Concurrent readout of sequence and base modifications from long unamplified DNA templates by Pacific Biosciences of California (PacBio) single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90–99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000–50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.

加利福尼亚太平洋生物科学公司 (PacBio) 单分子测序从长的未扩增 DNA 模板中同时读取序列和碱基修饰需要大量输入材料。在这里，我们采用 Tn5 转座来引入发夹寡核苷酸和限制数量的 DNA 片段（标签），以生成 PacBio 兼容的环状分子。我们开发了两种实现标签化的方法，并且比当前方案使用的输入量减少 90-99%：（1）通过标签化进行单分子实时测序（SMRT-Tag），它可以检测遗传变异和 CpG 甲基化； (2) 通过标记进行单分子腺嘌呤甲基化寡核小体测序测定 (SAMOSA-Tag)，它使用外源腺嘌呤甲基化添加第三个通道来探测染色质可及性。 40 ng 或更多人类 DNA（约 7,000 个细胞当量）的 SMRT-Tag 产生的数据可与金标准全基因组和亚硫酸氢盐测序相媲美。 30,000-50,000 个细胞核的 SAMOSA-Tag 解析了患者来源的前列腺癌异种移植物中的单纤维染色质结构、CTCF 结合和 DNA 甲基化，并发现了与转移相关的整体表观基因组紊乱。因此，标记有望为各种基础和临床应用提供灵敏、可扩展和多模式的单分子基因组学。