![]() The bacteria are maintained in natural transmission cycles between vertebrate reservoir hosts and tick vectors. belongs to a bacterial species complex that consists of more than 20 genospecies with differing propensity to cause Lyme borreliosis in humans. In the present work we compared different NGS methodologies for their utility in analyzing the very complex genome of Borrelia burgdorferi sensu stricto (s.s.). Costs of the sequencing methods vary considerably and thus, precision and economics are important factors for deciding which sequencing technology is best suited to investigate the pathogen in question. Although long read methods such as nanopore or SMRT sequencing may have less precision with respect to single nucleotide polymorphism accuracy, high sequence coverage with the latter method (>100×) has been reported to provide more accuracy at the single nucleotide. On the other hand Pacific Bioscience Systems’ Single Molecule Real-Time (SMRT) DNA sequencing and Oxford Nanopore Technologies’ nanopore sequencing methods generate long sequence reads (maximum length 20 kb and >100 kb, respectively) with enormous advantages for genome assembly and in particular for plasmids (e.g. Different sequencing methods are currently on the market with Illumina being notable for short sequencing read technology, but assembly of accessory regions of the genome may be particularly challenging from short read NGS data. Next generation sequencing (NGS) technologies have allowed a big step to be taken towards the goal of whole genome analysis. This may change pathogenicity as well as host, or vector specificity (e.g. Importantly, accessory regions often undergo horizontal gene transfer, probably promoted by mobile genome elements (phages, transposons, plasmids). Bacterial genomes can be divided into conserved core and less conserved accessory (or non-core) regions. A pre-requisite for comparative genomics is the identification of strains of differing pathogenicity and also the ability to assemble the whole of the genome, including accessory regions in genomes of pathogenic bacteria. This has important implications for the development of useful research strategies to monitor the risk of Lyme disease occurrence and how to medically manage it.Ĭomparative analyses of whole genome sequences have been shown to be vital for understanding the correlation between infecting pathogen and disease manifestation (e.g. An important conclusion from our work is that the evolution of Borrelia plasmids appears to be dynamic. However, a combination of short and long read sequencing methods is essential for proper assembly of all plasmids including cp32 and thus, for gaining an understanding of host- or vector adaptations. Short read methods are sufficient to assemble the main chromosome and many of the plasmids in B. ![]() strains investigated here, whilst having similar plasmid structures to each other (apart from fusion of cp32 plasmids), differed significantly from the reference strain B31-GB, especially in the case of cp32 plasmids. Using long and short read technologies together allowed us to show that the three B. ![]() The long read SMRT and nanopore sequences came, however, at the cost of indels (insertions or deletions) appearing in an unpredictable manner. While cp32 plasmids remained refractory to assembly using only short reads they were effectively assembled by long read sequencing methods. Inclusion of mate pair library reads improved the assembly in some plasmids as did prior enrichment of plasmids. We tested the effectiveness of different short read assemblers on Illumina sequences, and of long read generation methods on sequence data from Pacific Bioscience single-molecule real-time (SMRT) and nanopore (Oxford Nanopore Technologies) sequencing technology. Sequences of these strains were compared to the previously sequenced reference strain B31 (available in GenBank) to obtain proof-of-principle information on the suitability of next generation sequencing (NGS) library construction and sequencing methods on the assembly of bacterial plasmids. burgdorferi sensu stricto strain B31(−NRZ) and two closely related strains (PAli and PAbe) that were isolated from human patients. In this study we used three strains: a low passage isolate of B. For this reason, it is essential to explore methods for rapid and reliable characterisation of molecular level changes on plasmids. Genes on these plasmids are known to play essential roles in virulence and pathogenicity as well as host and vector associations. The number and structure of plasmids is variable even in strains within a single genospecies. Borrelia ( B.) burgdorferi sensu lato, including the tick-transmitted agents of human Lyme borreliosis, have particularly complex genomes, consisting of a linear main chromosome and numerous linear and circular plasmids.
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