Data from: EpiRADseq: scalable analysis of genome-wide patterns of methylation using next-generation sequencing

1. Research addressing the role of epigenetics in a diversity of experimental and natural systems is rapidly accumulating. Diverse methods have been developed to study epigenetic states, including bisulfite sequencing and AFLP-based approaches. However, existing methods are sometimes difficult to apply to non-traditional model organisms that lack genomic resources (bisulfite sequencing), and can fail to be economical and readily scalable to diverse research questions because of reliance on traditional Sanger sequencing (AFLP approaches). 2. Here we develop a reduced-representation library-based approach that is scalable and economical to quantitatively compare patterns of genome-wide methylation. This approach shares substantial similarity to the now widely used double digest restriction-site associated DNA sequencing-based method (ddRADseq), except that it utilizes a methylation-sensitive restriction enzyme. This method therefore identifies changes in the genomic methylation state of cytosine (to 5-methyl-cytosine; 5mC) by sampling loci (via next-generation sequencing) that are not methylated within a sample. We test this method to identify shifts in the epigenome of clonal water fleas (Daphnia ambigua) in response to exposure to fish predator cues, which are known to induce transgenerational changes in life history traits. 3. We found evidence for differential transgenerational responses (inferred via significant shifts in the methylation state of sampled loci) to predator cues among our treatment groups, and remarkably consistent responses within treatment groups. Our results demonstrate that this method is capable of producing highly repeatable results even without the use of a reference genome. 4. Applications of this general method are broad and diverse, and include the analysis of epigenetic shifts in both experimental and natural study systems.

1. 针对表观遗传学（epigenetics）在多种实验与自然系统中作用的相关研究正快速积累。目前已开发出多种用于研究表观遗传状态的方法，包括亚硫酸氢盐测序（bisulfite sequencing）及基于扩增片段长度多态性（Amplified Fragment Length Polymorphism, AFLP）的技术方案。然而，现有方法存在一定局限：亚硫酸氢盐测序难以应用于缺乏基因组资源的非传统模式生物；基于AFLP的技术因依赖传统桑格测序（Sanger sequencing），往往难以兼顾经济性与可扩展性，无法适配多样化的研究需求。2. 本研究开发了一种基于简化基因组文库的定量分析方法，可实现经济高效且可扩展的全基因组甲基化模式比较。该方法与当前广泛应用的双酶切限制性位点关联DNA测序（double digest restriction-site associated DNA sequencing, ddRADseq）技术高度相似，仅以甲基化敏感限制性内切酶替代了常规酶切步骤。此方法通过下一代测序（next-generation sequencing）捕获样本中未发生甲基化的基因座，从而识别胞嘧啶（cytosine）向5-甲基胞嘧啶（5-methyl-cytosine, 5mC）的基因组甲基化状态变化。我们以克隆型透明溞（Daphnia ambigua）为实验对象，验证该方法在鱼类捕食者信号暴露下的表观基因组变化检测能力——此前研究已证实此类信号可诱导透明溞产生跨代生活史性状改变。3. 实验结果显示，不同处理组的透明溞对捕食者信号存在差异化的跨代表观响应（通过采样基因座的甲基化状态显著变化推断得出），且同一处理组内的响应高度一致。本研究证实，即使不依赖参考基因组，该方法仍可产出重复性极佳的实验结果。4. 该通用方法的应用场景广泛多样，可用于分析实验与自然研究系统中的表观遗传状态变化。