Accurate detection of circulating tumor DNA using nanopore consensus sequencing

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 ~60x, enabling accurate detection of ​TP53 mutations at frequencies down to 0.02%. We demonstrate that 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.

Real-time analysis of the cancer genome and fragmentome from plasma and urine short and long cell-free DNA using Nanopore sequencing

“…Although not affecting the detection of large genomic events or fragmentomic analysis, one drawback of ONT sequencing is the single read base accuracy, which is currently inferior to that of conventional short-read approaches, and can affect SNV calling. Alternative approaches are under development to improve the recovery of SNV from plasma cfDNA using ONT sequencing (Marcozzi et al, 2021). Moreover, the pre-analytical conditions that are affecting cfDNA short-read sequencing and fragmentomic analysis could alter differently longer cfDNA fragments (van der Pol et al, 2022).…”

“…Nanopore sequencing using the ONT platform is a highly portable and deployable technology that is capable of sequencing quickly any length of amplified or native DNA fragment or even directly RNA molecules (Euskirchen et al, 2017; Katsman et al, 2022). Due to the higher rate of sequencing error in comparison to short-read sequencers, the ONT platform has rarely been used for cfDNA mutation analysis (Cheng et al, 2015; Marcozzi et al, 2021). However, preliminary works show that broader genomic events, like copy number aberrations, can be recovered and analyzed with ONT Nanopore sequencing of plasma cfDNA (Martignano et al, 2021).…”