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

Ticks pose substantial threats to public health. Blacklegged ticks () are responsible for most tick-borne diseases in the US, transmitting seven human pathogens. Molecular surveillance for tick-borne pathogens has been outpaced by their emergence, revealing a critical need to develop agnostic strategies that characterize emerging and putative 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 pooled infected and -uninfected ticks to evaluate the diagnostic capability of NAS. We found that NAS generates extensive datasets on tick-borne pathogens from individual ticks that aid in distinguishing true and false positive samples. Using a pooled 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 when present at the expense of an elevated false-negative rate. We found that quality-based filtering of sequence data has a profound influence on diagnostic metrics, emphasizing the need to optimize pooling strategy, wet-lab procedures, and bioinformatic pipelines to enhance the sensitivity of NAS for detecting tick-borne pathogens.