In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes.
In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes.
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DOI：
10.1093/nar/gks1467
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发表时间：
2013-04-01
影响因子：
14.9
通讯作者：
Milani L
中科院分区：
文献类型：
--
作者：
Ivanov M;Kals M;Kacevska M;Metspalu A;Ingelman-Sundberg M;Milani L
DNA甲基化是基因表达调控中最重要的表观遗传学改变之一。基因组DNA的亚硫酸氢盐测序是目前研究DNA甲基化模式的唯一方法。因此，亚硫酸氢盐转化的DNA的下一代测序是在全基因组范围内研究DNA甲基化谱的首选方法。然而，用于分析人类甲基化组的全基因组测序是昂贵的，并且用于靶向基因分析的方法将在主要兴趣限于一组基因的许多情况下提供良好的替代方案。在这里，我们报告了成功使用定制的Agilent SureSelect目标富集系统的混合捕获亚硫酸氢盐转化的DNA。我们制备了亚硫酸氢盐转化的下一代测序文库，该文库富集了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揭示，该方法允许对所选基因中CpG位点的甲基化水平进行可靠的定量，并且使用焦磷酸测序和Illumina 450K甲基化BeadChips对该方法进行验证揭示了良好的一致性。
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.
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影响因子：
46.9
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