Abstract
In recent decades the description of several new species of Brucella has expanded the known mammalian host diversity of the genus considerably. Of these more recently described species, B. microti, B. inopinata and B. vulpis have been described as ‘atypical’, exhibiting either atypical phenotypic traits (B. microti), or greater genetic diversity (B. inopinata and B. vulpis) than classically described Brucella species. There are also an increasing number of reports of the isolation of atypical Brucella sp. organisms from other vertebrate hosts, including fish, reptiles and particularly amphibians. Analyses based on whole genome sequencing (WGS) have indicated that, whilst such atypical isolates maintain a high degree of genetic homology with core Brucella species, they exhibit a degree of horizontal gene transfer, incorporating genomic regions with sequence identity to soil associated Alphaproteobacteria. The inclusion of genetic elements from outsidethe genus Brucella emphasises the need for robust and reliable WGS tools to accurately characterise the genomes of novel atypical strains. Long-read sequencing methods are particularly valuable for this, as they can be used to construct complete bacterial genomes more reliably than approaches based on the de novo assembly of short sequencing reads alone. However, the selection of suitable DNA extraction methods has been shown to be integral to the success of long-read sequencing approaches. Furthermore, as the pathogenic potential of atypical Brucella strains remains unclear, such DNA extraction methods should ideally be compatible with methods commonly used for release of inactivated bacteria from containment laboratories. Using inactivated cultures of three atypical Brucella species (B. microti, B. inopinata and B. vulpis) we evaluated DNA extraction methods for producing long-read sequencing data using Oxford Nanopore Technologies MinIon sequencing platform. We evaluated metrics of sequencing (e.g. read length N50 andread number) and compared de novo genome assemblies, in order to identify the most suitable method. The results of these analyses are presented here.
