Nanopore long-read sequencing technology greatly expands the capacity of long-range, single-molecule DNA-modification detection. A growing number of analytical tools have been developed to detect DNA methylation from nanopore sequencing reads. Here, we assess the performance of different methylation-calling tools to provide a systematic evaluation to guide researchers performing human epigenome-wide studies. 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. 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. The seven tools exhibit different performances across the evaluation criteria. 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. Furthermore, we demonstrate that 5hmC levels at least partly contribute to the discrepancy between bisulfite and nanopore sequencing. Lastly, we provide an online DNA methylation database ( https://nanome.jax.org ) to display the DNA methylation levels detected by nanopore sequencing and bisulfite sequencing data across different genomic contexts. Our study is the first systematic benchmark of computational methods for detection of mammalian whole-genome DNA modifications in nanopore sequencing. 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.

中文翻译：

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用于牛津纳米孔测序的 DNA 甲基化调用工具：一项调查和人类表观基因组范围的评估

纳米孔长读长测序技术极大地扩展了长距离、单分子DNA修饰检测的能力。越来越多的分析工具被开发出来，用于从纳米孔测序读数中检测 DNA 甲基化。在这里，我们评估不同甲基化调用工具的性能，以提供系统评估来指导研究人员进行人类表观基因组范围的研究。我们比较了七种分析工具，用于从全基因组规模的人类天然 DNA 生成的纳米孔长读长测序数据中检测 DNA 甲基化。我们评估了不同基因组背景下 CpG 甲基化预测的每次读取和每个位点性能、CpG 位点覆盖率以及每个工具消耗的计算资源。这七个工具在评估标准中表现出不同的性能。我们发现，对于 DNA 甲基化模式不一致的区域、基因间区域、低 CG 密度区域和重复区域的甲基化预测，所有工具都显示出改进的空间。此外，我们证明 5hmC 水平至少部分导致亚硫酸氢盐和纳米孔测序之间的差异。最后，我们提供了一个在线 DNA 甲基化数据库 (https://nanome.jax.org)，用于显示不同基因组背景下通过纳米孔测序和亚硫酸氢盐测序数据检测到的 DNA 甲基化水平。我们的研究是纳米孔测序中检测哺乳动物全基因组 DNA 修饰的计算方法的第一个系统基准。我们为跨平台标准化以及为使用纳米孔测序进行基因组规模修饰碱基检测而设计的分析工具的评估提供了广泛的基础。