小柯机器人

美国哈佛大学麻省总医院Bradley E. Bernstein和Volker Hovestadt小组合作研发了多样本和单细胞中基因调控元件的亚硫酸氢盐扩展表达测序。这一研究成果发表在2021年 5月6出版的国际学术期刊《自然-生物技术》上。

在本研究，研究人员研发了针对DNA甲基化区域分析的扩展表示亚硫酸氢盐测序（XRBS）方法。该设计在启动子、增强子和CTCF结合位点实现了扩展的调控元件覆盖范围和CpG二核苷酸丰富信号的平衡。在亚硫酸氢盐转化之前，将条形码化DNA片段聚集，从而可以在低样品条件下进行多重处理并保持参数一致性。XRBS在单个白血病细胞中的应用使研究人员能够评估单个细胞的基因拷贝数变异和甲基化改变。

研究分析表明，细胞间异染色质H3K9me3区的甲基化具有最高的变异性，这可能反映了这些晚期复制区的固有表观遗传学不稳定性，并且在采样时细胞所处的细胞周期也有所不同。

据悉，DNA甲基化的生物学作用已经通过全基因组或减少差异的亚硫酸氢盐测序分析方法得以阐明，但是这些方法不适用于哺乳动物基因组中的大量非编码调控元件。

附：英文原文

Title: Extended-representation bisulfite sequencing of gene regulatory elements in multiplexed samples and single cells

Author: Sarah J. Shareef, Samantha M. Bevill, Ayush T. Raman, Martin J. Aryee, Peter van Galen, Volker Hovestadt, Bradley E. Bernstein

Issue&Volume: 2021-05-06

Abstract: The biological roles of DNA methylation have been elucidated by profiling methods based on whole-genome or reduced-representation bisulfite sequencing, but these approaches do not efficiently survey the vast numbers of non-coding regulatory elements in mammalian genomes. Here we present an extended-representation bisulfite sequencing (XRBS) method for targeted profiling of DNA methylation. Our design strikes a balance between expanding coverage of regulatory elements and reproducibly enriching informative CpG dinucleotides in promoters, enhancers and CTCF binding sites. Barcoded DNA fragments are pooled before bisulfite conversion, allowing multiplex processing and technical consistency in low-input samples. Application of XRBS to single leukemia cells enabled us to evaluate genetic copy number variations and methylation variability across individual cells. Our analysis highlights heterochromatic H3K9me3 regions as having the highest cell-to-cell variability in their methylation, likely reflecting inherent epigenetic instability of these late-replicating regions, compounded by differences in cell cycle stages among sampled cells. DNA methylation is efficiently profiled in non-coding regulatory sequences in single cells.

DOI: 10.1038/s41587-021-00910-x

Source: https://www.nature.com/articles/s41587-021-00910-x

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