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.

Background: Bacteria of the Borrelia burgdorferi sensu lato (s.l.) complex are the causative agents of Lyme borreliosis. These pathogens are maintained in transmission cycles between tick vectors of the Ixodes ricinus–persulcatus species complex and vertebrate reservoir hosts. 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. Linear and circular plasmid genes are associated with host/vector interactions but assembling complete plasmids is challenging due to the high levels of complexity and sequence homology. Here, using recently developed high-fidelity (HiFi) PacBio sequencing, we explored strategies to obtain gap-free, complete and high quality Borrelia genomes. For this, we sequenced and assembled three isolates belonging to three species within the B. burgdorferi s.l. complex (B. bavariensis, B. garinii and B. valaisiana). We investigated if use of HiFi reads will lead to substantial improvement of genome assemblies. Results: Stand-alone sequencing and assembly methods are insufficient for the generation of complete and high quality Borrelia genomes. Optimizing genome assembly, quality control and refinement steps, we critically appraised existing techniques to create and ensemble an improved genome reconstruction pipeline. Conclusion: Our study demonstrates that, with HiFi sequencing 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.