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 Terhaard; Remco de Bree; Lot A Devriese; Stefan M Willems; Wigard P Kloosterman; Jeroen de Ridder

doi:10.1038/s41525-021-00272-y

Accurate detection of circulating tumor DNA using nanopore consensus sequencing

NPJ Genom Med. 2021 Dec 9;6(1):106. doi: 10.1038/s41525-021-00272-y.

Authors

Alessio Marcozzi^#^{1

2}, Myrthe Jager^#¹, Martin Elferink³, Roy Straver¹, Joost H van Ginkel^{4

5}, Boris Peltenburg^{6

7}, Li-Ting Chen¹, Ivo Renkens¹, Joyce van Kuik⁴, Chris Terhaard⁶, Remco de Bree⁷, Lot A Devriese⁸, Stefan M Willems^{4

9}, Wigard P Kloosterman^{10

11}, Jeroen de Ridder^{12

13}

Affiliations

¹ Center for Molecular Medicine and Oncode Institute, University Medical Center Utrecht, Utrecht University, Utrecht, CX, The Netherlands.
² Cyclomics, Utrecht, CG, The Netherlands.
³ Department of Genetics, University Medical Center Utrecht, Utrecht University, Utrecht, CX, The Netherlands.
⁴ Department of pathology, University Medical Center Utrecht, Utrecht University, Utrecht, CX, The Netherlands.
⁵ Department of Oral and Maxillofacial Surgery, University Medical Center Utrecht, Utrecht University, Utrecht, CX, The Netherlands.
⁶ Department of Radiotherapy, UMC Utrecht Cancer Center, University Medical Center Utrecht, Utrecht, The Netherlands.
⁷ Department of Head and Neck Surgical Oncology, UMC Utrecht Cancer Center, University Medical Center Utrecht, Utrecht, The Netherlands.
⁸ Department of Medical Oncology, UMC Utrecht Cancer Center, University Medical Center Utrecht, Utrecht, The Netherlands.
⁹ Department of Pathology and Medical Biology, University Medical Center Groningen, Rijksuniversiteit Groningen, Groningen, GZ, The Netherlands.
¹⁰ Cyclomics, Utrecht, CG, The Netherlands. wigard@cyclomics.com.
¹¹ Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, CX, The Netherlands. wigard@cyclomics.com.
¹² Center for Molecular Medicine and Oncode Institute, University Medical Center Utrecht, Utrecht University, Utrecht, CX, The Netherlands. J.deridder-4@umcutrecht.nl.
¹³ Cyclomics, Utrecht, CG, The Netherlands. J.deridder-4@umcutrecht.nl.

^# Contributed equally.

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.