UCL Cancer Institute, Paul O'Gorman Building, University College London, 72 Huntley Street, London WC1E 6BT, UK.
Methods. 2010 Nov;52(3):223-31. doi: 10.1016/j.ymeth.2010.04.003. Epub 2010 Apr 10.
DNA methylation is an epigenetic mark linking DNA sequence and transcription regulation, and therefore plays an important role in phenotypic plasticity. The ideal whole genome methylation (methylome) assay should be accurate, affordable, high-throughput and agnostic with respect to genomic features. To this end, the methylated DNA immunoprecipitation (MeDIP) assay provides a good balance of these criteria. In this Methods paper, we present AutoMeDIP-seq, a technique that combines an automated MeDIP protocol with library preparation steps for subsequent second-generation sequencing. We assessed recovery of DNA sequences covering a range of CpG densities using in vitro methylated λ-DNA fragments (and their unmethylated counterparts) spiked-in against a background of human genomic DNA. We show that AutoMeDIP is more reliable than manual protocols, shows a linear recovery profile of fragments related to CpG density (R(2)=0.86), and that it is highly specific (>99%). AutoMeDIP-seq offers a competitive approach to high-throughput methylome analysis of medium to large cohorts.
DNA 甲基化是一种将 DNA 序列与转录调控联系起来的表观遗传标记，因此在表型可塑性中发挥着重要作用。理想的全基因组甲基化（甲基组）检测方法应该具有准确性、经济性、高通量和对基因组特征的不可知性。为此，甲基化 DNA 免疫沉淀（MeDIP）检测提供了这些标准的良好平衡。在本方法论文中，我们提出了 AutoMeDIP-seq，这是一种将自动化 MeDIP 方案与文库制备步骤相结合的技术，用于随后的第二代测序。我们使用体外甲基化的 λ-DNA 片段（及其未甲基化的对应物）评估了覆盖一系列 CpG 密度的 DNA 序列的回收情况，这些片段以人基因组 DNA 为背景进行了掺入。我们表明，AutoMeDIP 比手动方案更可靠，表现出与 CpG 密度相关的片段的线性回收曲线（R(2)=0.86），并且具有高度特异性（>99%）。AutoMeDIP-seq 为中到大队列的高通量甲基组分析提供了一种有竞争力的方法。
