美国加州大学旧金山分校Vijay Ramani等研究人员合作发现，天然DNA的直接转位可实现灵敏的多模式单分子测序。相关论文于2024年5月9日在线发表在《自然—遗传学》杂志上。

研究人员利用Tn5转座来引入发夹式寡核苷酸和片段化（标记）有限数量的DNA，以生成与加州太平洋生物科学公司（PacBio）兼容的环状分子。研究人员开发了两种实现标记化（tagmentation）的方法，使用的投入量比现有方案少90-99%：(1) 通过标记化进行单分子实时测序（SMRT-Tag），可检测遗传变异和CpG甲基化；(2) 通过标记化进行单分子腺嘌呤甲基化寡核苷酸测序分析（SAMOSA-Tag），利用外源腺嘌呤甲基化增加第三个通道，以探测染色质的可及性。对40纳克或更多的人类DNA（约7000个细胞当量）进行SMRT-Tag，得到的数据与金标准的全基因组测序和亚硫酸氢盐测序相当。

对30000至50000个细胞核进行的SAMOSA-Tag分析了单纤维染色质结构、CTCF结合和患者来源前列腺癌异种移植物中的DNA甲基化，并发现了与转移相关的全局表观基因组紊乱。因此，标记化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。

据悉，PacBio的单分子测序技术需要大量输入材料，才能同时读出未扩增DNA长模板的序列和碱基修饰。

附：英文原文

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

Author: 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

Issue&Volume: 2024-05-09

Abstract: 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 40ng 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.

DOI: 10.1038/s41588-024-01748-0

Source: https://www.nature.com/articles/s41588-024-01748-0