Accurate detection of circulating tumor DNA using nanopore consensus sequencing

Marcozzi, Alessio; Jager, Myrthe; Elferink, Martin; Straver, Roy; van Ginkel, Joost H.; Peltenburg, Boris; Chen, Li-Ting; Renkens, Ivo; van Kuik, Joyce; Terhaard, Chris; de Bree, Remco; Devriese, Lot A.; Willems, Stefan M.; Kloosterman, Wigard P.; de Ridder, Jeroen

Levels of circulating tumor DNA (ctDNA) in liquid biopsies may serve as a sensitive biomarker for real-time, minimally-invasive tumor diagnostics and monitoring. However, detecting ctDNA is challenging, as much fewer than 5% of the cell-free DNA in the blood typically originates from the tumor. To detect lowly abundant ctDNA molecules based on somatic variants, extremely sensitive sequencing methods are required. Here, we describe a new technique, CyclomicsSeq, which is based on Oxford Nanopore sequencing of concatenated copies of a single DNA molecule. Consensus calling of the DNA copies increased the base-calling accuracy ∼60x, enabling accurate detection of TP53 mutations at frequencies down to 0.02%. We demonstrate that a TP53-specific CyclomicsSeq assay can be successfully used to monitor tumor burden during treatment for head-and-neck cancer patients. CyclomicsSeq can be applied to any genomic locus and offers an accurate diagnostic liquid biopsy approach that can be implemented in point-of-care clinical workflows.