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.

There have been several developments in the field of nanopore biosensor development and sequencing applications that 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 these platforms a mainstay in contemporary research labs. Equipped with the ability to provide long and short-read sequencing information with quick turn-around times and simple sample preparation, nanopore sequencers are quickly 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 conclude with examples of important clinical applications to give perspective on the potential future of nanopore sequencing in the field of molecular diagnostics.

Tuberous sclerosis complex (TSC) is caused by mutations in TSC1 or TSC2 genes and affects multiple organs. TSC proteins control cell growth by regulating the activity of the mechanistic target of rapamycin complex 1. Extracellular vesicles (EVs) are membrane-bound particles produced by cells that mediate cellular communication, function, and growth. Although extensive studies regarding the genetic basis of TSC exist, the exact mechanism contributing to its pathogenesis remains unresolved. It has been proposed that EVs generated by renal cyst epithelia of mice and cells with Tsc gene mutations contain factors that alter the function and proliferation of TSC-sufficient cells. To test this, EVs from the kidneys and kidney explants of wildtype and Tsc1KO mice were isolated and characterized by Western blotting, transmission electron microscopy, dynamic light scattering, and fluorescent nanoparticle tracking. Our results show an enrichment in EV-associated markers and particle sizes of similar ranges. RNA-seq and proteomic analyses identified EV shuttle factors. EV RNA and protein shuttle factors showed significant differences. Furthermore, EVs isolated from Tsc1KO mice inhibited the proliferation of M-1 cells. Understanding the role of EVs in cell proliferation and cystogenesis in TSC may lead to the development of new approaches for the treatment of this disease.