A streamlined hybrid-capture and genome-wide multi-omic platform for highly sensitive ctDNA minimal residual disease monitoring.

Background: Circulating tumor DNA (ctDNA) analysis has revolutionized minimal residual disease (MRD) monitoring, but conventional tumor-informed amplicon-based sequencing (AMP) is limited by the narrow variant capacity and diversity. Hybrid capture-based sequencing (HYB) is more versatile and enables both tumor-informed and tumor-naïve liquid biopsy profiling. Methods: We analytically validated the performance of our novel HYB workflow and VarSURE variant calling pipeline, using reference standards (n = 6), plasma samples of cancer patients (n = 75) and healthy donors (n = 90). Genome-wide (GW) non-mutation features including copy number alterations, fragmentomics, and end-motif signatures were also evaluated to enhance ctDNA-MRD detection. Clinical performance was directly compared against our legacy AMP method (K-TRACK, Gene Solutions), using pre-treatment blood samples across multiple cancers (n = 290) and longitudinal cohorts of colorectal cancer (CRC, n = 64), and hepatocellular carcinoma (HCC, n = 47). Results: Optimal parameters to maximize assay performance included single-stranded DNA ligation technology, cfDNA input ≥ 15 ng, post-UMI sequencing depth ≥ 2500X, and high number of tracked mutations. In the tumor-informed setting, the HYB workflow was modestly better than the AMP method in detection of pre-treatment ctDNA; addition of GW features was marginally beneficial except in lung cancer. Surveillance ctDNA determined by the HYB workflow had superior sensitivity to predict recurrence in both CRC (AMP: 90.0%, HYB: 100%) and HCC (AMP: 80.0%, HYB: 96.0%). In the tumor-naïve setting, the performance gap widened significantly, and the combined HYB and GW workflow showed the highest performance in baseline ctDNA detection across all cancers, and achieved sensitivity of 90.0% and 92.0% to detect recurrence in CRC and HCC respectively. Conclusions: The new methodology offers a streamlined and scalable solution for both comprehensive liquid biopsy profiling and longitudinal MRD tracking in routine clinical practice.