BACKGROUND:Bacteria of the Borrelia burgdorferi sensu lato (s.l.) complex can cause Lyme borreliosis. Different B. burgdorferi s.l. genospecies vary in their host and vector associations and human pathogenicity but the genetic basis for these adaptations is unresolved and requires completed and reliable genomes for comparative analyses. The de novo assembly of a complete Borrelia genome is challenging due to the high levels of complexity, represented by a high number of circular and linear plasmids that are dynamic, showing mosaic structure and sequence homology. Previous work demonstrated that even advanced approaches, such as a combination of short-read and long-read data, might lead to incomplete plasmid reconstruction. Here, using recently developed high-fidelity (HiFi) PacBio sequencing, we explored strategies to obtain gap-free, complete and high quality Borrelia genome assemblies. Optimizing genome assembly, quality control and refinement steps, we critically appraised existing techniques to create a workflow that lead to improved genome reconstruction.RESULTS:Despite the latest available technologies, stand-alone sequencing and assembly methods are insufficient for the generation of complete and high quality Borrelia genome assemblies. We developed a workflow pipeline for the de novo genome assembly for Borrelia using several types of sequence data and incorporating multiple assemblers to recover the complete genome including both circular and linear plasmid sequences.CONCLUSION:Our study demonstrates that, with HiFi data and an ensemble reconstruction pipeline with refinement steps, chromosomal and plasmid sequences can be fully resolved, even for complex genomes such as Borrelia. The presented pipeline may be of interest for the assembly of further complex microbial genomes.

Lyme borreliosis (LB) is the most common arthropod-borne disease in Europe and North America and is caused by members of the Borrelia burgdorferi sensu lato (Bbsl) species complex. These bacteria are transmitted by ixodid tick vectors and therefore human LB risk is influenced by the prevalence and distribution of Bbsl genospecies within tick vectors throughout the wild. These distributions can easily change over spatiotemporal scales and, to understand LB risk fully, up to date information on prevalence and distribution of Bbsl is required. The last survey of Bbsl in southern Germany, including parts of the Munich metropolitan area, was completed in 2006 and new data is needed. Ixodid ticks were collected in seven plots located in and around Munich, Germany, from March to July 2019 and were screened for Bbsl. Borrelia burgdorferi s. l. positive ticks (52 adults, 158 nymphs) were found in all plots and adults (0–61.5 % Bbsl positive/plot) and nymphs (17.4–59.5 % Bbsl positive/plot) did not differ significantly in their overall Bbsl prevalence. The number of Bbsl positive nymphs did vary significantly between plots but the number of positive adults did not. In total, six Bbsl genospecies were located with B. afzelii and B. garinii dominating. Additionally, the relapsing-fever species B. miyamotoi was found in two sampling plots. Our results highlight the variability in Bbsl prevalence and genospecies distribution over short geographic distances and aid in understanding LB risk in and around the Munich metropolitan area.