A novel one-step multiplex PCR method (OS-MPCR) for efficient NGS library preparation in cancer
- 1Department of Pathology, Zibo First Hospital, Zibo, Shandong, China
- 2Shanghai Yijian Health Technology Co., Ltd., Shanghai, China
Abstract
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.
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BibTeX
@article{feng2026novel,
title = {{A novel one-step multiplex PCR method (OS-MPCR) for efficient NGS library preparation in cancer}},
author = {Haiyan Feng and Xiao Hu and Lei Zhang and Qi Zhang and Shuocun Wang and Bingqing Lv and Yufeng Zhang and Dong Shi and Ping Zhou},
journal = {PLOS One},
year = {2026},
month = sep,
doi = {10.1371/journal.pone.0357776},
url = {https://doi.org/10.1371/journal.pone.0357776},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - A novel one-step multiplex PCR method (OS-MPCR) for efficient NGS library preparation in cancer
AU - Haiyan Feng
AU - Xiao Hu
AU - Lei Zhang
AU - Qi Zhang
AU - Shuocun Wang
AU - Bingqing Lv
AU - Yufeng Zhang
AU - Dong Shi
AU - Ping Zhou
PY - 2026
DA - 2026/09/15
JO - PLOS One
DO - 10.1371/journal.pone.0357776
UR - https://doi.org/10.1371/journal.pone.0357776
AB - 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.
SN - 1932-6203
ER -