Direct RNA Sequencing of Foot-and-mouth Disease Virus Genome Using a Flongle on MinION.

Foot-and-mouth disease (FMD) is a severe and extremely contagious viral disease of cloven-hoofed domestic and wild animals, which leads to serious economic losses to the livestock industry globally. FMD is caused by the FMD virus (FMDV), a positive-strand RNA virus that belongs to the genus Aphthovirus, within the family Picornaviridae. Early detection and characterization of FMDV strains are key factors to control new outbreaks and prevent the spread of the disease. Here, we describe a direct RNA sequencing method using Oxford Nanopore Technology (ONT) Flongle flow cells on MinION Mk1C (or GridION) to characterize FMDV. This is a rapid, low cost, and easily deployed point of care (POC) method for a near real-time characterization of FMDV in endemic areas or outbreak investigation sites. Key features • Saves ~35 min of the original protocol time by omitting the reverse transcription step and lowers the costs of reagents and consumables. • Replaces the GridION flow cell from the original protocol with the Flongle, which saves ~90% on the flow cell cost. • Combines the NGS benchwork with a modified version of our African swine fever virus (ASFV) fast analysis pipeline to achieve FMDV characterization within minutes. Graphical overview Schematic of direct RNA sequencing of foot-and-mouth disease virus (FMDV) process, which takes ~50 min from extracted RNA to final loading, modified from the ONT SQK-RNA002 protocol (Version: DRS_9080_v2_revO_14Aug2019).

Rapid Detection and Quick Characterization of African Swine Fever Virus Using the VolTRAX Automated Library Preparation Platform.

African swine fever virus (ASFV) is the causative agent of a severe and highly contagious viral disease affecting domestic and wild swine. The current ASFV pandemic strain has a high mortality rate, severely impacting pig production and, for countries suffering outbreaks, preventing the export of their pig products for international trade. Early detection and diagnosis of ASFV is necessary to control new outbreaks before the disease spreads rapidly. One of the rate-limiting steps to identify ASFV by next-generation sequencing platforms is library preparation. Here, we investigated the capability of the Oxford Nanopore Technologies' VolTRAX platform for automated DNA library preparation with downstream sequencing on Nanopore sequencing platforms as a proof-of-concept study to rapidly identify the strain of ASFV. Within minutes, DNA libraries prepared using VolTRAX generated near-full genome sequences of ASFV. Thus, our data highlight the use of the VolTRAX as a platform for automated library preparation, coupled with sequencing on the MinION Mk1C for field sequencing or GridION within a laboratory setting. These results suggest a proof-of-concept study that VolTRAX is an effective tool for library preparation that can be used for the rapid and real-time detection of ASFV.