Evaluating the detection capabilities of nanopore sequencing for Borrelia burgdorferi detection in blacklegged ticks.

Ticks pose substantial threats to public health. Blacklegged ticks (Ixodes scapularis) are responsible for most tick-borne diseases in the US, transmitting seven human pathogens, including the etiological agent of Lyme disease, Borrelia burgdorferi. Molecular surveillance for tick-borne pathogens has been outpaced by their emergence, revealing a critical need to develop agnostic strategies that characterize expanding and reemerging pathogens. Oxford Nanopore Technology’s nanopore adaptive sampling (NAS), an approach that selectively enriches or depletes for target genomes or genetic loci, provides an opportunity to generate real-time genomic insights into tick-borne pathogens. In the current study, we performed PCR and NAS on individual Borrelia burgdorferi-infected and -uninfected ticks to evaluate the capability of NAS for tick-borne pathogen surveillance. We found that NAS generates real-time genetic datasets on B. burgdorferi that can supplement infectivity status ascertained via PCR. Using a multiplexing approach consisting of whole genomic DNA from 168 total ticks multiplexed over seven sequencing experiments, our results indicated that NAS is extremely specific (0.97 [95% CI: 0.93, 1.00]) with moderate sensitivity (0.48 [95% CI: 0.41, 0.55]), suggesting a strong capacity to confirm B. burgdorferi when present at the expense of an elevated false-negative rate. We found that quality-based filtering of sequence data has a substantial influence on detection metrics, emphasizing the need to optimize our multiplexing strategy, wet-lab procedures, and bioinformatic pipelines to enhance the sensitivity of NAS for detecting tick-borne pathogens.