Capture sequencing enables enhanced genome recovery of Borrelia burgdorferi in mice

The Lyme disease spirochete, Borrelia burgdorferi , maintains a low burden during vertebrate infection. This limitation has historically proven to be particularly challenging for genome-based studies of B. burgdorferi infection and spirochete presence after antibiotic treatment. Here, we used TBDCapSeq, an oligo capture-based next- generation sequencing assay, to demonstrate its utility for genome recovery of B. burgdorferi directly from C3H- and BALB/c-infected mice. Tissue fragments from the ear, heart, tarsus, and infection-site skin were collected at 3 and 6 months post-tick infection from antibiotic-treated and untreated animals. We evaluated 59 tissues from 20 untreated mice and 48 tissues from 17 doxycycline-treated mice for spirochete burden and extent of genome recovery. In the untreated mice, genome recovery from each tissue was determined by the amount of borrelial DNA present in the sequencing library and was highest in the ear and heart, followed by the skin, and lowest in the tarsus. We established that approximately 50% of the genome could be recovered with as few as 10 spirochetes per assay and >90% recovery could be achieved with only 35 spirochete genomes. In the antibiotic-treated mice, trace quantities of fragmented Borrelia DNA were still present in tissues at 3 and 6 months. However, none of the tissue samples from these mice yielded an intact B. burgdorferi genome characteristic of a viable spirochete. Combined, our findings demonstrate that capture sequencing enables genomic analyses of B. burgdorferi directly within animal hosts. IMPORTANCE Lyme disease, caused by the spirochete Borrelia burgdorferi , is the most common tick-borne infection in the United States. Low pathogen burden is a major limitation of B. burgdorferi studies in animal hosts and humans that has impacted a wide range of studies ranging from diagnostics, evolutionary analyses, and pathogenesis. It has also contributed to the controversies regarding B. burgdorferi persistence after antibiotic treatment. We show that a next-generation capture sequencing-based approach can readily overcome the current limitations of B. burgdorferi genomic analyses within animal hosts. We successfully recovered abundant genomic information directly from mouse tissues, and we did not find genetic evidence of intact bacteria present after antibiotic treatment. This approach can be employed to gain further insights into B. burgdorferi and provide critical genome data during animal and human infection.