Taylor Kristen H, Kramer Robin S, Davis J Wade, Guo Juyuan, Duff Deiter J, Xu Dong, Caldwell Charles W, Shi Huidong
Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center, University of Missouri-Columbia, Columbia, Missouri, USA.
Cancer Res. 2007 Sep 15;67(18):8511-8. doi: 10.1158/0008-5472.CAN-07-1016.
We developed a novel approach for conducting multisample, multigene, ultradeep bisulfite sequencing analysis of DNA methylation patterns in clinical samples. A massively parallel sequencing-by-synthesis method (454 sequencing) was used to directly sequence >100 bisulfite PCR products in a single sequencing run without subcloning. We showed the utility, robustness, and superiority of this approach by analyzing methylation in 25 gene-related CpG rich regions from >40 cases of primary cells, including normal peripheral blood lymphocytes, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and mantle cell lymphoma (MCL). A total of 294,631 sequences was generated with an average read length of 131 bp. On average, >1,600 individual sequences were generated for each PCR amplicon far beyond the few clones (<20) typically analyzed by traditional bisulfite sequencing. Comprehensive analysis of CpG methylation patterns at a single DNA molecule level using clustering algorithms revealed differential methylation patterns between diseases. A significant increase in methylation was detected in ALL and FL samples compared with CLL and MCL. Furthermore, a progressive spreading of methylation was detected from the periphery toward the center of select CpG islands in the ALL and FL samples. The ultradeep sequencing also allowed simultaneous analysis of genetic and epigenetic data and revealed an association between a single nucleotide polymorphism and the methylation present in the LRP1B promoter. This new generation of methylome sequencing will provide digital profiles of aberrant DNA methylation for individual human cancers and offers a robust method for the epigenetic classification of tumor subtypes.
我们开发了一种新方法，用于对临床样本中的DNA甲基化模式进行多样本、多基因、超深度亚硫酸氢盐测序分析。采用大规模平行合成测序法（454测序），在一次测序运行中直接对100多个亚硫酸氢盐PCR产物进行测序，无需亚克隆。通过分析40多例原代细胞（包括正常外周血淋巴细胞、急性淋巴细胞白血病（ALL）、慢性淋巴细胞白血病（CLL）、滤泡性淋巴瘤（FL）和套细胞淋巴瘤（MCL））中25个基因相关的富含CpG区域的甲基化情况，我们展示了该方法的实用性、稳健性和优越性。共生成了294,631条序列，平均读长为131 bp。平均而言，每个PCR扩增子产生了超过1600条个体序列，远远超过传统亚硫酸氢盐测序通常分析的少数克隆（<20个）。使用聚类算法在单个DNA分子水平上对CpG甲基化模式进行综合分析，揭示了疾病之间的差异甲基化模式。与CLL和MCL相比，ALL和FL样本中检测到甲基化显著增加。此外，在ALL和FL样本中，检测到甲基化从选定CpG岛的周边向中心逐渐扩散。超深度测序还允许同时分析遗传和表观遗传数据，并揭示了一个单核苷酸多态性与LRP1B启动子中存在的甲基化之间的关联。这种新一代的甲基化组测序将为个体人类癌症提供异常DNA甲基化的数字图谱，并为肿瘤亚型的表观遗传分类提供一种稳健的方法。
