Thus, the requirement for high coverage and inability of direct single-molecule 5mC detection by SMRT is a limitation. However, this detection is not the result of direct 5mC detection at single-molecule resolution but rather the aggregation of the subtle impact of 5mC on polymerase kinetics signals during DNA synthesis. SMRT sequencing can detect 5mC modifications based on polymerase kinetics at 250× coverage.
Recently, third-generation sequencing technologies, including single-molecule real-time (SMRT) sequencing by Pacific Biosciences (PacBio), and nanopore sequencing by Oxford Nanopore Technologies (ONT), have overcome the read-length limitation to achieve ultra-long read, single-base detection at a genome-wide level. DNA methylation measurement has traditionally depended on the combination of bisulfite conversion (which can damage DNA) and next-generation sequencing (which detects only short-range methylation patterns). Several DNA modifications, such as N6-methyladenine (6 mA), N4-methylcytosine (4mC), and 5-methylcytosine (5mC) and its oxidative derivatives, i.e., 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), are diversely distributed in genomes and play important roles in genomic imprinting, chromatin-structure modulation, transposon inactivation, stem cell pluripotency and differentiation, inflammation, and transcription-repression regulation. We provide a broad foundation for cross-platform standardization and an evaluation of analytical tools designed for genome-scale modified base detection using nanopore sequencing.ĭNA methylation, the process by which methyl groups are added to DNA molecules, is a fundamental epigenetic modification process in gene transcription regulation. Our study is the first systematic benchmark of computational methods for detection of mammalian whole-genome DNA modifications in nanopore sequencing. Lastly, we provide an online DNA methylation database ( ) to display the DNA methylation levels detected by nanopore sequencing and bisulfite sequencing data across different genomic contexts.
Furthermore, we demonstrate that 5hmC levels at least partly contribute to the discrepancy between bisulfite and nanopore sequencing. We show that the methylation prediction at regions with discordant DNA methylation patterns, intergenic regions, low CG density regions, and repetitive regions show room for improvement across all tools. The seven tools exhibit different performances across the evaluation criteria. We evaluate the per-read and per-site performance of CpG methylation prediction across different genomic contexts, CpG site coverage, and computational resources consumed by each tool. We compare seven analytic tools for detecting DNA methylation from nanopore long-read sequencing data generated from human natural DNA at a whole-genome scale. Here, we assess the performance of different methylation-calling tools to provide a systematic evaluation to guide researchers performing human epigenome-wide studies. A growing number of analytical tools have been developed to detect DNA methylation from nanopore sequencing reads.
Nanopore long-read sequencing technology greatly expands the capacity of long-range, single-molecule DNA-modification detection.
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