(405b) A Simplified Purpose-Built Microfluidic Platform for Nanopore Sequencing Library Preparation of Viral Samples

The rapid and real-time sequencing brought about by nanopore technology can serve as an excellent molecular diagnostic tool to evaluate and combat threats to US agriculture and livestock. However, the portability and ease of use afforded by these small sequencing devices is often hampered by sample preparation requirements such as equipment that typically isn’t found outside of a laboratory setting. Additionally, sample preparation in the field adds significant risk of contamination during user operation, limiting diagnostic quality.

This study evaluated a semi-automated, field deployable system for the rapid handling of viral samples including RNA extraction, library preparation, and sequencing using the Oxford Nanopore MinION device. At the heart of the platform is a pump operated microfluidic device which facilitates sample amplification and library preparation in a closed system, significantly minimizing risk of contamination. This device incorporates multiple reaction and mixing chambers and has been expanded and optimized to simultaneously process up to four samples with minimal crossover, reducing the reagent cost and library production time. The device utilizes specially designed built-in structures for passive mixing as well as paramagnetic beads controlled by a magnet for active mixing. Along with adding increased mixing capabilities to the device, the paramagnetic beads also allow for isolation and precise movement of nucleic acids on-chip for sample control beyond what the automated syringe pumps offers. All the device inlets have been designed with integrated bubble traps to limit undesirable diffusion into the device and minimize sample loss from gaseous expansion during heating. All heating on the device is facilitated by a simple hot plate.

The endemic Senecavalley Virus A (SVA) was used to model pathogenic FMDV. Loop mediated isothermal amplification (LAMP) was utilized to increase starting quantity of RNA recovered from nucleic acid extraction protocols in order to meet minimum requirements of tagmentation based library preparation. A multiplex LAMP primer set (6 total primer sets) was designed to evenly amplify 3 distinct regions covering 16% of the 7300bp genome without necessitating a thermocycler. Our device produces libraries with an alignment rate of 51%, and generates enough aligned reads to provide positive diagnosis within the first 27 minutes of sequencing across all three targeted regions. We are currently evaluating the device efficacy on vesicular fluid samples extracted from SVA positive pigs.