Direct whole-genome sequencing of Coxiella burnetii from environmental samples using a two-step selective enrichment approach

Abstract

Current high-throughput sequencing platforms enable routine whole-genome sequencing of bacteria via clonal amplification from primary samples, followed by DNA extraction, library preparation, and sequencing. This approach enriches bacterial DNA while removing contaminating nucleic acids, allowing efficient genomic sequencing using minimal sequencing resources per sample. However, for fastidious or obligate intracellular bacteria, such as Coxiella burnetii, cultivation is often impractical, leaving pathogen DNA heavily diluted by host and environmental nucleic acids. In such cases, sequencing requires substantially greater throughput per sample, highlighting the need for culture-independent methods to support genomic epidemiology and surveillance. Here, we report a novel two-stage DNA enrichment strategy applied to C. burnetii-contaminated sheep wool samples. Such samples feature very low pathogen biomass and a high host-DNA background. Our approach integrates (i) selective whole-genome amplification of complex DNA extracts, followed by (ii) in-solution hybridization capture using custom double-stranded DNA baits. Critically, we developed a WGA-based method to synthesize whole-genome dsDNA baits from minimal input DNA, enabling efficient bait production even in the absence of cultured isolates. Notably, we demonstrate that baits derived from C. burnetii DNA contaminated with an unrelated host (e.g., chicken) remain effective for enriching C. burnetii from samples derived from a different host species (e.g., sheep), indicating robust cross-host applicability. Using this workflow, we achieved up to 95% C. burnetii enrichment and generated six genomic assemblies suitable for downstream phylogenomic and epidemiological analyses. This cultivation-free, two-stage enrichment framework can be applied for phylogenetics and epidemiology and may significantly expand the feasibility of genomic studies for intracellular and otherwise uncultivable bacterial pathogens.