Analytical validation of a highly accurate and reliable next-generation sequencing-based urine assay

Urinary tract infections (UTIs) are diagnosed based on symptoms and confirmed by urine culture, despite its limitations in sensitivity. False-negative cultures can lead to inappropriate antimicrobial use or urosepsis in high-risk patients. Next-generation sequencing (NGS)-based metagenomics offers a comprehensive and precise alternative but is rarely applied clinically. We developed and validated BIOTIA-ID, a clinical-grade NGS-based diagnostic pipeline for pathogen detection in urine. Remnant clinical and spiked urine samples underwent extraction, metagenomic library preparation, and Illumina NextSeq 550 sequencing. We trained and applied a bioinformatic pipeline that uses machine learning to identify pathogens and resistance markers. BIOTIA-DX was intentionally designed and trained to increase stringency and reduce false positive detection of urogenital commensals or opportunistic microbes present at colonization levels. Internal controls ensured standardized, high-stringency results. The assay was validated on 1,470 urine specimens evaluating over 14.5k analytes. The clinical validation achieved a 97.2% sensitivity and 99.6% specificity with a limit of detection (LoD) of <15,000 CFU/mL for most bacterial species and <5,000 CFU/mL for fungal species. Discordant results were reconciled by target-specific qPCR or 16S Sanger sequencing, and 87% of the NGS results were concordant with the comparator. A subset of 332 clinical specimens was tested and validated for antimicrobial resistance (AMR). sul and blaSHV genes were commonly associated with Escherichia coli and Klebsiella pneumoniae, while cfxA was found in Prevotella and Pseudomonas spp. detected by BIOTIA-ID. Overall, these data demonstrate that BIOTIA-ID is a comprehensive, highly accurate end-to-end diagnostic assay with notable advantages over current culture-based diagnostics.IMPORTANCEUrinary tract infections (UTIs) are among the most common infections, yet current diagnostic methods, including urine culture, often fail to detect pathogens accurately, leading to delayed treatment and inappropriate antimicrobial use. Clinical metagenomics offers a powerful alternative, especially in complicated cases. BIOTIA-ID is a validated, clinical-grade next-generation sequencing (NGS)-based assay that provides highly accurate pathogen identification and antimicrobial resistance profiling. By incorporating machine learning and stringent quality controls, BIOTIA-ID minimizes false positives and enhances diagnostic precision. Our study demonstrates its superior performance over culture, with potential to improve UTI diagnostics, guide targeted therapy, and support antimicrobial stewardship. The implementation of urine metagenomic diagnostics could support recurrent and complicated UTI patient management, providing a more reliable alternative to traditional methods.