Adaptive sampling-based enrichment enables genome reconstruction of intracellular symbionts despite host background and reference divergence

Recovering genomes of intracellular microbes from host-dominated samples remains a major challenge in microbial genomics, due to low target abundance, overwhelming host DNA, and the inability to culture these organisms independently. Despite extensive interest in Wolbachia, efficient genome recovery directly from host tissues remains limited by the inefficiency of host-dominated sequencing and the constraints of existing enrichment strategies. Here, we demonstrate that Oxford Nanopore adaptive sampling (AS) enables efficient, real-time enrichment of target DNA directly from complex host tissues, providing a culture-free approach for genome recovery in such systems. To our knowledge, this represents the first application of enrichment-mode adaptive sampling to achieve de novo reconstruction of an intracellular endosymbiont genome in a mosquito system. Using Aedes aegypti mosquitoes infected with a locally derived wAlbB-like strain, we applied enrichment-mode AS to selectively sequence Wolbachia DNA. This resulted in an increase from <1% Wolbachia reads in conventional shotgun data to [~]90% under adaptive sampling. De novo assembly of AS-enriched long reads yielded a near-complete genome ([~]1.5 Mb) in two contigs with >96-99% completeness. Comparative analyses revealed multiple large-scale chromosomal rearrangements relative to the reference wAlbB genome, demonstrating that adaptive sampling does not impose reference-dependent genome structure. Annotation further identified three prophage-associated regions, including two strain-specific expansions absent from the reference genome. Notably, cytoplasmic incompatibility genes (cifA and cifB) were identified adjacent to one of these regions, consistent with their known genomic association with prophage elements. Importantly, adaptive sampling remained effective despite substantial structural divergence between the reference and target genomes, revealing an unexpectedly robust application of this approach beyond its presumed operating conditions. Together, these results establish enrichment-mode adaptive sampling as a robust and scalable strategy for genome-resolved analysis of intracellular bacteria in host-associated systems.