A low-cost, ultra-fast, low-input amplicon NGS workflow for rapid cancer profiling

Background: Liquid biopsy-based analysis of cell-free DNA (cfDNA) from plasma and urine has emerged as a powerful, non-invasive strategy for identifying clinically actionable oncogenic mutations. However, rapid and accurate detection of low-frequency variants remains technically challenging, particularly when working with limited input DNA. Predicine has developed an amplicon-based NGS platform to overcome these barriers through high analytical sensitivity, superior error suppression, and an accelerated workflow to support precision oncology applications. Methods: A streamlined amplicon-based NGS workflow was established for sensitive detection of clinically relevant alterations in FGFR (16 variants) , PIK3CA (62 variants) , ESR1 (25 variants) , KRAS (21 variants), EGFR (62 variants) as well as additional variants commonly implicated in breast, prostate, bladder, lung and GIST cancers. The assay leverages Predicine's proprietary error-correction technology to suppress sequencing artifacts and enhance variant-calling accuracy. Sequencing-ready libraries were generated within three hours using a protocol optimized for low-input cfDNA. Analytical performance, including sensitivity and specificity, was systematically evaluated. Results: This study demonstrated high analytical performance for detecting mutations in FGFR, PIK3CA, ESR1, KRAS, EGFR, and other clinically actionable targets across plasma and urine cfDNA. The streamlined workflow delivered ≥98% uniform coverage and >95% on-target rates across all target regions. Low-frequency variants were consistently detected at allele frequencies as low as ≤0.25% using 30 ng of cfDNA. The assay features a rapid library preparation process with a short hands-on time, enabling sequencing-ready samples to be generated within three hours, further supporting fast turnaround for liquid biopsy analysis. Specificity exceeded 98% in healthy donor cfDNA samples, indicating minimal background noise and low false-positive rates. While the evaluation focused on liquid biopsy specimens, the same rapid and efficient workflow is also compatible with FFPE-derived DNA, extending its applicability to tissue-based mutation profiling. Conclusions: We report a low-cost, rapid, and high-throughput NGS solution for cfDNA analysis from plasma, urine and FFPE tissue DNA, offering a unified approach for mutation profiling across both liquid biopsy and tissue samples. This flexibility, combined with exceptional sensitivity, minimized error rates, low DNA input requirements, and fast turnaround time, positions this amplicon-based NGS assay as a powerful tool for clinical research, cancer diagnostics, and longitudinal disease monitoring.