Nanda Arjun S, Wu Ke, Irkliyenko Iryna, Woo Brian, Ostrowski Megan S, Clugston Andrew S, Sayles Leanne C, Xu Lingru, Satpathy Ansuman T, Nguyen Hao G, Alejandro Sweet-Cordero E, Goodarzi Hani, Kasinathan Sivakanthan, Ramani Vijay
Gladstone Institute for Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA, USA.
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA.
Nat Genet. 2024 Jun;56(6):1300-1309. doi: 10.1038/s41588-024-01748-0. Epub 2024 May 9.
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.
通过加利福尼亚太平洋生物科学公司（PacBio）的单分子测序技术从长的未扩增DNA模板中同时读出序列和碱基修饰需要大量的输入材料。在这里，我们采用Tn5转座来引入发夹寡核苷酸，并对有限量的DNA进行片段化（转座标签化），以生成与PacBio兼容的环状分子。我们开发了两种实现转座标签化的方法，与当前方案相比，其输入量减少了90-99%：（1）转座标签化单分子实时测序（SMRT-Tag），可用于检测遗传变异和CpG甲基化；（2）转座标签化单分子腺嘌呤甲基化寡核小体测序分析（SAMOSA-Tag），利用外源腺嘌呤甲基化增加第三个通道来探测染色质可及性。40 ng或更多的人类DNA（约7000个细胞当量）的SMRT-Tag产生的数据与金标准全基因组测序和亚硫酸氢盐测序相当。30000-50000个细胞核的SAMOSA-Tag解析了患者来源的前列腺癌异种移植中的单纤维染色质结构、CTCF结合和DNA甲基化，并揭示了转移相关的全基因组表观基因组紊乱。因此，转座标签化有望为各种基础和临床应用实现灵敏、可扩展和多模态的单分子基因组学。
