A high fidelity approach to assembling the complex Borrelia genome

Abstract

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.

Unprecedented genetic variability of PFam54 paralogs among Eurasian Lyme borreliosis‐causing spirochetes

“…We also observed the complete absence of specific, reference genes from all analyzed isolates ( pko2069 , pko2070 , zqa73 ) (Table S2 ). Noteworthy, our analysis was based on short‐read sequencing technologies which have been shown in recent years to not capture the entirety of the complex Borrelia genome, with the use of long‐read technologies (e.g., PacBio) providing much better, but also not perfect, resolution (Combs et al., 2022 ; Hepner et al., 2023 ; Margos et al., 2017 ). This could explain the absence of some genes in our analysis and also the presence of many, singular architecture types potentially due to lower resolution in specific samples.…”

Salmonella Typhimurium with Eight Tandem Copies of blaNDM-1 on a HI2 Plasmid

“…By employing state-of-the-art PacBio HiFi sequencing methods, we revealed the evolution process of drug resistance in Salmonella due to the acquisition of the HI2 plasmid carrying the bla NDM-1 gene, along with eight tandem copies of IS CR1 unit (IS CR1 - dsbD - trpF - ble - bla NDM-1 -IS Aba125 Δ1- sul1 Δ1) on the HI2 plasmid. This sequencing method can provide more accurate and complete genome assembly results, which is of great significance for the study of complex genomes [ 32 ]. In China, S. Typhimurium is one of the main serotypes of NTS that cause human infection, often causing diarrhea in patients [ 33 ].…”

Analysis of theBorreliaceaePangenome Reveals a Conserved Genomic Archictecture Across Phylogenetic Scales

“…I next assessed the spatial organization of homology groups within Borreliaceae genomes. Because many assemblies are incomplete due to the difficulty of generating complete assemblies through short-read sequencing [24,25,43] , I conducted a provisional analysis of draft genome organization by aligning a representative of each pangenome homology group to well-annotated reference genomes. Lipoproteins are preferentially encoded on plasmids throughout Borreliaceae (Figure 4).…”