Internally Calibrated Chromatin Immunoprecipitation Using Barcoded Nucleosomes

 描述(申请人提供)：染色质免疫沉淀(ChIP)是研究基因表达的表观遗传调控的核心方法之一。定量聚合酶链式反应、微阵列和下一代测序(NGS)等分析方法增加了吞吐量，但并没有提高芯片数据集的可靠性。使用最先进的试剂，CHIP充其量是半定量的，最差的是不能代表体内翻译后修饰(PTM)的密度。为了解决这一缺陷，EpiCypher公司与芝加哥大学的Alex Ruthenburg博士合作，后者开创了一种内部标准化芯片实验的方法，以便随后标准化和量化表观遗传标记。这种方法在这里被称为内部校准免疫沉淀测序，或ICeChIP-Seq，最近获得了EpiCypher公司的独家许可，依赖于EpiCypher和Ruthenburg实验室合作开发和制造的设计核小体。这些核小体用感兴趣的表观遗传标记进行修饰，并包裹在包含唯一的识别条形码序列的DNA序列中。条形码设计的核小体以不同的浓度加入芯片反应中，含有纯化的染色质和针对感兴趣的表观遗传标记的珠状下拉抗体。在免疫沉淀之后，分析下一代测序数据以1)每个条形码和2)免疫沉淀的输入DNA检测到的读取次数，所得到的比率用于定量计算IP富集度。然后，可以将读数归一化为每个条形码核小体的输入浓度，从而为样本DNA读数的定量提供标准曲线。条形码核小体用作校准器，因为它们受到与样本染色质在芯片反应过程中经历的相同的变异性来源的影响，并且它们精确地类似于IP的目标。该分析的概念验证开发侧重于以下几个方面：条形码核小体的合成；其质量分析；H3K4me3分析的开发；分析算法；表征信噪比；最低染色质输入需求和日常可变性。鉴于到目前为止产生的强劲数据，以及对改进芯片序列分析的迫切需要，我们提出了一项直接到第二阶段的工作计划，以实现这一强大方法的商业化。第一阶段的报告提供了令人信服的证据，证明该方法是对现有方法的重大改进，第二阶段的目标是制造H3K4me3分析试剂的试剂盒，对该分析进行外部验证，并将PTM特异性ICeChIP-Seq试剂的菜单扩展到其他表观遗传标记。

 DESCRIPTION (provided by applicant): Chromatin immunoprecipitation (ChIP) is one of the core methods in the study of epigenetic regulation of gene expression. Analytical methods, such as qPCR, microarray and next generation sequencing (NGS) have increased throughput, but not the reliability of ChIP datasets. Using state-of-the-art reagents, ChIP is at best semi-quantitative, and at worst unrepresentative of in vivo post-translational modification (PTM) density. To address this short-coming, EpiCypher, Inc. has partnered with Dr. Alex Ruthenburg of the University of Chicago who has pioneered a method to internally-standardize ChIP experiments for subsequent normalization and quantitation of epigenetic marks. The method, referred to here as Internally Calibrated Immunoprecipitation Sequencing, or ICeChIP-Seq, was recently licensed on an exclusive basis by EpiCypher, Inc. and relies on designer nucleosomes collaboratively developed and manufactured by EpiCypher and the Ruthenburg lab. These nucleosomes are modified with an epigenetic mark of interest and wrapped in a DNA sequence containing a unique, identifying barcode sequence. Barcoded designer nucleosomes are added to the ChIP reaction at various concentrations, containing purified chromatin and a bead-attached pull-down antibody against the epigenetic mark of interest. After immunoprecipitation, next generation sequencing data is analyzed for the number of reads detected for 1) each barcode and 2) immunoprecipitated input DNA with the resulting ratio used to compute IP enrichment quantitatively. Read number can then be normalized to input concentration for each barcoded nucleosome, providing a standard curve for quantitation of sample DNA reads. The barcoded nucleosomes serve as calibrators because they are subjected to the same sources of variability the sample chromatin experiences during the ChIP reaction, and they precisely resemble the target of the IP. Proof-of-concept development of the assay has focused on the following: synthesis of barcoded nucleosomes; analysis of their quality; development of an H3K4me3 assay; analysis algorithms; characterizing signal-to-noise ratio; minimum chromatin input demands and day-to-day variability. Given the strong data generated to date, and the urgent need for an improved ChIP-Seq assay, we have proposed a Direct to Phase II workplan toward the commercialization of this powerful method. The Phase I report provides compelling evidence that the method is a dramatic improvement over existing methods, with Phase II aims focused on manufacturing a kit for the H3K4me3 assay reagents, external validation of this assay and expanding the menu of PTM-specific ICeChIP-Seq reagents to other epigenetic marks.