Abstract
The adoption of Oxford Nanopore Technologies (ONT) sequencing as a tool in plant virology has been relatively slow despite its promise in more recent years to yield large quantities of long nucleotide sequences in real time without the need for prior amplification. The portability of the MinION and Flongle platforms combined with lowering costs and continued improvements in read accuracy make ONT an attractive method for both low- and high-scale virus diagnostics. Here, we provide a detailed step-by-step protocol using the ONT Flongle platform that we have developed for the routine application on a range of symptomatic post-entry quarantine and domestic surveillance plant samples. The aim of this methods paper is to highlight ONT’s feasibility as a valuable component to the diagnostician’s toolkit and to hopefully stimulate other laboratories towards the eventual goal of integrating high-throughput sequencing technologies as validated plant virus diagnostic methods in their own right.
Highlights
In 1989, while thinking about synthetic lipid membranes, David Deamer sketched his idea of an artificial pore through which DNA would thread its way, altering the ionic current in a manner that was specific for each nucleotide base
Oxford Nanopore Technologies (ONT) sequencing is especially useful in detecting mixed infections, new host associations and previously undescribed species
The cost to run a single sample on a Flongle flow cell, including ribodepletion and ds cDNA synthesis is approximately $220 USD
Summary
In 1989, while thinking about synthetic lipid membranes, David Deamer sketched his idea of an artificial pore through which DNA would thread its way, altering the ionic current in a manner that was specific for each nucleotide base. We provide step-by-step details of a protocol that we routinely employ using the ONT MinION sequencing device with Flongle flow cells for diagnostic testing of symptomatic post-entry quarantine and domestic surveillance samples This method has update on ONT, where they concluded that it is the “most readily applicable to (plant) viral diagnostics”. This method has been successfully used on a whole range of different plant species to detect both visuccessfully used on a whole range of different plant species to detect both ruses and viroidsbeen as well as phytoplasmas and liberibacters and is a considered an viruses and viroids as well as phytoplasmas and liberibacters and is a considered an essential essential method in our diagnostic toolkit.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
