Critical assessment of nanopore sequencing for the detection of multiple forms of DNA modifications.

While nanopore sequencing is increasingly used for mapping DNA modifications, it is important to recognize false positive calls as they can mislead biological interpretations. To assist biologists and methods developers, we describe a framework for rigorous evaluation that highlights the use of false discovery rate with rationally designed negative controls capturing both general background and confounding modifications. Our critical assessment across multiple forms of DNA modifications highlights that while nanopore sequencing performs reliably for high-abundance modifications, including 5-methylcytosine (5mC) at CpG sites in mammalian cells and 5-hydroxymethylcytosine (5hmC) in mammalian brain cells, it makes a significant proportion of false positive detections for low-abundance modifications, such as 5mC at CpH sites, 5hmC and N6-methyldeoxyadenine (6mA) in most mammal cell types. This study highlights the urgent need to incorporate this framework in future methods development and biological studies, and advocates prioritizing nanopore sequencing for mapping abundant over rare modifications in biomedical applications.