Centre for Personalised Nanomedicine, Australian Institute for Nanoengineering and Biotechnology, University of Queensland, Brisbane, Australia.
Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Melbourne, Victoria, 8006, Australia.
Sci Rep. 2017 Mar 22;7:45096. doi: 10.1038/srep45096.
Multiplex bisulfite-PCR sequencing is a convenient and scalable method for the quantitative determination of the methylation state of target DNA regions. A challenge of this application is the presence of CpGs in the same region where primers are being placed. A common solution to the presence of CpGs within a primer-binding region is to substitute a base degeneracy at the cytosine position. However, the efficacy of different substitutions and the extent to which bias towards methylated or unmethylated templates may occur has never been evaluated in bisulfite multiplex sequencing applications. In response, we examined the performance of four different primer substitutions at the cytosine position of CpG's contained within the PCR primers. In this study, deoxyinosine-, 5-nitroindole-, mixed-base primers and primers with an abasic site were evaluated across a series of methylated controls. Primers that contained mixed- or deoxyinosine- base modifications performed most robustly. Mixed-base primers were further selected to determine the conditions that induce bias towards methylated templates. This identified an optimized set of conditions where the methylated state of bisulfite DNA templates can be accurately assessed using mixed-base primers, and expands the scope of bisulfite resequencing assays when working with challenging templates.
多重亚硫酸氢盐-PCR 测序是一种定量测定目标 DNA 区域甲基化状态的便捷且可扩展的方法。这种应用的一个挑战是在引物放置的区域中存在 CpG。在引物结合区域内存在 CpG 的常见解决方案是在胞嘧啶位置替代碱基简并性。然而，不同替代物的效果以及偏置甲基化或未甲基化模板的程度在亚硫酸氢盐多重测序应用中从未得到评估。作为回应，我们研究了在包含在 PCR 引物中的 CpG 的胞嘧啶位置处的四种不同引物替代物的性能。在这项研究中，评估了脱氧肌苷、5-硝基吲哚、混合碱基引物和带有无碱基位点的引物在一系列甲基化对照物中的性能。含有混合或脱氧肌苷碱基修饰的引物表现出最稳健的性能。进一步选择混合碱基引物来确定诱导偏向甲基化模板的条件。这确定了一组优化的条件，其中可以使用混合碱基引物准确评估亚硫酸氢盐 DNA 模板的甲基化状态，并在使用具有挑战性的模板时扩展了亚硫酸氢盐重测序测定的范围。
