Ultrasensitive multiomic platform using epitope-targeted DNA methylation mapping

项目总结 基因表达受DNA甲基化(DNAME)之间复杂的分子串扰调节 以及其他染色质特征：例如，组蛋白翻译后修饰(PTM)和与染色质相关的 蛋白质(CHAP；转录因子和染色质重构体)。值得注意的是，染色质的变化 景观可以对DNAME模式产生深远的影响(反之亦然)，这些变化是相互关联的 从癌症到神经性疾病(从癌症到神经紊乱)。然而，我们的 了解DNAME如何与其他染色质特征共生/协调以控制基因 由于缺乏可靠的基因组工具，表达受到限制。在这里，EpiCypher正在与New England Biolabs合作 (Neb)开发靶向酶甲基化测序(TEM-seqTM)，这是一种超敏感的多组体 提供表位定义的高分辨率DNAME谱(5mC/5hmC)的映射技术 染色质特征。EpiCypher正在领导超灵敏基因组图谱分析的开发，该分析使用 使用Cut&Run/Cut&Tag方法(在Cutana®平台下)生成真正量化的数据 大大减少了单元输入和测序深度(与芯片序列相比，每个参数节省了10倍)。 Cutana分析由EpiCypher专有的尖峰设计器核小体(DNuc)技术支持，以 实现技术监测和量化标准化。TEMSEQ项目的关键创新是 与EpiCypher的定量切割和运行相结合的新型多组体工作流的开发 技术，使用NEB的酶甲基序列(EM-SEQ)方法进行无偏DNAME分析。EM-SEQ 利用DNAME(5mC/5hmC)的酶转化，提供了亚硫酸氢盐亟需的替代品 测序(BS；一种降解DNA并具有系统性序列偏差的化学处理)以产生高水平的 分辨率、无偏DNAME配置文件，样本输入减少约10倍(与BS相比)。在第一阶段目标1中，我们将严格执行 在三个单元中验证我们的TEM-SEQ工作流程，对照标准CUT&RUN和EM-SEQ进行基准测试 分析，并进一步开发EpiCypher的尖峰控制，以与TEM-Seq兼容。我们将前进到 第二阶段，当我们证明TEM-seq产生与组蛋白相关的高度可靠的DNAME图谱时 使用&lt；50k单元和&lt；10M读取的PTM和CHAP。在第二阶段目标2中，我们将扩大尖峰插入的发展 控制面板并为广泛的染色质特征制定可靠的协议(使用经过验证的抗体) 和样品处理方法(新鲜、冷冻和固定)，包括药物治疗时程实验 支持临床应用。在第二阶段目标3中，我们将开发/验证一套TEM-seq测试版，并创建 数据分析门户和自动化分析，以加速商业采用并实现高吞吐量 提供服务。TEM-SEQ将提供一个强大的新工具来扩大我们对复杂染色质的理解 信号，进一步释放表观遗传学靶向药物和诊断的潜力。

PROJECT SUMMARY Gene expression is regulated by the complex molecular cross-talk between DNA methylation (DNAme) and other chromatin features: e.g. histone post-translational modifications (PTMs) and chromatin associated proteins (ChAPs; transcription factors and chromatin remodelers). Significantly, changes in the chromatin landscape can have a profound impact on DNAme patterning (and vice versa), and these changes are connected to development as well as a broad range of diseases (from cancer to neurological disorders). However, our understanding of how DNAme co-occurs / coordinates with additional chromatin features to control gene expression is limited by a lack of reliable genomic tools. Here, EpiCypher is partnering with New England Biolabs (NEB) to develop Targeted Enzymatic Methylation-sequencing (TEM-seqTM), an ultra-sensitive multiomic mapping technology that delivers high resolution DNAme profiles (5mC/5hmC) at epitope-defined chromatin features. EpiCypher is leading the development of ultra-sensitive genomic mapping assays that use CUT&RUN / CUT&Tag methods (under the CUTANA® platform) to generate truly quantitative data using dramatically reduced cell input and sequencing depth (>10-fold savings on each parameter vs. ChIP-seq). CUTANA assays are supported by EpiCypher’s proprietary spike-in designer nucleosome (dNuc) technology to enable technical monitoring and quantitative normalization. The key innovation of the TEM-seq project is the development of a novel multiomic workflow that marries EpiCypher’s quantitative CUTANA CUT&RUN technology with unbiased DNAme analysis using NEB’s enzymatic methyl-seq (EM-seq) approach. EM-seq utilizes the enzymatic conversion of DNAme (5mC / 5hmC) and provides a much-needed alternative to bisulfite sequencing (BS; a chemical treatment that degrades DNA and has systemic sequence biases) to generate high resolution, unbiased DNAme profiles with ~10-fold less sample input (vs. BS). In Phase I Aim 1, we will rigorously validate our TEM-seq workflow in three cell lines, benchmark results against standard CUT&RUN and EM-seq assays, and further develop EpiCypher’s spike-in controls for compatibility with TEM-seq. We will advance to Phase II when we demonstrate that TEM-seq generates highly reliable DNAme maps associated with histone PTMs and ChAPs using <50k cells and <10M reads. In Phase II Aim 2, we will expand development of spike-in control panels and develop robust protocols for a wide panel of chromatin features (using validated antibodies) and sample processing methods (fresh, frozen, and fixed), including drug treatment time-course experiments to enable clinical applications. In Phase II Aim 3, we will develop / validate a TEM-seq beta kit, and also create a data analysis portal and automated assays to accelerate commercial adoption and enable a high-throughput service offering. TEM-seq will provide a powerful new tool to expand our understanding of complex chromatin signaling, further unlocking the potential of epigenetics-targeted drugs and diagnostics.