Direct and real-time metagenomic sequencing of target-amplified genomes (mTAGs) for public health surveillance

Metagenomic sequencing is transforming microbiology but remains limited for pathogen surveillance due to poor sensitivity for low-abundance organisms in complex samples. Here, we present metagenomic target-amplified genome (mTAG) sequencing, a portable, culture-free workflow that combines algorithmically designed probe panels with long-read sequencing to selectively enrich pathogen DNA. Applied to poultry cecal samples, mTAG achieved up to 307-fold improvements in pathogen coverage depth over standard shotgun metagenomics at matched throughput. This step-change in culture-independent diagnostics enables real-time detection of foodborne pathogens, even in settings where traditional laboratory infrastructure is unavailable. By providing highly targeted, information-rich genomic data, mTAG bridges the gap between rapid field-deployable tests and comprehensive laboratory sequencing. It has broad utility for microbiologists, epidemiologists, and public health agencies seeking scalable, sensitive, and rapid methods for pathogen monitoring across clinical, agricultural, and environmental contexts.