Recovery and microbial host assignment of mobile genetic elements in complex microbiomes: insights from a spiked gut sample

ABSTRACT Mobile genetic elements (MGEs) are major drivers of horizontal gene transfer, including the spread of antimicrobial resistance (AMR) genes. However, determining the microbial host of an MGE in complex microbiomes remains challenging. Here, we spike a niche-aspecific Bacillus velezensis strain carrying a plasmid and linear phage-plasmid into a batch bioreactor simulating the human gut, and use it as a spike-in control to assess the performance of Hi-C sequencing and Oxford Nanopore Technologies (ONT)-enabled DNA methylation detection to identify MGE-host pairs. To improve recovery of low-abundance genomes, we used a novel ONT adaptive sampling (AS) strategy that depletes de novo assembled, sample-specific high-abundance contigs, rather than relying on reference genomes. This approach led to an approximately twofold enrichment of low-abundance replicons, including the spike-in strain. Methylation-based host assignment failed for the B. velezensis MGEs, likely due to the absence of DNA methylation. In contrast, Hi-C successfully linked the phage-plasmid to its host, but not the plasmid, likely due to non-intact cells, and only after removing artefactual signals through bioinformatic processing. For a native Escherichia coli strain, Hi-C and methylation data linked it to two plasmids. Selective isolation and whole-genome sequencing of both the native E. coli and spike-in B. velezensis then confirmed the metagenomic observations. Our results highlight that Hi-C and methylation data can provide powerful insights into MGE-host associations, but their interpretation requires careful computational analysis and biological validation. Moreover, our AS strategy offers a cost-efficient method to boost coverage of low-abundance genomes, improving metagenomic investigation of MGEs in complex microbiomes. IMPORTANCE Mobile genetic elements are important contributors to horizontal gene transfer, including of antimicrobial resistance genes. Understanding which microbes carry these mobile elements is vital to assess the spread of resistance. Here, we use a nanopore adaptive sampling approach to increase detection of low-abundance bacteria and mobile elements and use DNA methylation detection and Hi-C sequencing to determine mobile element hosts. By introducing a known bacterium and isolating a native strain, we could evaluate the performance of these methods, indicating that although powerful, they require careful experimental design, interpretation, and validation. However, when combined, these approaches enable a comprehensive investigation of mobile elements and gene transfer dynamics in complex environments.