Massively Parallel Sequencing Technology for Cancer Epigenome.

描述(申请人提供)：甲基化CpG岛扩增(MCA)是一种DNA文库构建技术，允许在整个基因组中扩增146,148个甲基化CpG富集区，覆盖所有基因的76%和所有真正的CpG岛的70%。当与微阵列结合时，这项技术已被证明是高度特异的，对于正常细胞和癌症细胞全基因组甲基化的高通量分析非常有用。然而，探针性能不均匀、交叉反应、归一化困难以及相关控制问题等问题阻碍了MCA的充分定量潜力的实现。我们建议通过将MCA与下一代Solexa 1G测序技术(MCA-Seq)相结合来开发和验证DNA甲基化分析的高分辨率工具。在项目的R21阶段，我们将应用各种策略来优化MCA-Seq，以提高覆盖率并将所需的初始DNA数量降至最低。此外，我们将为MCA-Seq建立质量控制，并开发用于数据分析的优化算法。然后，我们将验证我们优化的MCA-Seq协议，并使用一种模拟甲基化生物变异的创新方法来评估其敏感性、特异性和定量准确性。我们建议的研究将导致开发一种简单、健壮和可靠的全基因组DNA甲基化检测方法，这将在癌症研究中具有广泛的实用价值。我们的长期目标是利用这项技术来测试生物学假说。因此，在该项目的R33阶段，我们计划通过在一组特征良好的肿瘤细胞系和原发癌症患者样本中应用MCA-Seq来全面实施这项新兴技术。这些未来的研究将进一步验证MCA-Seq准确描述高度可变和异常的癌症表观基因组并产生初步生物学数据的能力。MCA-SEQ的开发和验证将使我们在理解导致癌症的基本生物学机制方面取得重大进展，并可能有助于未来癌症治疗的设计。

DESCRIPTION (provided by applicant): Methylated CpG island amplification (MCA) is a DNA library construction technique that permits amplification of 146,148 methylated CpG rich regions throughout the whole genome, covering 76% of all genes and 70% of all bona fide CpG islands. When coupled with microarrays, this technique has proven to be highly specific and very useful for the high-throughput analysis of genome-wide methylation in normal and cancer cells. Problems such as non-uniform probe performance, cross-reactivity, difficulties of normalization, and issues of relevant controls, however, prevent the full quantitative potential of MCA from being realized. We propose to develop and validate a high-resolution tool for DNA methylation profiling by coupling MCA with 'next generation' Solexa 1G sequencing technology (MCA-Seq). During the R21 phase of the project, we will apply various strategies to optimize MCA-Seq to improve coverage and minimize the quantity of initial DNA required. Additionally, we will build quality controls for MCA-Seq and develop optimized algorithms for data analysis. We will then validate our optimized MCA-Seq protocols, and evaluate the sensitivity, specificity, and quantitative accuracy using an innovative approach that simulates biological variation in methylation. Our proposed research will result in the development of a simple, robust, and reliable genome-wide assay for DNA methylation which will have broad utility in cancer research. Our long term goal is to utilize this technology to test biological hypotheses. Therefore, in an R33 phase of this project, we plan to fully implement this emerging technology by applying MCA-Seq in a set of well characterized tumor cell lines and primary cancer patient samples. These future studies will further validate the ability of MCA-Seq to accurately profile highly variable and aberrant cancer epigenomes, and generate preliminary biological data. The development and validation of MCA-Seq will enable major advances in our understanding of the basic biological mechanisms that contribute to cancer, and likely contribute to the design of future cancer therapies.