How to improve the performance of circulating tumor DNA (ctDNA) signal acquisition and the accuracy to authenticate ultra low-frequency mutation are major challenges of minimal residual disease (MRD) detection in solid tumors. In this study, we developed a new MRD bioinformatics algorithm, namely multi-variant joint confidence analysis (MinerVa), and tested this algorithm both in contrived ctDNA standards and plasma DNA samples of patients with early non-small cell lung cancer (NSCLC). Our results showed that the specificity of multi-variant tracking of MinerVa algorithm ranged from 99.62% to 99.70%, and when tracking 30 variants, variant signals could be detected as low as 6.3 × 10?5 variant abundance. Furthermore, in a cohort of 27 NSCLC patients, the specificity of ctDNA-MRD for recurrence monitoring was 100%, and the sensitivity was 78.6%. These findings indicate that the MinerVa algorithm can efficiently capture ctDNA signals in blood samples and exhibit high accuracy in MRD detection.
Objective To explore a new method of extending circulating tumor DNA (ctDNA) by adapter ligation to adapt it to nanopore sequencing. Methods A RC adapter was designed to extend ctDNA fragments, and reaction conditions including end repair, dA-tailing, and ligation were optimized. A dual-barcode and RC-adapter-based sample splitting workflow was established, leading to the development of a novel nanopore sequencing based method for ctDNA methylation detection, named RCnano. Results Agarose gel electrophoresis and sequencing results showed that the 48 bp adapter was the optimal length for RCnano. Secondary sample splitting based on RC adapter sequences recovered 42% of unclassified reads, equivalent to a 4%-5% increase in total data yield. Compared with standard nanopore sequencing library preparation, the optimized RCnano workflow increased sequencing output by approximately 6-fold. Among four nanopore methylation callers, Nanopolish performed best, with a mean absolute error of 0.021. RCnano showed good correlation with bisulfite amplicon sequencing (r2=0.952) and was able to detect methylation sites at an abundance as low as 0.1%. Conclusions This study demonstrates a novel application of nanopore sequencing for ultrasensitive ctDNA methylation analysis, which could enhance noninvasive cancer diagnostics and real-time epigenetic monitoring.