Nanopore-based consensus sequencing enables accurate multimodal tumor cell-free DNA profiling

Shallow genome-wide cell-free DNA (cfDNA) sequencing holds great promise for non-invasive cancer monitoring by providing reliable copy number alteration (CNA) and fragmentomic profiles. Single nucleotide variations (SNVs) are, however, much harder to identify with low sequencing depth due to sequencing errors. Here we present Nanopore Rolling Circle Amplification (RCA)-enhanced Consensus Sequencing (NanoRCS), which leverages RCA and consensus calling based on genome-wide long-read nanopore sequencing to enable simultaneous multimodal tumor fraction estimation through SNVs, CNAs, and fragmentomics. Efficacy of NanoRCS is tested on 18 cancer patient samples and 3 healthy controls, demonstrating its ability to reliably detect tumor fractions as low as 0.24%. In vitro experiments confirm that SNV measurements are essential for detecting tumor fractions below 2%. NanoRCS provides the opportunity for cost-effective and rapid processing, which aligns well with clinical needs, particularly in settings where quick and accurate cancer monitoring is essential for personalized treatment strategies. ### Competing Interest Statement AM, DM, WK and JdR declare competing financial interests. AM, WK and JdR filed patents and founded a company (Cyclomics) related to the technique. AM, DM, WK and JdR work (partially) at Cyclomics. FM is co-inventor on patents related to cfDNA analysis and has consulted for RocheDX. HvL has consultant or advisory role at Amphera, Anocca, Astellas, AstraZeneca, Beigene, Boehringer, Daiichi-Sankyo, Dragonfly, MSD, Myeloid, Servier Research funding, medication supply, and/or other research support at Auristone, Incyte, Merck, ORCA, Servier and speaker role for: Astellas, Beigene, Benecke, BMS, Daiichi-Sankyo, JAAP, Medtalks, Novartis, Springer, Travel Congress Management B.V.. SD has a consultant or advisory role for BMS (related to checkpoint inhibitors); research funding and medication supply, from Incyte (related to checkpoint inhibitors); and speaker roles for Servier, BMS, and Benecke. The other authors declare no conflicts of interest.