The impact of methodology on the reproducibility and rigor of DNA methylation data.

Epigenetic modifications are crucial for normal development and implicated in disease pathogenesis. While epigenetics continues to be a burgeoning research area in neuroscience, unaddressed issues related to data reproducibility across laboratories remain. Separating meaningful experimental changes from background variability is a challenge in epigenomic studies. Here we show that seemingly minor experimental variations, even under normal baseline conditions, can have a significant impact on epigenome outcome measures and data interpretation. We examined genome-wide DNA methylation and gene expression profiles of hippocampal tissues from wild-type rats housed in three independent laboratories using nearly identical conditions. Reduced-representation bisulfite sequencing and RNA-seq respectively identified 3852 differentially methylated and 1075 differentially expressed genes between laboratories, even in the absence of experimental intervention. Difficult-to-match factors such as animal vendors and a subset of husbandry and tissue extraction procedures produced quantifiable variations between wild-type animals across the three laboratories. Our study demonstrates that seemingly minor experimental variations, even under normal baseline conditions, can have a significant impact on epigenome outcome measures and data interpretation. This is particularly meaningful for neurological studies in animal models, in which baseline parameters between experimental groups are difficult to control. To enhance scientific rigor, we conclude that strict adherence to protocols is necessary for the execution and interpretation of epigenetic studies and that protocol-sensitive epigenetic changes, amongst naive animals, may confound experimental results.

表观遗传修饰对正常发育至关重要，并与疾病发病机制有关。虽然表观遗传学在神经科学中仍然是一个新兴的研究领域，但跨实验室数据可重复性相关的未解决问题仍然存在。在表观基因组研究中，将有意义的实验变化与背景变异性区分开来是一项挑战。在此我们表明，即使在正常基线条件下，看似微小的实验差异也会对表观基因组结果测量和数据解释产生重大影响。我们使用几乎相同的条件检查了来自三个独立实验室饲养的野生型大鼠海马组织的全基因组DNA甲基化和基因表达谱。简化代表性亚硫酸氢盐测序和RNA测序分别在实验室之间鉴定出3852个差异甲基化基因和1075个差异表达基因，即使在没有实验干预的情况下也是如此。难以匹配的因素，如动物供应商以及一部分饲养和组织提取程序，在三个实验室的野生型动物之间产生了可量化的差异。我们的研究表明，即使在正常基线条件下，看似微小的实验差异也会对表观基因组结果测量和数据解释产生重大影响。这对于动物模型中的神经学研究尤其有意义，因为在这些研究中，实验组之间的基线参数难以控制。为了提高科学严谨性，我们得出结论，严格遵守方案对于表观遗传学研究的实施和解释是必要的，并且在未处理的动物中，对方案敏感的表观遗传变化可能会混淆实验结果。