Crary-Dooley Florence K, Tam Mitchell E, Dunaway Keith W, Hertz-Picciotto Irva, Schmidt Rebecca J, LaSalle Janine M
a Department of Medical Microbiology and Immunology , Genome Center , University of California , Davis , CA , USA.
b Department of Public Health Sciences , University of California , Davis , CA , USA.
Epigenetics. 2017 Mar 4;12(3):206-214. doi: 10.1080/15592294.2016.1276680. Epub 2017 Jan 5.
DNA methylation is an epigenetic mark at the interface of genetic and environmental factors relevant to human disease. Quantitative assessments of global DNA methylation levels have therefore become important tools in epidemiology research, particularly for understanding effects of environmental exposures in complex diseases. Among the available methods of quantitative DNA methylation measurements, bisulfite sequencing is considered the gold standard, but whole-genome bisulfite sequencing (WGBS) has previously been considered too costly for epidemiology studies with high sample numbers. Pyrosequencing of repetitive sequences within bisulfite-treated DNA has been routinely used as a surrogate for global DNA methylation, but a comparison of pyrosequencing to WGBS for accuracy and reproducibility of methylation levels has not been performed. This study compared the global methylation levels measured from uniquely mappable (non-repetitive) WGBS sequences to pyrosequencing assays of several repeat sequences and repeat assay-matched WGBS data and determined uniquely mappable WGBS data to be the most reproducible and accurate measurement of global DNA methylation levels. We determined sources of variation in repetitive pyrosequencing assays to be PCR amplification bias, PCR primer selection bias in methylation levels of targeted sequences, and inherent variability in methylation levels of repeat sequences. Low-coverage, uniquely mappable WGBS showed the strongest correlation between replicates of all assays. By using multiplexing by indexed bar codes, the cost of WGBS can be lowered significantly to improve the accuracy of global DNA methylation assessments for human studies.
DNA甲基化是一种表观遗传标记，处于与人类疾病相关的遗传和环境因素的交叉点上。因此，对全基因组DNA甲基化水平的定量评估已成为流行病学研究中的重要工具，特别是对于理解环境暴露在复杂疾病中的影响。在现有的定量DNA甲基化测量方法中，亚硫酸氢盐测序被认为是金标准，但全基因组亚硫酸氢盐测序（WGBS）此前被认为对于样本数量众多的流行病学研究来说成本过高。对亚硫酸氢盐处理后的DNA中的重复序列进行焦磷酸测序已被常规用作全基因组DNA甲基化的替代方法，但尚未对焦磷酸测序与WGBS在甲基化水平的准确性和可重复性方面进行比较。本研究将从唯一可映射（非重复）的WGBS序列测得的全基因组甲基化水平与几种重复序列的焦磷酸测序分析以及与重复分析匹配的WGBS数据进行了比较，并确定唯一可映射的WGBS数据是全基因组DNA甲基化水平最具可重复性和准确性的测量方法。我们确定重复焦磷酸测序分析中的变异来源为PCR扩增偏差、靶向序列甲基化水平中的PCR引物选择偏差以及重复序列甲基化水平的固有变异性。低覆盖度、唯一可映射的WGBS在所有分析的重复样本之间显示出最强的相关性。通过使用索引条形码进行多重分析，可以显著降低WGBS的成本，以提高人类研究中全基因组DNA甲基化评估的准确性。
