The Oxford Nanopore (ONT) platform provides portable and rapid genome sequencing, and its ability to natively profile DNA methylation without complex sample processing is attractive for point-of-care real-time sequencing. We recently demonstrated ONT shallow whole-genome sequencing to detect copy number alterations (CNAs) from the circulating tumor DNA (ctDNA) of cancer patients. Here, we show that cell type and cancer-specific methylation changes can also be detected, as well as cancer-associated fragmentation signatures. This feasibility study suggests that ONT shallow WGS could be a powerful tool for liquid biopsy. Graphical Abstract

Base editors are dedicated engineered deaminases that enable directed conversion of specific bases in the genome or transcriptome in a precise and efficient manner, and hold promise for correcting pathogenic mutations. A major concern limiting application of this powerful approach is the issue of off-target edits. Several recent studies have shown substantial off-target RNA activity induced by base editors and demonstrated that off-target mutations may be suppressed by improved deaminases versions or optimized guide RNAs. Here we describe a new class of off-target events that are invisible to the established methods for detection of genomic variations, and were thus far overlooked. We show that much of the off-target activity of the deaminases is nonspecific, seemingly stochastic, affecting a large number of sites throughout the genome or the transcriptome and accounting for the majority of off-target activity. We develop and employ a different, complementary, approach that is sensitive to the stochastic off-targets activity, and use it to quantify the abundant off-target RNA mutations due to current optimized deaminase editors. Engineered base editors enable directed manipulation of the genome or transcriptome at single-base resolution. We believe that implementation of this computational approach would facilitate design of more specific base editors. We provide a computational tool to quantify global off-target activity, which can be used to optimize future base editors.

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.

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