Rapid Response, High-Throughput Extraction of Biosafety Level 4 Viral Nucleic Acids

Abstract

Recent outbreaks of Ebola and Lassa virus have highlighted the need for real-time monitoring of virus evolution and spread using genome sequencing to enable rapid response efforts. For these category A pathogens, this effort requires development of well vetted protocols that assure complete virus inactivation while preserving the quality of nucleic acids. Such methods will allow for safe and effective high-throughput sequencing work outside of biosafety level 4 (BSL-4) facilities, and can ease the burden of manual sample processing, which can create bottlenecks during outbreaks. The currently preferred methods of viral nucleic acid inactivation and extraction are low to medium throughput at best, involving manual manipulation through every step of the process. In response, I optimized an automated protocol using the KingFisher Flex Magnetic Particle Processor for high-throughput nucleic acid extractions with MagMAXTM bead-based kits, and compare its RNA yield and sequencing coverage against the commonly used Qiagen spin column kit. I also automated many aspects of the extraction and library construction protocol using the Bravo Liquid Handler in order to further increase throughput. One of the most repeated and time-consuming steps in the library construction process is nucleic acid purification using SPRI beads, I thoroughly tested a protocol to automate high-throughput nucleic acid purification on the Bravo Liquid Handler. I found comparable yields from extractions performed by the KingFisher Flex Magnetic Particle Processor using the MagMax Pathogen Kit and Qiagen spin column, in terms of RNA yield and sequencing coverage, as quantified by RT-qPCR and Illumina next-generation sequencing coverage. I also saw comparable results between high-throughput automated and manual nucleic acid purification assays. Together, I have developed an effective high-throughput protocol to inactivate and process clinical samples that can enable real-time genome sequencing during viral outbreaks.