There has been significant progress made in the field of nanopore biosensor development and sequencing applications, which address previous limitations that restricted widespread nanopore use. These innovations, paired with the large-scale commercialization of biological nanopore sequencing by Oxford Nanopore Technologies, are making the platforms a mainstay in contemporary research laboratories. Equipped with the ability to provide long- and short read sequencing information, with quick turn-around times and simple sample preparation, nanopore sequencers are rapidly improving our understanding of unsolved genetic, transcriptomic, and epigenetic problems. However, there remain some key obstacles that have yet to be improved. In this review, we provide a general introduction to nanopore sequencing principles, discussing biological and solid-state nanopore developments, obstacles to single-base detection, and library preparation considerations. We present examples of important clinical applications to give perspective on the potential future of nanopore sequencing in the field of molecular diagnostics.

Sequencing of long coding RNAs informs about the abundance and the novelty in the transcriptome, while sequencing of short coding RNAs (e.g., microRNAs) or long non-coding RNAs informs about the epigenetic regulation of the transcriptome. Currently, each of these goals is addressed by separate sequencing experiments given the different physical characteristics of RNA species from biological samples. Sequencing of both short and long RNAs from the same experimental run has not been reported for long-read Nanopore sequencing to date and only recently has been achieved for short-read (Illumina) methods. We propose a library preparation method capable of simultaneously profiling short and long RNA reads in the same library on the Nanopore platform and provide the relevant bioinformatics workflows to support the goals of RNA quantification. Using a variety of synthetic samples we demonstrate that the proposed method can simultaneously detect short and long RNAs in a manner that is linear over 5 orders of magnitude for RNA abundance and three orders of magnitude for RNA length. In biological samples the proposed method is capable of profiling a wider variety of short and long non-coding RNAs when compared against the existing Smart-seq protocols for Illumina and Nanopore sequencing.