Revisiting phage-host inference from metagenome-assembled genomes reveals the need for scaffold-level validation

Abstract

Accurate phage-host prediction underpins ecological interpretation and experimental validation of uncultivated phages, yet most current approaches rely heavily on metagenome-assembled genomes (MAGs). This reliance implicitly assumes that MAG-level quality metrics, such as completeness and contamination, ensure scaffold-level correctness. Here, we demonstrate that this assumption is frequently violated. Tetranucleotide frequency (TNF) analyses reveal that CRISPR-associated regions often diverge compositionally from their host genomes, increasing the risk of misbinning, while experimentally validated phage-host genome pairs show consistent TNF separation between phages and their hosts, challenging compositional binning assumptions. Through controlled simulations, we further show that the incorporation of substantial amounts of TNF-matched, non-host phage sequences into high-quality MAGs does not substantially alter standard completeness and contamination estimates, rendering such errors largely undetectable. Together, these results expose a systematic and underappreciated source of bias in MAG-based phage-host inference and highlight the need for explicit scaffold-level validation beyond conventional genome-quality metrics. We further propose a practical scaffold-level validation framework that integrates structural linkage and taxonomic consistency to improve the reliability of phage-host inference from MAGs.