Recent advances in the detection of base modifications using the Nanopore sequencer

Abstract

DNA and RNA modifications have important functions, including the regulation of gene expression. Existing methods based on short-read sequencing for the detection of modifications show difficulty in determining the modification patterns of single chromosomes or an entire transcript sequence. Furthermore, the kinds of modifications for which detection methods are available are very limited. The Nanopore sequencer is a single-molecule, long-read sequencer that can directly sequence RNA as well as DNA. Moreover, the Nanopore sequencer detects modifications on long DNA and RNA molecules. In this review, we mainly focus on base modification detection in the DNA and RNA of mammals using the Nanopore sequencer. We summarize current studies of modifications using the Nanopore sequencer, detection tools using statistical tests or machine learning, and applications of this technology, such as analyses of open chromatin, DNA replication, and RNA metabolism.

Fig. 1

Modified base detection using Nanopore sequencing and general methods. Schema of modified base detection using the Nanopore sequencer (a) and through bisulfite conversion (b), immunoprecipitation of nucleic acids (c), and SMRT sequencing (d)

Fig. 2

Tools for modified base detection using Nanopore sequencing. Differences in the squiggles (a) and mismatch rates (b) of an artificial bromouridine base are shown. The direct RNA-seq reads of synthetic RNA were aligned using minimap2 [58]. The squiggle plot was drawn using Tombo [57]. The aligned reads were visualized using IGV [79]

Fig. 3

Applied methods for modified base detection using Nanopore sequencing. a Detection of open chromatin regions using DNA methyltransferase accessibility. DNA in open chromatin regions is specifically methylated. Open chromatin regions are detected as highly methylated regions by Nanopore sequencing. b Detection of DNA replications using base analogs. The replicated regions are labeled by pulse labeling during S phase. The replicated regions are detected as labeled regions by Nanopore sequencing. c Detection of nascent RNA using base analogs. Nascent RNAs are labeled by pulse labeling using base analogs. Nascent and preexisting RNAs are classified by the existence of base analogs