Metagenomic Sequencing Enables Clinically Relevant Identification of 176 Targets from Faecal Samples

Background Robust identification of pathogens is essential for managing patients with symptomatic infection, yet conventional diagnostic methods are limited to a small subset of clinically relevant pathogens and genes. Metagenomic next generation sequencing (mNGS) can fill this gap, as it can comprehensively assess the pathogens and genes in a sample. This study evaluates the clinical (22 targets), analytical (19 targets), and in silico (176 targets) performance of a faecal mNGS assay on clinically relevant bacterial, eukaryotic, viral, virulence factor, and antimicrobial resistance gene targets. Methods Clinical performance was evaluated relative to conventional pathology testing using 510 clinical faecal samples from patients presenting with gastrointestinal symptoms. Simulated samples were used to assess analytical performance and establish the assay’s limit of detection by adding cells to a faecal matrix. In silico faecal samples containing targets reflecting the limit of detection of the assay were used to evaluate performance across all 176 targets. Results Clinical specificity was ≥96% for all targets and median sensitivity was 91% for pathogens. Gene targets were the most challenging to identify with 58.7% median sensitivity. Application of the mNGS assay to triplicate biological samples of 158 clinical specimens found the assay to be highly reproducible with 99.5% of test results being concordant. Results for in silico faecal samples confirmed strong performance across all 176 targets, with the majority identified at just above the limit of detection. Conclusions The faecal mNGS assay performed on par with existing diagnostic techniques while identifying a significantly broader range of clinically relevant targets in parallel.