Yang Yao, Scott Stuart A
Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1498, New York, NY, 10029, USA.
Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1497, New York, NY, 10029, USA.
Methods Mol Biol. 2017;1654:125-134. doi: 10.1007/978-1-4939-7231-9_8.
For the past two decades, bisulfite sequencing has been a widely used method for quantitative CpG methylation detection of genomic DNA. Coupled with PCR amplicon cloning, bisulfite Sanger sequencing allows for allele-specific CpG methylation assessment; however, its time-consuming protocol and inability to multiplex has recently been overcome by next-generation bisulfite sequencing techniques. Although high-throughput sequencing platforms have enabled greater accuracy in CpG methylation quantitation as a result of increased bisulfite sequencing depth, most common sequencing platforms generate reads that are similar in length to the typical bisulfite PCR size range (~300-500 bp). Using the Pacific Biosciences (PacBio) sequencing platform, we developed single molecule real-time bisulfite sequencing (SMRT-BS), which is an accurate targeted CpG methylation analysis method capable of a high degree of multiplexing and long read lengths. SMRT-BS is reproducible and was found to be concordant with other lower throughput quantitative CpG methylation methods. Moreover, the ability to sequence up to ~1.5-2.0 kb amplicons, when coupled with an optimized bisulfite-conversion protocol, allows for more thorough assessment of CpG islands and increases the capacity for studying the relationship between single nucleotide variants and allele-specific CpG methylation.
在过去二十年中，亚硫酸氢盐测序一直是用于基因组DNA定量CpG甲基化检测的广泛使用的方法。结合PCR扩增子克隆，亚硫酸氢盐桑格测序允许进行等位基因特异性CpG甲基化评估；然而，其耗时的操作流程和无法多重分析的缺点最近已被新一代亚硫酸氢盐测序技术克服。尽管高通量测序平台由于亚硫酸氢盐测序深度增加而在CpG甲基化定量方面实现了更高的准确性，但大多数常见的测序平台产生的读长与典型的亚硫酸氢盐PCR大小范围（约300-500bp）相似。我们使用太平洋生物科学公司（PacBio）的测序平台开发了单分子实时亚硫酸氢盐测序（SMRT-BS），这是一种准确的靶向CpG甲基化分析方法，能够进行高度多重分析并具有长读长。SMRT-BS具有可重复性，并且被发现与其他较低通量的定量CpG甲基化方法一致。此外，当与优化的亚硫酸氢盐转化方案相结合时，对长达约1.5-2.0kb扩增子进行测序的能力允许对CpG岛进行更全面的评估，并增加了研究单核苷酸变异与等位基因特异性CpG甲基化之间关系的能力。
