HiFi based metagenomic assembly strategy provides accuracy near isolated genome resolution in MAG assembly

Recovering high-contiguity, circular bacterial genomes from complex microbiomes (e.g., gut) is challenged by limitations of short-read and error-prone long-read sequencing. This study comprehensively compares PacBio High-Fidelity (HiFi) sequencing-based metagenome-assembled genomes (MAGs) against Illumina MAGs, Oxford Nanopore Technologies (ONT) MAGs, and isolate whole-genome sequencing genomes from the same sample. HiFi sequencing yielded 31 high-quality MAGs, including 10 complete circular genomes. HiFi MAGs demonstrated significantly higher completeness, continuity, and lower contamination than Illumina or ONT MAGs (<i>p-</i>adj < 0.05). Crucially, HiFi MAGs exhibited closer genomic proximity to corresponding isolates at both single-nucleotide polymorphism and gene presence/absence levels. This benchmarking establishes HiFi as a robust approach for generating MAGs rivaling isolated genome quality, providing critical insights for accurate microbial genomic studies.