Volden, R. et al. Proc. Natl. Acad. Sci. USA 115, 9726–9731 (2018).

Long-read sequencing technology offers an opportunity to decipher full-length RNA transcript isoforms. However, long-read sequencers typically suffer from high error rates, low throughput and high cost. To improve read accuracy, Volden et al. harnessed rolling-circle amplification to generate concatemeric cDNAs for sequencing by a nanopore sequencer. They split the raw reads into subreads and applied custom algorithms to obtain full-length cDNA consensus reads. The principle is similar to the circular consensus sequencing applied by a PacBio sequencer. The researchers termed this method Rolling Circle Amplification to Concatemeric Consensus (R2C2) and demonstrated that it can generate more accurate reads of RNA transcript isoforms in bulk or single-cell samples. They applied R2C2 to characterize the transcriptomes of single B cells and identified multiple isoforms of the CD19 receptor, a target for CAR-T cell therapy. Although R2C2 still cannot achieve both high throughput and high accuracy, it provides a way to analyze full-length transcriptomes at a reasonable cost.