Benchmarking novel nanopore-based methods for targeted vector-borne pathogen surveillance and genomic vector and host characterization

Abstract

Pathogens transmitted by arthropod vectors are emerging and re-emerging on a global scale with severe consequences to human and animal health. Over recent decades, vector-borne diseases, particularly those spread by ticks and mosquitoes, have seen dramatic increases in incidence across the U.S. and worldwide. In the U.S. alone, reported cases of tick-borne diseases have more than doubled over the last two decades and cases across all vector-borne diseases have increased roughly three-fold. Controlling the spread of vector-borne pathogens is challenging due to the complex ecological dynamics involved in their transmission. Vector-borne pathogens often cycle between multiple vector and wildlife host species, and individual vectors can exhibit co-infections with multiple pathogens concurrently. Prompt and accurate identification of vectors, wildlife hosts, and their pathogens is critical; however, traditional vector biosurveillance methods are often reliant on aging technologies and are incapable of producing robust genomic information for species identification and pathogen detection. This thesis describes findings from three studies aimed at benchmarking novel nanopore-based sequencing methods—specifically using a targeted long-read sequencing strategy known as nanopore adaptive sampling (NAS)—for advancing genomic identification of potential arthropod vectors and wildlife hosts, and for performing targeted metagenomic pathogen surveillance. In Chapter 1, I provide a brief review of the next-generation sequencing landscape with a focus on recent advancements in single-molecule nanopore sequencing. I discuss the NAS approach for bioinformatics-driven targeted sequencing, some of its applications to date, and its potential to overcome many limitations inherent to other traditional vector surveillance methodologies. In the first study, detailed in Chapter 2, I describe a suite of experiments that specifically leverage the portable and field-ready capabilities of targeted nanopore sequencing. Here, NAS in combination with a portable laboratory was evaluated across two remote field sites in Guyana, South America for the purposes of real-time molecular species barcoding and molecular systematics. Across a variety of field-collected animal taxa—including mosquitoes, a phlebotomine sand fly, bats, and other small mammals—I demonstrate how NAS can be used to target and sequence key mitochondrial barcoding genes and provide in situ molecular identifications. The use of NAS as part of the portable laboratory allowed for bypassing of traditional amplification-based approaches, greatly streamlining sample preparation steps in the field, and enabling production of high-quality barcoding gene consensus sequences, mitochondrial genome assemblies, and maximum likelihood phylogenetic analyses. Expanding from these findings, the second study, described in Chapter 3, demonstrates how NAS can be utilized for more detailed mitogenomic characterization across blood-feeding arthropods. In this chapter, I detail findings from a collection of NAS sequencing experiments on blood-fed specimens of several potential insect vector species, targeting mitochondrial reads of both arthropods and their potential vertebrate bloodmeal sources. Here, NAS enabled sequencing, assembly, and annotation of high-quality new mitochondrial genomes for four mosquito species (Aedes trivittatus, Aedes vexans, Culex restuans, and Culex territans) and a hematophagous deer fly (Chrysops niger). NAS greatly improved recovery of target mitochondrial reads in comparison to control experiments without NAS, and also enabled dual identification of insect bloodmeal sources. Finally, in Chapter 4, I describe findings from a study aimed at benchmarking the NAS approach as a novel tool for metagenomic pathogen surveillance in tick vectors. Using wild Ixodes scapularis ticks—the primary vector of numerous tick-borne pathogens—I demonstrate how NAS can be used to selectively sequence multiple bacterial tick-borne pathogens. Across eight field-collected I. scapularis ticks evaluated, use of NAS enabled detection of four bacterial tick-borne pathogens: Borrelia burgdorferi s.s., Borrelia miyamotoi, Anaplasma phagocytophilum, and Ehrlichia muris eauclariensis. I provide in-depth comparisons of NAS experiments utilizing an enrichment strategy, targeting whole bacterial pathgoen genomes, and a depletion strategy, rejecting Ixodes host reads. Across both strategies, the implementation of NAS yielded a roughly two-fold read enrichment across all bacterial pathogens detected in comparison to unenriched control sequencing experiments. Collectively, the results from these studies provide key baseline data detailing some of the important potential applications of targeted sequencing with NAS for vector-borne pathogen surveillance. The findings from these chapters help to document how novel NAS-based methodologies can be readily adapted to enhance molecular species identification workflows, enable real-time genomics in remote settings, and produce robust genomic resources for understanding vector-borne pathogen transmission dynamics. These findings highlight the importance of integrating targeted sequencing methods like NAS into public health surveillance and species monitoring efforts in order to more effectively monitor and control emerging vector-borne disease threats.