molecules in their native form with direct RNA sequencing, eliminating PCR bias and enabling base modifications to be identified alongside the nucleotide sequence. cDNA-PCR sequencing is optimised for highest throughput with very low PCR bias. In addition, a protocol is available utilising the Ligation Sequencing Kit for direct, PCR-free sequencing of cDNA. Each approach provides both quantitative and qualitative data (Figure 2): fusion transcripts can be detected and counted with confidence from a single dataset. Rapid turnaround times can be achieved through real-time sequencing and analysis on the portable, cost-effective MinION or Flongle devices, the flexible GridION device, or scaled up for maximum output and throughput on powerful PromethION devices. Samples can also be sequenced in multiplex to batch samples as needed and further reduce cost per sample.

Enrich and characterise fusion transcripts with semi-specific RT-PCR

In some instances, fusion transcripts may be produced by a fusion gene resulting from one of several translocation events; the identity of both genes may not be known, meaning that primers cannot be designed to target both ends of the transcript. Here, semi-specific RT-PCR can be used to enrich for fusion transcripts where the fusion partner is unknown (Figure 3). First, the total RNA in a library is reverse-transcribed via a VN primer, which hybridises to any poly(A)-tailed RNA and is tailed with a primer site for subsequent amplification. Semi-specific PCR is then performed, amplifying transcripts using one primer complementary to the tailed cDNA molecules and one targeting the known end of the transcript. In this way, full-length wildtype and fusion transcripts can be enriched and sequenced with high depth of coverage, enabling accurate detection of fusions.

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Figure 3. Semi-specific RT-PCR and long-read nanopore sequencing enables detection of full-length fusion transcripts where only one end of the fusion is known. The q12 region of human chromosome 22 can be involved in several different translocation events (a). In each, a fusion gene is formed; these lead to different types of cancer. Semi-specific RT-PCR was used to reverse-transcribe RNA from a clinical research sample of a known translocation affecting the EWSR1 gene (b). Sequencing of the enriched amplicons enabled precise breakpoint identification and identification of the fusion partner (c).

Case study

Characterising somatic structural variation in colorectal cancer with long nanopore reads

A novel gene fusion RNF38-RAD51B is also identified, and we find it functionally acts to enhance migration, invasion, and metastasis capabilities of colorectal cancer cells

Xu et al. PLoS Genetics 19(2): e1010514 (2023)

Elucidating genetic mechanisms behind disease states, such as cancer, has the potential to help in the development of new therapeutic strategies. Noting that gene fusions are involved in the development of approximately 16% of all cancer types, Xu et al. used long nanopore sequencing reads to detect novel genetic rearrangements. Large genomic fusion events are challenging to detect using short-read sequencing due to their size, leading the authors to suggest that ‘nanopore sequencing may serve as a new strategy for detecting oncogenic gene fusions’.

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cancer research using nanopore sequencing

Identification of novel transcripts using targeted nanopore sequencing

Ailsa MacCalman (University of Exeter, UK) used targeted nanopore whole-transcript sequencing to characterise 330 disease-associated genes in clinical research pancreatic samples. At NCM 2022, she described how this work resulted in the discovery of novel transcripts, not present in existing gene annotations, including fusion transcripts. This data will provide insights into the landscape of the transcriptome across pancreatic development.

Sequencing workflow

How do I detect fusion transcripts with nanopore sequencing?

Enrichment of fusion transcripts can be performed using sequence-specific (where both ends of the transcript are known) or semi-specific (where one end of the transcript is known) RT-PCR. Preparation of libraries using the PCR-cDNA Sequencing Kit, followed by sequencing on one MinION Flow Cell, delivers ~18 million cDNA reads, for very high depth of coverage. Sequencing can be scaled down further on smaller Flongle Flow Cells for cost-effective long-read sequencing. Both the MinION and the GridION devices are compatible with MinION and Flongle Flow Cells; the portable MinION device is ideal for sequencing at the point of sampling, whilst the GridION enables sequencing on up to five individually addressable flow cells for flexible, on-demand analysis. A number of robust tools are available for analysing full-length nanopore RNA sequencing reads, both from Oxford Nanopore and the Nanopore Community. Oxford Nanopore provides a wide range of intuitive EPI2ME workflows to support nanopore data analysis for all levels of expertise.

Find out more about nanopore data analysis

RNA sequencing white paper

Discover more about the advantages of full-length transcript sequencing with Oxford Nanopore in the RNA sequencing white paper.

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Detect fusion transcripts in clinical research samples with long-read nanopore sequencing


cDNA-PCR Sequencing Kit

Analysis: wf-transcriptomes


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