High definition profiling of mammalian DNA methylation by array capture and single molecule bisulfite sequencing
High definition profiling of mammalian DNA methylation by array capture and single molecule bisulfite sequencing
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DOI：
10.1101/gr.095190.109
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发表时间：
2009-09-01
期刊：
影响因子：
7
通讯作者：
Hicks, James B.
中科院分区：
文献类型：
--
作者：
Hodges, Emily;Smith, Andrew D.;Hicks, James B.
DNA甲基化稳定发育程序化的基因表达状态。异常甲基化与疾病进展相关，是癌症基因组的常见特征。目前，很少有方法能够在复杂哺乳动物基因组的所需区域中定量、大规模、单碱基分辨率定位DNA甲基化状态。在这里，我们提出了一种结合基于阵列的杂交选择和大规模平行亚硫酸氢盐测序的方法来分析跨越数十万个碱基的基因组区域中的DNA甲基化。这种单分子策略使得能够以高采样精度定量检查甲基化可变位置。使用亚硫酸氢盐捕获，我们评估了324个随机选择的CpG岛（CGI）的甲基化模式，代表超过25，000个CpG位点。Illumina测序的单泳道允许明确调用>90%的靶位点的甲基化状态。使用常规亚硫酸氢盐毛细管测序从选定区域的子集扩增的克隆PCR产物来验证杂交选择方法的准确性。这证实了即使是部分甲基化的状态也可以被成功地调用。人原代细胞和癌细胞的比较揭示了多个差异甲基化区域。超过25%的岛屿显示出复杂的甲基化模式，部分甲基化状态定义了整个CGI，或者在岛屿内的特定区域块中出现了对比甲基化状态。我们观察到甲基化状态的转变通常与基因组标志相关，包括转录起始位点和内含子-外显子连接。甲基化，沿着特定的组蛋白标记，在外显子区域富集，表明染色质状态可以预示成熟mRNA的含量。
DNA methylation stabilizes developmentally programmed gene expression states. Aberrant methylation is associated with disease progression and is a common feature of cancer genomes. Presently, few methods enable quantitative, large-scale, single-base resolution mapping of DNA methylation states in desired regions of a complex mammalian genome. Here, we present an approach that combines array-based hybrid selection and massively parallel bisulfite sequencing to profile DNA methylation in genomic regions spanning hundreds of thousands of bases. This single molecule strategy enables methylation variable positions to be quantitatively examined with high sampling precision. Using bisulfite capture, we assessed methylation patterns across 324 randomly selected CpG islands (CGI) representing more than 25,000 CpG sites. A single lane of Illumina sequencing permitted methylation states to be definitively called for >90% of target sties. The accuracy of the hybrid-selection approach was verified using conventional bisulfite capillary sequencing of cloned PCR products amplified from a subset of the selected regions. This confirmed that even partially methylated states could be successfully called. A comparison of human primary and cancer cells revealed multiple differentially methylated regions. More than 25% of islands showed complex methylation patterns either with partial methylation states defining the entire CGI or with contrasting methylation states appearing in specific regional blocks within the island. We observed that transitions in methylation state often correlate with genomic landmarks, including transcriptional start sites and intron-exon junctions. Methylation, along with specific histone marks, was enriched in exonic regions, suggesting that chromatin states can foreshadow the content of mature mRNAs.