Scaffold-level validation is needed for phage–host inference from metagenome-assembled genomes

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 can be violated under particular challenging conditions. 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.