Modification mapping by nanopore sequencing

Abstract

Next generation sequencing (NGS) has provided biologists with an unprecedented view into biological processes and their regulation over the past 2 decades, fueling a wave of development of high throughput methods based on short read DNA and RNA sequencing. For nucleic acid modifications, NGS has been coupled with immunoprecipitation, chemical treatment, enzymatic treatment, and/or the use of reverse transcriptase enzymes with fortuitous activities to enrich for and to identify covalent modifications of RNA and DNA. However, the majority of nucleic acid modifications lack commercial monoclonal antibodies, and mapping techniques that rely on chemical or enzymatic treatments to manipulate modification signatures add additional technical complexities to library preparation. Moreover, such approaches tend to be specific to a single class of RNA or DNA modification, and generate only indirect readouts of modification status. Third generation sequencing technologies such as the commercially available “long read” platforms from Pacific Biosciences and Oxford Nanopore Technologies are an attractive alternative for high throughput detection of nucleic acid modifications. While the former can indirectly sense modified nucleotides through changes in the kinetics of reverse transcription reactions, nanopore sequencing can in principle directly detect any nucleic acid modification that produces a signal distortion as the nucleic acid passes through a nanopore sensor embedded within a charged membrane. To date, more than a dozen endogenous DNA and RNA modifications have been interrogated by nanopore sequencing, as well as a number of synthetic nucleic acid modifications used in metabolic labeling, structure probing, and other emerging applications. This review is intended to introduce the reader to nanopore sequencing and key principles underlying its use in direct detection of nucleic acid modifications in unamplified DNA or RNA samples, and outline current approaches for detecting and quantifying nucleic acid modifications by nanopore sequencing. As this technology matures, we anticipate advances in both sequencing chemistry and analysis methods will lead to rapid improvements in the identification and quantification of these epigenetic marks.

Long-Read Structural and Epigenetic Profiling of a Kidney Tumor-Matched Sample with Nanopore Sequencing and Optical Genome Mapping

“…Accurately detecting DNA modifications with ONT requires training data encompassing the modification across diverse sequence contexts. Such datasets can be generated through experimental validation with established methods or in-situ approaches (as for 5mC) (White and Hesselberth 2022). Generating in-situ 5hmC residues is chemically challenging compared to 5mC, and the Remora-based 5hmC caller hasn’t been benchmarked and peer-reviewed to date.…”

“…In OGM, an additional color may be used to chemically tag modifications of interest and create a hybrid genetic/epigenetic physical map of the molecules (Jeffet et al 2021; Heck et al 2019; Sharim et al 2019; Gabrieli et al 2018; Margalit et al 2021). ONT, on the other hand, does not require any additional preparative steps for calling epigenetic modifications as it relies solely on the electrical contrast generated by the native chemical structure of the modified base (White and Hesselberth 2022). Nevertheless, accurate modification calling requires a complete training set, which is not trivial for most base modifications.…”

Direct sequencing of totalSaccharomyces cerevisiaetRNAs by LC–MS/MS

“…While we apply this bottom-up RNA sequencing platform to confirm previously identified modified positions within S. cerevisiae total tRNA, this assay can be further implemented for de novo identification of previously undescribed modified sites. Particularly, this bottom-up RNA sequencing pipeline can be used in tandem with RNA-seq ( Motorin and Marchand 2021 ; Watkins et al 2022 ), direct nanopore sequencing (DRS) ( Thomas et al 2021 ; White and Hesselberth 2022 ), and global ribonucleoside modification profiling (GRMP) ( Jones et al 2023a ) to provide an overarching view of what modifications are present, where they are present, and how abundant the modifications are. Currently, the tRNA modification landscapes of many model organisms remain uncharacterized (e.g., M. musculus and D. melanogaster ) ( Boccaletto et al 2018 ) despite the increasing recognition that tRNA modifications play significant roles in cellular stress and disease ( Huber et al 2019 ; Suzuki 2021 ).…”

Nanopore Direct RNA Sequencing for Modified Uridine Nucleotides Yields Signals Dependent on the Physical Properties of the Modified Base

“…The addition of more negative charge, which we studied with FU, ho 5 U, and 6‐azaU, can yield broad ionic current levels, as observed for 6‐azaU but not for FU or ho 5 U, which is not easily predicted without conducting the experiment. Application of modification‐aware base call algorithms is a proposed solution to sequence for RNA modifications; [47] however, there exist >140 known modifications, and the chance each produces a unique nanopore sequencing signature that a computer can deconvolute is questionable. The utility of adducts to shift the signal of a target RNA modification when sequenced and compared to a control can directly permit differentiation of two modifications in which they have similar nanopore signatures.…”