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

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• Mobile genetic elements (MGEs) drive horizontal gene transfer, facilitating the spread of antimicrobial resistance (AMR) genes.
• Identifying the microbial hosts of MGEs in complex microbiomes is essential for tracking AMR dissemination.

Purpose of the Study:

• To evaluate Hi-C sequencing and DNA methylation detection for identifying MGE-host pairs.
• To assess a novel Oxford Nanopore Technologies (ONT) adaptive sampling (AS) strategy for enhancing the recovery of low-abundance genomes and MGEs.
• To investigate MGE-host associations in a simulated human gut microbiome using spike-in controls and native strains.

Main Methods:

• Utilized a spike-in control of *Bacillus velezensis* with a plasmid and phage-plasmid in a bioreactor.
• Employed ONT adaptive sampling (AS) to enrich low-abundance replicons by depleting high-abundance contigs.
• Applied Hi-C sequencing and DNA methylation detection to determine MGE-host associations.
• Confirmed findings through selective isolation and whole-genome sequencing of native *Escherichia coli* and spike-in *B. velezensis*.

Main Results:

• ONT adaptive sampling (AS) achieved approximately twofold enrichment of low-abundance replicons, including the spike-in strain.
• Hi-C successfully linked a phage-plasmid to its *B. velezensis* host but failed for the plasmid alone.
• DNA methylation-based host assignment was unsuccessful for *B. velezensis* MGEs.
• Hi-C and methylation data successfully linked a native *Escherichia coli* strain to two plasmids.
• Subsequent isolation and sequencing validated the metagenomic MGE-host assignments.

Conclusions:

• Hi-C and methylation data offer powerful insights into MGE-host associations but require careful computational analysis and biological validation.
• The developed AS strategy is a cost-efficient method to boost coverage of low-abundance genomes, enhancing microbiome research.
• Combined approaches enable comprehensive investigation of MGEs and gene transfer dynamics in complex environments.