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.
