Direct transposition of native DNA for sensitive multimodal single-molecule sequencing IV

Concurrent readout of sequence and base modifications from long unamplified DNA templates by 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. Chromatin profiling with m6dA methyltransferase footprinting of mESCs, followed by transposase-mediated PCR-free library construction ex situ (SAMOSA-Tag ex situ) and single molecule sequencing.

利用PacBio单分子测序（PacBio single-molecule sequencing）从未扩增的长DNA模板中同时读取序列与碱基修饰信息，需投入大量起始材料。本研究对Tn5转座（Tn5 transposition）进行改造，以引入发夹寡核苷酸（hairpin oligonucleotides），并对有限量DNA实施转座酶标签片段化（tagmentation），从而生成适配PacBio测序的环状分子。我们开发了两种实现转座片段化的方法，其所需起始材料较现有方案减少90%~99%：其一为转座酶标签片段化单分子实时测序（SMRT-Tag），可用于检测遗传变异与CpG甲基化；其二为转座酶标签片段化单分子腺嘌呤甲基化寡核小体测序分析（SAMOSA-Tag），该方法借助外源腺嘌呤甲基化开辟第三检测通道，用以探究染色质可及性。针对40 ng及以上的人类DNA（约7000个细胞当量）开展SMRT-Tag测序，所得数据可与金标准全基因组测序及亚硫酸氢盐测序相媲美。对30000~50000个细胞核进行SAMOSA-Tag测序，可解析患者来源前列腺癌异种移植模型中的单纤维染色质结构、CTCF结合位点与DNA甲基化，并揭示了转移相关的全局表观基因组紊乱。因此，转座酶标签片段化技术有望为多样化的基础与临床应用提供灵敏、可扩展且多模态的单分子基因组学研究方案。以m6dA甲基转移酶（m6dA methyltransferase）足迹分析对小鼠胚胎干细胞（mESCs）开展染色质谱分析（chromatin profiling），随后进行转座酶介导的体外无PCR文库构建（SAMOSA-Tag ex situ）与单分子测序。