Chromosomal aberration detection in FFPE tissue using proximity ligation sequencing

摘要 染色体异常的检测是血液肿瘤谱系的一线诊断。 染色体异常，如易位、倒位、缺失和插入，在历史上都是 使用细胞遗传学方法或最近通过应用长阅读测序或光学方法进行鉴定 测绘技术。这些方法在实体肿瘤研究和诊断中的适用性较低 因为它们要么需要活细胞，要么需要高分子量DNA。绝大多数实体瘤 活检组织存储在福尔马林固定的石蜡包埋(FFPE)块中，这一过程高度碎裂 基因组DNA。在这项建议中，我们描述了一种与FFPE组织兼容的低成本和可扩展的方法，该方法 能够使用邻近连接测序检测染色体异常。 邻近连接方法，如染色体构象捕获(3C)和Hi-C可用于排序和 定位基因组片段，重建端到端的染色体序列。当序列偏离时 根据预期的顺序或取向，例如在染色体异常的情况下，序列 在Hi-C热图上显示为明显的非对角线信号，用于识别染色体 反常是一种自动化的过程. 我们建议将邻近连接作为一种细胞基因组学方法来检测染色体的宽度 高分辨率、低成本的像差。这份提案勾勒出了一条通向商业化产品的道路 和服务，这将建立一种高度验证的方法，用于研究并最终用于诊断 布景。这将通过以下方式来实现：1)设计一种易于使用、支持多井的FFPE Hi-C协议 平板处理，2)建立一个强大的自动化平台，可重复调用来自Hi-C的染色体异常 3)在真实世界样本上验证了这些方法的有效性和重复性。这一切的结果是 努力将是一种新的癌症细胞遗传学方法，称为SequencingTM(KBS)的核型分析。

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).