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.
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, the flexible GridION, 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.
Characterising somatic structural variation in colorectal cancer with long nanopore reads
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’.
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.
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. Visit the Bioinformatics section of the Nanopore Community for data analysis tutorials.
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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