Chromosomal aberration detection in FFPE tissue using proximity ligation sequencing

使用邻近连接测序检测 FFPE 组织中的染色体畸变

• 批准号: 10759887
• 负责人: Stephen Matthew Eacker
• 金额: $ 85.83万
• 依托单位: PHASE GENOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-25 至 2025-08-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10759887
• 关键词: Address; Biological Markers; Biopsy; Cell Line; Cell Survival; Characteristics; Chromatin; Chromosome abnormality; Chromosomes; Clinical; Commercial grade; Complex; Cytogenetics; DNA; Data; Data Set; Decision Making; Detection; Development; Diagnosis; Diagnostic; Formaldehyde; Formalin; Freezing; Funding; Future; Genomic DNA; Genomic Segment; Genomics; Goals; Grant; Hi-C; Hydration status; Industrialization; Investigation; Karyotype determination procedure; Letters; Libraries; Ligation; Liquid substance; Malignant Neoplasms; Maps; Methodology; Methods; Modernization; Molecular Weight; Nature; Online Systems; Optics; Organic solvent product; Paraffin Embedding; Performance; Phase; Process; Prognostic Marker; Protocols documentation; Recovery; Reporting; Reproducibility; Research; Research Personnel; Resolution; Resources; Sampling; Services; Signal Transduction; Single Nucleotide Polymorphism; Small Business Innovation Research Grant; Solid Neoplasm; Specimen; Stratification; System; Technology; Testing; Time; Tissue Embedding; Validation; biomarker discovery; blood neoplasm; chromosome conformation capture; clinically relevant; cohort; computational platform; cost; design; diagnostic biomarker; genome analysis; improved; informatics tool; insertion/deletion mutation; next generation sequencing; novel strategies; pre-clinical; preservation; prognostic; success; tumor; virtual

ABSTRACT The detection of chromosomal aberrations is a frontline diagnostic for the spectrum of blood neoplasms. Chromosomal aberrations, such as translocations, inversions, deletions and insertions, have been historically identified using cytogenetic methods or more recently through application of long read sequencing or optical mapping technologies. These methods have been less applicable in solid tumor research and diagnostics because they require either viable cells or high-molecular weight DNA. The vast majority of solid tumor biopsies are stored in formalin-fixed paraffin-embedded (FFPE) blocks, a process that highly fragments genomic DNA. In this proposal we describe a low-cost and scalable method compatible with FFPE tissue that enables the detection of chromosomal aberration using proximity ligation sequencing. Proximity ligation methods such as chromosome conformation capture (3C) and Hi-C can be used to order and orient segments of genomes, reconstructing end-to-end chromosome sequences. When a sequence deviates from the expected order or orientation, such as is in the case of chromosomal aberrations, the sequence appears as an obvious off-diagonal signal on a Hi-C heatmap, making identification of chromosomal abnormalities an automatable process. We propose to apply proximity ligation as a cytogenomic method to detect the breadth of chromosomal aberrations at high resolution and low cost. This proposal outlines a path to a commercially available product and service, which will establish a highly validated method for use in research and eventually in a diagnostic setting. This will be accomplished by 1) designing an easy to use FFPE Hi-C protocol amenable to multiwell plate handling, 2) building a robust automated platform to reproducibly call chromosome aberrations from Hi-C data, and 3) proving the validity and reproducibility of these methods on real world sample. The result of these efforts will be a new cancer cytogenetics methodology called Karyotyping by SequencingTM (KBS).

抽象的 染色体畸变的检测是血液肿瘤光谱的一线诊断。 从历史上看 通过使用长读取测序或光学的应用，使用细胞遗传学方法或更多 映射技术。这些方法不太适用于实体瘤研究和诊断 因为它们需要生存的细胞或高分子重量DNA。绝大多数实体瘤 活检存储在福尔马林固定的石蜡包裹（FFPE）块中，这一过程高度碎片 基因组DNA。在此提案中，我们描述了一种与FFPE组织兼容的低成本和可扩展方法 使用接近连接测序可以检测染色体像差。 接近连接方法，例如染色体构象捕获（3C）和HI-C，可用于订购和 基因组的东方段，重建端到端染色体序列。当序列偏离 从预期的顺序或方向，例如在染色体畸变的情况下，序列 在HI-C热图上显示为明显的非对角线信号，鉴定染色体 异常可自动化过程。 我们建议将接近连接作为细胞基因组方法来检测染色体的广度 高分辨率和低成本的畸变。该提案概述了通往市售产品的途径 和服务，该服务将建立一种高度验证的研究方法，并最终在诊断中 环境。这将通过1）设计易于使用的FFPE HI-C协议适合Multiwell 板处理，2）构建一个可重复调用HI-C的染色体畸变的强大自动化平台 数据，以及3）证明这些方法在现实世界样本上的有效性和可重复性。这些结果 努力将是一种新的癌症细胞遗传学方法论，称为SequencingTM（KBS）的核分型。