The most common nonstandard nucleotides found in genomic DNA are ribonucleotides. Although ribonucleotides are frequently incorporated into DNA by replicative DNA polymerases, very little is known about the distribution and signatures of ribonucleotides incorporated into DNA. Recent advances in high-throughput ribonucleotide sequencing can capture the exact locations of ribonucleotides in genomic DNA. Ribose-Map is a user-friendly, standardized bioinformatics toolkit for the comprehensive analysis of ribonucleotide sequencing experiments. It allows researchers to map the locations of ribonucleotides in DNA to single-nucleotide resolution and identify biological signatures of ribonucleotide incorporation. In addition, it can be applied to data generated using any currently available high-throughput ribonucleotide sequencing technique, thus standardizing the analysis of ribonucleotide sequencing experiments and allowing direct comparisons of results. This protocol describes in detail how to use Ribose-Map to analyze ribonucleotide sequencing data, including preparing the reads for analysis, locating the genomic coordinates of ribonucleotides, exploring the genome-wide distribution of ribonucleotides, determining the nucleotide sequence context of ribonucleotides and identifying hotspots of ribonucleotide incorporation. Ribose-Map does not require background knowledge of ribonucleotide sequencing analysis and assumes only basic command-line skills. The protocol requires less than 3 h of computing time for most datasets and ~30 min of hands-on time. Ribose-Map is available at https://github.com/agombolay/ribose-map.

在基因组 DNA 中发现的最常见的非标准核苷酸是核糖核苷酸。尽管核糖核苷酸经常通过复制性 DNA 聚合酶掺入 DNA 中，但对掺入 DNA 中的核糖核苷酸的分布和特征知之甚少。高通量核糖核苷酸测序的最新进展可以捕获基因组 DNA 中核糖核苷酸的确切位置。Ribose-Map 是一个用户友好的标准化生物信息学工具包，用于全面分析核糖核苷酸测序实验。它允许研究人员将 DNA 中核糖核苷酸的位置映射到单核苷酸分辨率，并识别核糖核苷酸掺入的生物学特征。此外，它可以应用于使用任何当前可用的高通量核糖核苷酸测序技术生成的数据，从而使核糖核苷酸测序实验的分析标准化并允许直接比较结果。该协议详细描述了如何使用 Ribose-Map 分析核糖核苷酸测序数据，包括准备用于分析的读数、定位核糖核苷酸的基因组坐标、探索核糖核苷酸的全基因组分布、确定核糖核苷酸的核苷酸序列上下文和识别热点核糖核苷酸掺入。Ribose-Map 不需要核糖核苷酸测序分析的背景知识，只需要基本的命令行技能。对于大多数数据集，该协议需要不到 3 小时的计算时间和约 30 分钟的动手时间。核糖图可在 https://github.com/agombolay/ribose-map 获得。