Accurate detection of circulating tumor DNA using nanopore consensus sequencing
Alessio Marcozzi, Myrthe Jager, Martin Elferink, Roy Straver, Joost H. van Ginkel, Boris Peltenburg, Li‐Ting Chen, Ivo Renkens, Joyce van Kuik, Chris H.J. Terhaard, Remco de Bree, Lot A. Devriese, Stefan M. Willems, Wigard P. Kloosterman, Jeroen de Ridder
Abstract
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 ~60×, 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 clinical workflows.