University of Exeter Medical School, University of Exeter, Exeter EX2 5DW, UK.
Cellular & Gene Editing Research, Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton CB10 1RQ, UK.
Hum Mol Genet. 2022 Sep 10;31(18):3181-3190. doi: 10.1093/hmg/ddac112.
Most epigenetic epidemiology to date has utilized microarrays to identify positions in the genome where variation in DNA methylation is associated with environmental exposures or disease. However, these profile less than 3% of DNA methylation sites in the human genome, potentially missing affected loci and preventing the discovery of disrupted biological pathways. Third generation sequencing technologies, including Nanopore sequencing, have the potential to revolutionize the generation of epigenetic data, not only by providing genuine genome-wide coverage but profiling epigenetic modifications direct from native DNA. Here we assess the viability of using Nanopore sequencing for epidemiology by performing a comparison with DNA methylation quantified using the most comprehensive microarray available, the Illumina EPIC array. We implemented a CRISPR-Cas9 targeted sequencing approach in concert with Nanopore sequencing to profile DNA methylation in three genomic regions to attempt to rediscover genomic positions that existing technologies have shown are differentially methylated in tobacco smokers. Using Nanopore sequencing reads, DNA methylation was quantified at 1779 CpGs across three regions, providing a finer resolution of DNA methylation patterns compared to the EPIC array. The correlation of estimated levels of DNA methylation between platforms was high. Furthermore, we identified 12 CpGs where hypomethylation was significantly associated with smoking status, including 10 within the AHRR gene. In summary, Nanopore sequencing is a valid option for identifying genomic loci where large differences in DNAm are associated with a phenotype and has the potential to advance our understanding of the role differential methylation plays in the etiology of complex disease.
迄今为止，大多数表观遗传学流行病学研究都利用微阵列来识别基因组中 DNA 甲基化变异与环境暴露或疾病相关的位置。然而，这些方法只能识别人类基因组中不到 3%的 DNA 甲基化位点，可能会遗漏受影响的基因座，并阻止发现被破坏的生物学途径。第三代测序技术，包括纳米孔测序，有可能彻底改变表观遗传学数据的生成方式，不仅可以提供真正的全基因组覆盖范围，还可以直接从天然 DNA 中分析表观遗传修饰。在这里，我们通过与最全面的微阵列（Illumina EPIC 阵列）定量的 DNA 甲基化进行比较，评估了纳米孔测序在流行病学中的可行性。我们实施了一种 CRISPR-Cas9 靶向测序方法，与纳米孔测序相结合，以分析三个基因组区域中的 DNA 甲基化，试图重新发现现有技术已经表明在吸烟者中存在差异甲基化的基因组位置。使用纳米孔测序读数，在三个区域的 1779 个 CpG 上定量了 DNA 甲基化，与 EPIC 阵列相比，提供了更精细的 DNA 甲基化模式分辨率。两个平台之间估计的 DNA 甲基化水平的相关性很高。此外，我们确定了 12 个 CpG，其中低甲基化与吸烟状态显著相关，包括 AHRR 基因内的 10 个。总之，纳米孔测序是一种识别与表型相关的 DNAm 差异较大的基因组基因座的有效选择，并有潜力推进我们对差异甲基化在复杂疾病病因学中的作用的理解。
