Legault Lisa-Marie, Chan Donovan, McGraw Serge
CHU Sainte-Justine Research Center, Montréal, Quebec, Canada.
Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec, Canada.
Bio Protoc. 2019 Feb 20;9(4):e3171. doi: 10.21769/BioProtoc.3171.
DNA methylation is a common mechanism of epigenetic regulation involved in transcriptional modulation and genome stability. With the evolution of next-generation sequencing technologies, establishing quantitative genome-wide DNA methylation profiles is becoming routine in many laboratories. However, many of these approaches take several days to accomplish and use subjective PCR methods to amplify sequencing libraries, which can induce amplification bias. Here we propose a rapid Reduced Representation Bisulfite Sequencing (rRRBS) protocol to minimize PCR amplification bias and reduce total time of multiplexed library construction. In this modified approach, the precise quantification of the final library amplification step is accomplished and monitored by qPCR, instead of using standard PCR and gel electrophoresis, to determine the appropriate number of cycles to perform. The main advantages of this rRRBS method are: i) Reduced amount of amplification enzyme used for library prep, ii) Reduced number of PCR cycles resulting in less PCR amplification bias, and iii) Preparation of quality multiplexed rRRBS libraries in only ~2 days.
DNA甲基化是一种常见的表观遗传调控机制，参与转录调控和基因组稳定性。随着下一代测序技术的发展，在许多实验室中建立全基因组范围的定量DNA甲基化图谱已成为常规操作。然而，这些方法中的许多都需要几天时间才能完成，并且使用主观的PCR方法来扩增测序文库，这可能会导致扩增偏差。在此，我们提出了一种快速简化代表性亚硫酸氢盐测序（rRRBS）方案，以尽量减少PCR扩增偏差并缩短多重文库构建的总时间。在这种改进的方法中，通过qPCR而不是使用标准PCR和凝胶电泳来完成和监测最终文库扩增步骤的精确定量，以确定合适的循环次数。这种rRRBS方法的主要优点包括：i）用于文库制备的扩增酶用量减少；ii）PCR循环次数减少，导致PCR扩增偏差较小；iii）仅需约2天即可制备高质量的多重rRRBS文库。
