AM‑DMF-Lib-prep: A clinical ready lib-prep-on-a-chip platform with active-matrix digital microfluidics

Clinical adoption of metagenomic next‑generation sequencing (mNGS) is limited by costly, labor-intensive library preparation requiring substantial DNA input. Here, we present an active‑matrix digital microfluidics library preparation platform (AM‑DMF-Lib-prep) that overcomes these barriers by automating the “sample‑in, library‑out” workflow in an enclosed, digitalized lab-on-a-chip system. The platform accommodates a wide DNA input range (1–25 ng), producing high-quality libraries with exceptional reproducibility (inter-chip R² = 0.9998) and 25.6% lower variability than conventional manual methods. It achieves ~90% reagent reduction while enhancing detection sensitivity for low-abundance pathogens by 43.7–76.7% in microbial community standard spike-in sample. Validation across 94 diverse clinical specimens confirmed high concordance with reference diagnostics. Collectively, AM‑DMF-Lib-prep establishes a precise, cost‑effective, and scalable solution with demonstrated clinical readiness, directly addressing the pre‑analytical bottleneck in routine mNGS.