Work

Evaluation and validation of a reproducible mNGS workflow for gut Microbiome analysis

Year: 2026

Type: article

Abstract: Technical variability in pre-analytical and analytical steps significantly hinders the reproducibility of gut metagenomic next-generation sequencing (mNGS). To address this, we developed and validated a practical mNGS workflow. First, by evaluating three commercial preservation buffers at 37°C, we identified Buffer C as the most effective for maintaining microbiome integrity under simulated transport stress. Second, we introduced a cost-effective, self-prepared inhibitor removal buffer (Self-IB) for automated extraction, which yielded species abundances highly concordant with the manual reference method (R² > 0.998). Finally, the optimized workflow demonstrated high intra- and inter-batch reproducibility, with most species-level coefficients of variation below 10%. This integrated, end-to-end pipeline (Buffer C + Self-IB + automated workflow) facilitates high data fidelity and robustness, offering a practical strategy for industrial and clinical applications requiring longitudinal consistency, such as dynamic monitoring following fecal microbiota transplantation (FMT).

Source: Scientific Reports

DOI: 10.1038/s41598-026-70557-8