Research Center for Child Mental Development, University of Fukui, Fukui, Japan.
Division of Developmental Higher Brain Functions, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University, University of Fukui, Osaka, Japan.
Clin Epigenetics. 2023 Aug 28;15(1):138. doi: 10.1186/s13148-023-01544-3.
The pooled sample method is used in epigenomic research and expression analysis and is a cost-effective screening approach for small amounts of DNA. Evaluation of the pooled sample method in epigenomic studies is performed using the Illumina Infinium Methylation 450K BeadChip array; however, subsequent reports on the updated 850K array are lacking. A previous study demonstrated that the methylation levels obtained from individual samples were accurately replicated using pooled samples but did not address epigenome-wide association study (EWAS) statistics. The DNA quantification method, which is important for the homogeneous mixing of DNA in the pooled sample method, has since become fluorescence-based, and additional factors need to be considered including the resolution of batch effects of microarray chips and the heterogeneity of the cellular proportions from which the DNA samples are derived. In this study, four pooled samples were created from 44 individual samples, and EWAS statistics for differentially methylated positions (DMPs) and regions (DMRs) were conducted for individual samples and compared with the statistics obtained from the pooled samples.
The methylation levels could be reproduced fairly well in the pooled samples. This was the case for the entire dataset and when limited to the top 100 CpG sites, consistent with a previous study using the 450K BeadChip array. However, the statistical results of the EWAS for the DMP by individual samples were not replicated in pooled samples. Qualitative analyses highlighting methylation within an arbitrary candidate gene were replicable. Focusing on chr 20, the statistical results of EWAS for DMR from individual samples showed replicability in the pooled samples as long as they were limited to regions with a sufficient effect size.
The pooled sample method replicated the methylation values well and can be used for EWAS in DMR. This method is sample amount-effective and cost-effective and can be utilized for screening by carefully understanding the effective features and disadvantages of the pooled sample method and combining it with candidate gene analyses.
聚合样本方法被用于表观基因组学研究和表达分析，是一种针对少量 DNA 进行经济高效筛选的方法。在表观基因组学研究中，使用 Illumina Infinium Methylation 450K BeadChip 芯片评估聚合样本方法；然而，后续关于更新的 850K 芯片的报告却很少。先前的一项研究表明，使用聚合样本可以准确复制来自单个样本的甲基化水平，但并未解决全基因组关联研究（EWAS）的统计问题。自那时以来，对于聚合样本方法中 DNA 均匀混合非常重要的 DNA 定量方法已成为基于荧光的方法，并且需要考虑其他因素，包括微阵列芯片批次效应的分辨率和 DNA 样本来源的细胞比例的异质性。在这项研究中，从 44 个个体样本中创建了四个聚合样本，并对个体样本进行了差异甲基化位置（DMP）和区域（DMR）的 EWAS 统计分析，并将其与从聚合样本中获得的统计数据进行了比较。
在聚合样本中可以相当好地重现甲基化水平。这在整个数据集以及当限制在前 100 个 CpG 位点时都是如此，这与之前使用 450K BeadChip 芯片进行的研究一致。然而，个体样本中 DMP 的 EWAS 的统计结果在聚合样本中无法复制。突出显示任意候选基因内甲基化的定性分析是可复制的。关注 chr20，个体样本中 DMR 的 EWAS 的统计结果在聚合样本中具有可复制性，只要它们限于具有足够效应大小的区域。
聚合样本方法很好地复制了甲基化值，并且可以用于 DMR 的 EWAS。这种方法在样本量和成本方面都很有效，可以通过仔细了解聚合样本方法的有效特征和缺点并将其与候选基因分析相结合来进行筛选。
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