Development of a capture sequencing assay for enhanced detection and genotyping of tick-borne pathogens

Komal Jain,Santiago Sanchez-Vicente,Vishal Kapoor,Linden Hu,Stephen Sameroff,W Ian Lipkin,Thomas Briese,Adriana Marques,Brian Fallon,Alper Gokden,Teresa Tagliafierro,Rafal Tokarz,Nischay Mishra,Luis A Marcos,Cheng Guo

doi:10.1038/s41598-021-91956-z

Abstract

Inadequate sensitivity has been the primary limitation for implementing high-throughput sequencing for studies of tick-borne agents. Here we describe the development of TBDCapSeq, a sequencing assay that uses hybridization capture probes that cover the complete genomes of the eleven most common tick-borne agents found in the United States. The probes are used for solution-based capture and enrichment of pathogen nucleic acid followed by high-throughput sequencing. We evaluated the performance of TBDCapSeq to surveil samples that included human whole blood, mouse tissues, and field-collected ticks. For Borrelia burgdorferi and Babesia microti, the sensitivity of TBDCapSeq was comparable and occasionally exceeded the performance of agent-specific quantitative PCR and resulted in 25 to > 10,000-fold increase in pathogen reads when compared to standard unbiased sequencing. TBDCapSeq also enabled genome analyses directly within vertebrate and tick hosts. The implementation of TBDCapSeq could have major impact in studies of tick-borne pathogens by improving detection and facilitating genomic research that was previously unachievable with standard sequencing approaches.

Highlights

Inadequate sensitivity has been the primary limitation for implementing high-throughput sequencing for studies of tick-borne agents
We primarily focused on samples that were infected with B. burgdorferi s.s. or B. microti, as these agents are among the most frequent causes of tick-borne illness in the US
To evaluate the utility of TBDCapSeq with respect to unbiased high-throughput sequencing (UHTS) for testing tissue samples, we examined heart, ear, and bladder tissues from three C3H mice infected with 1 05 spirochetes of the N40 D10E9 strain of B. burgdorferi s.s. (Table 2)

Summary

Introduction

Inadequate sensitivity has been the primary limitation for implementing high-throughput sequencing for studies of tick-borne agents. The implementation of TBDCapSeq could have major impact in studies of tick-borne pathogens by improving detection and facilitating genomic research that was previously unachievable with standard sequencing approaches. Assay limitations have prevented comprehensive genomic analyses of B. burgdorferi s.s. in human specimens and, at present, there is a great paucity of B. burgdorferi s.s. genomic data obtained directly from patient samples This limits our understanding of how strain diversity could influence the development of Lyme disease-associated syndromes such as neuroborreliosis, Lyme arthritis and post-treatment Lyme disease syndrome. We demonstrate that TBDCapSeq is capable of enhanced targeted detection of all major agents of TBD found in the United States that provides invaluable genomic data that can be used to augment our understanding of strain variation and its importance to tick-borne disease

Methods

Results

Conclusion