A novel one-step multiplex PCR method (OS-MPCR) for efficient NGS library preparation in cancer

Background Next-generation sequencing (NGS) is essential for precision oncology, yet its clinical implementation is often hindered by limited sample quantity, complex workflows, and cross-contamination risks. We evaluated a modified One-Step Multiplex PCR (OS-MPCR) assay designed to streamline library construction while maintaining high analytical sensitivity and diagnostic accuracy. Methods The OS-MPCR method integrates adapter ligation and library amplification into a single, closed-tube reaction, eliminating intermediate open-tube manipulations. The analytical performance was evaluated using standard reference materials targeting SNVs, Indels, and fusions. Clinical validation was performed on retrospective formalin-fixed paraffin-embedded (FFPE) specimens (DNA, N = 76; RNA, N = 34) from lung cancer patients, using well-established liquid-phase hybrid capture assays as the clinical verification standard. Results OS-MPCR streamlined the workflow, reducing hands-on time to just 20 minutes. The assay enabled reliable variant detection at 1% frequency with as little as 1 ng of DNA for SNVs/Indels and 5 ng of total RNA for fusions, with low-input quantification limits inherently bounded by absolute molecular copy numbers. In the clinical cohort, OS-MPCR demonstrated 100% diagnostic concordance with the verification method. Quantitative analysis revealed a strong correlation in variant allele frequencies (Spearman r = 0.9777, 95% CI: 0.9668–0.9851, P < 0.0001), with libraries consistently achieving highly reproducible yields and stable, near 1.0 target rates across the entire cohort. Conclusion OS-MPCR offers a streamlined and low-input alternative to conventional multi-step multiplex PCR methods. By combining reduced hands-on time and intrinsic contamination control with high analytical accuracy, this approach represents a practical refinement for targeted amplicon sequencing in diagnostic workflows.