A guide to cDNA sequencing with Oxford Nanopore
The ability to sequence RNA has provided invaluable insight into the study of living things across numerous applications, shedding light on the dynamics of the transcriptome, from single cells to whole tissues.
In medicine, the identification of differentially spliced isoforms and fusion transcripts can inform disease diagnosis and treatment.
RNA sequencing provides a rapid, effective method of virus identification, and in developmental biology the ability to track transcriptional changes over time helps to resolve the developmental mechanisms at play. Transcriptomics also has applications in environmental and agricultural science, such as in strategies for pest management. Despite the numerous advances made in transcriptome analysis using short-read cDNA sequencing technologies, these methods have several shortcomings.
Transcripts are generally several kilobases long: the typical human gene contains 12 exons each with an average length of 236 base pairs and alternative splicing has been observed in 95% of human genes. Short reads only partially cover a transcript’s length, making accurate isoform assembly a difficult process reliant on computational reconstruction; short reads also exhibit high rates of multimapping.
With nanopore sequencing, there is no upper read length limit: size selection is not necessary and whole transcripts can be sequenced end-to-end in single reads, enabling simple, accurate assembly, with the ability to distinguish between highly similar isoforms, identify novel transcripts, and detect fusions. Furthermore, rates of multimapping are significantly lower.