A Framework for Analytical Validation of Genomic DNA-Based Shotgun Metagenomic Next-Generation Sequencing Workflows

The article presents a methodology for analytical validation of metagenomic next-generation sequencing (mNGS), increasingly used as a rapid and unbiased alternative to culture-based pathogen detection. The research team evaluated the performance of the PaRTI-Seq mNGS workflow, which includes host DNA depletion, DNA extraction, library preparation, and bioinformatic analysis. Analytical sensitivity at the library level was tested using DNA from five microorganisms including Enterobacter hormaechei, Candida albicans, and Mycobacterium smegmatis. Results showed that the workflow achieves limits of detection ranging from 1 CFU for E. hormaechei to 3,702 CFU for M. smegmatis. For microbiome applications, the workflow maintained consistent bacterial diversity down to 5 ng input DNA. The study provides a practical and generalizable framework for analytical validation of mNGS workflows in diagnostic and microbiome applications.