A next-generation method for cytogenomics using Hi-C proximity ligation sequencing

摘要 生殖基因测试是一种不同的方法集合，可以识别特定类别的突变 已知会干扰配子发生、受孕和胎儿存活。解决问题的测试的数量和种类 这一临床需求反映了与生殖医学相关的遗传损害的规模，范围从 全染色体非整倍体到单核苷酸变异。在本提案中，我们描述了一个高度可扩展的 生殖基因测试，能够以高分辨率、低成本和更快的速度提供跨越这种规模的结果 周转时间比目前的测试更短。 我们的方法使用了一种称为Hi-C的邻近连接DNA测序方法，它捕获了超长距离的 使用无处不在的短读测序和台式分子生物学的基因组邻接性信息。我们 已经广泛证明了Hi-C数据可以用来排序和定位基因组组合， 重建端到端的染色体序列。这种方法还可以鉴定结构基因组 重排，包括平衡易位、倒置和其他典型的不能检测到的异常 测序方法。 我们建议应用高通量邻近连接作为一种细胞基因组学方法来检测 以高分辨率和低成本进行染色体畸变。这份提案勾勒出了一条通向商业 可用的产品和服务，这将建立一种高度有效的方法，用于研究并最终使用 在诊断环境中。这将通过1)设计一种易于使用的Hi-C协议来实现 多孔板处理，2)建立一个健壮的计算平台，可重复地调用染色体异常 3)在真实世界样本上证明了这些方法的有效性和重复性。这个 由此产生的试剂盒和软件产品将是一种新的细胞基因组学方法，称为SequencingTM(KBS)的核型分析 我们将作为生殖测试市场的精准医学测试部署。

ABSTRACT Reproductive genetic tests are a disparate collection of methodologies with identify specific classes of mutations known to interfere with gametogenesis, conception and fetal viability. The number and variety of tests addressing this clinical need reflect the scale of genetic lesions that are relevant to reproductive medicine, ranging from whole chromosome aneuploidy to single nucleotide variants. In this proposal, we describe a highly scalable reproductive genetic test that can deliver results that span this scale at high resolution, lower cost, and faster turnaround time than current tests. Our approach uses a proximity ligation DNA sequencing method called Hi-C, which captures ultra-long-range genomic contiguity information using ubiquitious short-read sequencing and benchtop molecular biology. We have demonstrated extensively that Hi-C data can be used to order and orient genome assemblies, reconstructing end-to-end chromosome sequences. This method also identifies structural genomic rearrangements including balanced translocations, inversions, and other aberrations not detectable by typical sequencing approaches. We propose to apply high-throughput 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 Hi-C protocol amenable to multiwell plate handling, 2) building a robust computational platform to reproducibly call chromosome aberrations from Hi-C data, and 3) proving the validity and reproducibility of these methods on real world samples. The resulting kit and software product will be a new cytogenomic method called Karyotyping by SequencingTM (KBS) that we will deploy as a Precision Medicine test for the reproductive testing market.