Detecting cell-of-origin and cancer-specific methylation features of cell-free DNA from Nanopore sequencing

Abstract

DNA methylation (5mC) is a promising biomarker for detecting circulating tumor DNA (ctDNA), providing information on a cell's genomic regulation, developmental lineage, and molecular age. Sequencing assays for detecting ctDNA methylation involve pre-processing steps such as immunoprecipitation, enzymatic treatment, or the most common method, sodium bisulfite treatment. These steps add complexity and time that pose a challenge for clinical labs, and bisulfite treatment in particular degrades input DNA and can result in loss of informative ctDNA fragmentation patterns. In this feasibility study, we demonstrate that whole genome sequencing of circulating cell-free DNA using conventional Oxford Nanopore Technologies (ONT) sequencing can accurately detect cell-of-origin and cancer-specific 5mC changes while preserving important fragmentomic information. The simplicity of this approach makes it attractive as a liquid biopsy assay for cancer as well as non-cancer applications in emergency medicine.

Detecting cell-of-origin and cancer-specific methylation features of cell-free DNA from Nanopore sequencing

“…Source code for methylation deconvolution is available at https:// github. com/ methy lgram marlab/ cfdna-ont [47]. An archive of all source code is deposited in Zenodo DOI:10.5281/zenodo.6641763 [48].…”

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

“…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).…”

Enhancing clinical potential of liquid biopsy through a multi-omic approach: A systematic review

“…Finally, a recently developed technological approach for detecting cancer-specific methylation and cancer-associated fragmentation signatures, without disrupting bisulfite conversion, is native Nanopore sequencing (Katsman et al, 2022;Lau et al, 2022). In this assay, PCR-free libraries are read at a single molecule level by passing through nanopores generating an alteration of electric signal which is recorded by the sequencer.…”

“…By abolishing PCR, the biases in molecule quantification are strongly reduced. With this approach it is possible to cover many informative cfDNA CpG sites, even with a shallow coverage, still obtaining significant discrimination on aberrant methylation state (Lau et al, 2022) and retain cell-of-origin information (Katsman et al, 2022).…”