DNA methylation is an important epigenetic modification in many species that is critical for development, and implicated in ageing and many complex diseases, such as cancer. Many cost-effective genome-wide analyses of DNA modifications rely on restriction enzymes capable of digesting genomic DNA at defined sequence motifs. There are hundreds of restriction enzyme families but few are used to date, because no tool is available for the systematic evaluation of restriction enzyme combinations that can enrich for certain sites of interest in a genome. Herein, we present customised Reduced Representation Bisulfite Sequencing (cuRRBS), a novel and easy-to-use computational method that solves this problem. By computing the optimal enzymatic digestions and size selection steps required, cuRRBS generalises the traditional MspI-based Reduced Representation Bisulfite Sequencing (RRBS) protocol to all restriction enzyme combinations. In addition, cuRRBS estimates the fold-reduction in sequencing costs and provides a robustness value for the personalised RRBS protocol, allowing users to tailor the protocol to their experimental needs. Moreover, we show in silico that cuRRBS-defined restriction enzymes consistently out-perform MspI digestion in many biological systems, considering both CpG and CHG contexts. Finally, we have validated the accuracy of cuRRBS predictions for single and double enzyme digestions using two independent experimental datasets.

中文翻译：

---

cuRRBS：用于减少表示方法的酶组合的简单而强大的评估

DNA甲基化是许多物种中重要的表观遗传修饰，对发育至关重要，并涉及衰老和许多复杂疾病，例如癌症。DNA修饰的许多具有成本效益的全基因组分析都依赖能够在定义的序列基序上消化基因组DNA的限制酶。限制酶家族有数百种，但迄今为止很少使用，因为没有工具可用于系统评估可以丰富基因组中某些特定位点的限制酶组合。在这里，我们提出了定制的简化表示亚硫酸氢盐测序（cuRRBS），这是一种新颖且易于使用的计算方法，可以解决此问题。通过计算所需的最佳酶消化和大小选择步骤，cuRRBS将传统的基于MspI的简化表示亚硫酸氢盐测序（RRBS）方案推广到所有限制酶组合。此外，cuRRBS估计了测序成本的降低，并为个性化RRBS协议提供了稳健性值，从而使用户可以根据自己的实验需求定制协议。此外，我们展示在计算机上，考虑到CpG和CHG的情况，在许多生物系统中cuRRBS定义的限制性内切酶始终优于MspI消化。最后，我们使用两个独立的实验数据集验证了针对单酶和双酶消化的cuRRBS预测的准确性。