Ultra-deep metagenomic sequencing enables reconstruction of diverse, high quality microbial genomes from human urine samples

Urinary tract infections (UTIs) represent a major global health challenge, necessitating the precise identification of causative pathogens for effective diagnosis and treatment. While metagenomic next-generation sequencing (mNGS) has emerged as a powerful diagnostic tool, its clinical application has been limited by the lack of high-quality microbial genomes from urine samples. We reconstructed 223 reliable quality MAGs spanning bacterial, fungal, mycoplasmal, and viral, including the first demonstration of multiple Escherichia coli subpopulations within individual urine samples. The collection comprised six fungal genomes, ten mycoplasma genomes, and eight completed viral genomes. Comparative analysis showed 72% concordance with culture while detecting additional pathogens in 30% of cases, including fastidious organisms missed by conventional methods. This study establishes an optimized mNGS framework that overcomes current diagnostic limitations in UTIs through high-depth sequencing and minimal host contamination, enabling unprecedented resolution of the urinary microbiota, including the first identification of intra-sample E. coli subpopulations. The comprehensive MAG collection provides a valuable resource for advancing UTI diagnostics, mechanistic research, and personalized treatment strategies.