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

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 evaluated a practical workflow for mNGS. By evaluating three commercial preservation buffers (A/B/C), we found that PBB exhibited the highest fidelity to the original microbiome composition, while PBC demonstrated the strongest robustness among the three buffers tested against community disturbance, maintaining structural consistency over nine days of incubation at 37 °C. Subsequently, we introduced a cost-effective, self-prepared inhibitor removal buffer (Self-IB) for automated extraction, which yielded species abundances highly correlated with the manual comparator method (R 2 > 0.998), indicating comparable performance. The optimized workflow showed satisfactory intra- and inter-batch reproducibility, as evidenced by the high inter-operator correlation (R 2 > 0.99) and stable Shannon diversity index across replicates. Depending on the application requirement—whether prioritizing initial community fidelity or long-term stability under thermal stress—either PBB or PBC can be selected accordingly. Collectively, this study provides an integrated workflow for gut microbiome research, covering sample preservation to nucleic acid extraction, and contributes to improving the reliability and robustness of mNGS data.