Cell-free DNA (cfDNA) sequencing with Nanopore technology allows direct interrogation of genetic information in circulating DNA without PCR amplification. This real-time protocol enables methylation status analysis. It offers insights into cfDNA’s genomic landscape, benefiting clinical diagnostics like cancer detection, treatment monitoring, and minimal residual disease identification. The protocol involves cfDNA extraction, library preparation, loading onto a Nanopore sequencing device, real-time sequencing, and data analysis for variant calling, copy number and methylation status analysis.
The library preparation involves repairing DNA ends, dA-tailing, and ligating sequencing adapters. The kit ensures high sequencing accuracies (Q20+) on nanopore Flowcells R10.4.1, with updates for enhanced DNA capture and fuel fix technology for longer runs. The protocol is optimized for short DNA fragments recovery, based on a modified long-reads protocol.