Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm 17177, Sweden.
Nucleic Acids Res. 2013 Apr 1;41(6):e72. doi: 10.1093/nar/gks1467. Epub 2013 Jan 15.
DNA methylation is one of the most important epigenetic alterations involved in the control of gene expression. Bisulfite sequencing of genomic DNA is currently the only method to study DNA methylation patterns at single-nucleotide resolution. Hence, next-generation sequencing of bisulfite-converted DNA is the method of choice to investigate DNA methylation profiles at the genome-wide scale. Nevertheless, whole genome sequencing for analysis of human methylomes is expensive, and a method for targeted gene analysis would provide a good alternative in many cases where the primary interest is restricted to a set of genes. Here, we report the successful use of a custom Agilent SureSelect Target Enrichment system for the hybrid capture of bisulfite-converted DNA. We prepared bisulfite-converted next-generation sequencing libraries, which are enriched for the coding and regulatory regions of 174 ADME genes (i.e. genes involved in the metabolism and distribution of drugs). Sequencing of these libraries on Illumina's HiSeq2000 revealed that the method allows a reliable quantification of methylation levels of CpG sites in the selected genes, and validation of the method using pyrosequencing and the Illumina 450K methylation BeadChips revealed good concordance.
DNA 甲基化是参与基因表达调控的最重要的表观遗传改变之一。亚硫酸氢盐测序的基因组 DNA 是目前唯一的方法来研究 DNA 甲基化模式在单核苷酸分辨率。因此，下一代测序的亚硫酸氢盐转化 DNA 是研究在全基因组范围内的 DNA 甲基化模式的首选方法。然而，全基因组测序分析人类甲基组是昂贵的，靶向基因分析的方法将提供一个很好的替代在许多情况下，主要利益局限于一组基因。在这里，我们报告成功地使用定制的安捷伦 SureSelect 目标富集系统杂交捕获亚硫酸氢盐转化 DNA。我们准备了亚硫酸氢盐转化下一代测序文库，富集的 174 个 ADME 基因（即参与药物代谢和分布的基因）的编码和调控区。在 Illumina 的 HiSeq2000 上对这些文库进行测序表明，该方法可以可靠地定量选择基因中 CpG 位点的甲基化水平，使用焦磷酸测序和 Illumina 450K 甲基化 BeadChips 验证该方法显示出良好的一致性。
