New tools for tick-borne pathogen surveillance

Project Summary Tick and rodent surveillance for pathogens requires substantial technical expertise and laboratory infrastructure. Most surveillance activities focus on detection of one or two potential pathogens in the vector or reservoir population and non-target pathogens remain undetected. To overcome the current limitations associated with traditional tick surveillance methods, we are proposing a novel dual metagenomic and metatranscriptomic sequencing solution using cutting-edge nanopore adaptive sampling (NAS) protocols. The NAS method leverages Oxford Nanopore Technologies sequencing technology (a portable MinION sequencer) and a recently developed bioinformatic pipeline that facilitates the immediate mapping of individual nucleotide molecules (DNA, cDNA, or RNA) to a given reference as each molecule is sequenced. User-defined thresholds then specify the retention or rejection of specific molecules, informed by the real-time reference mapping results as they are physically passing through a given sequencing nanopore. Hundreds to thousands of individual sequencing pores are controlled in real-time using a powerful Graphics Processing Unit (GPU) and the NAS software with retain/reject decisions made in less than a second for each individual pore. This allows for sensitive detection of a very wide range of targeted pathogen and host species barcoding sequences without becoming swamped in the sea of non- target host and symbiotic bacterial nucleic acids. The system can be field deployed and requires minimal infrastructure. An internet connection is not required. In the course of this proposed study, we will test NAS for diagnostic accuracy (sensitivity, specificity, and threshold of detection), test for detection of a variety of in vitro grown DNA and RNA-based pathogens, differentiate closely related pathogen species and strains, develop and ground truth protocols for field testing on wild ticks and rodents from a well-characterized region (Minnesota), and comprehensively field test the methods and protocols in a region predicted to have the one of the highest densities of emerging rodent-associated zoonoses in the US (central Kansas). We expect NAS to become an important and affordable tool with a wide variety of surveillance applications.