Advanced sequencing methods for repeat expansion disorders: exploring the dark matter of next-generation sequencing

短串联重复序列，也称为微卫星，是由重复序列组成的相对不稳定的DNA元件， 亚基的长度通常为2-6 bp。短串联重复序列中的大致病性扩增（数百至 数千个核苷酸）与迄今为止超过40种遗传疾病有关，这些疾病统称为 重复扩张性疾病。虽然重复扩增疾病代表了一组不同的遗传疾病， 通常涉及神经系统，并以神经系统、神经退行性、神经肌肉和/或 发育异常目前用于发现重复扩增障碍的遗传基础的方法是 在多个方面具有挑战性，往往限制他们的研究：他们依赖于大家庭的可用性， 多个受影响的个体，并采用低通量和费力的分子生物学方法。遗传 对许多疾病的研究现在已经转移到使用“下一代”测序方法， 在全基因组范围内检测变异。然而，由于下一代测序读数通常相当复杂， 短（100-250个核苷酸 核苷酸），使用这种方法识别和准确分型扩展的串联重复序列存在技术障碍。 approach.在这里，我们提出了一种新的，下一代测序方法，这将使扩大短串联 使用短的下一代测序读段有效地富集和分型重复序列。在我们的方法中，人工 遗传多样性通过合成杂交的结合被引入到单调重复序列中 含有独特的随机DNA标签（独特的分子标识符）的探针。在这些探针与靶杂交后， DNA、共价连接和PCR扩增转换的重复序列DNA，DNA片段化的标准方案 和下一代测序文库制备。重复片段，现在包含整合的分子标签， 然后进行大规模平行测序在分析过程中，跨越相邻分子标签的读段是 计算组装在一起，成为一个更大的连续序列，使重建转换道上 这些合成的高多样性区域的基础。在计算去除分子标签后， 可以精确地分辨原始道。在我们的第一个目标中，我们将开发和优化这项技术， 在几种涉及不同的神经束的代表性重复扩增疾病中扩增的重复序列的准确大小测定 长度和组成。在我们的第二个目标中，我们将调整我们的方法，以开发一个多路复用的探针组 靶向人类基因组中的所有短串联重复序列，从而实现廉价、高通量的全基因组筛选 对于已知的和以前未知的扩展重复疾病。可靠的、具有成本效益的、定量的、 并且用于检测和表征扩增重复序列紊乱的普遍适用的工具将提供增强的， 在这些疾病的诊断和遗传研究中的变革能力。因此，这些方法 极大地促进了重复扩增疾病遗传发现和研究，并将应用于对其他 基因组中的重复元件，包括着丝粒和端粒。

Short tandem repeats, also known as microsatellites, are relatively unstable DNA elements comprised of repeating subunits typically ranging from 2-6 bp in length. Large pathogenic expansions in short tandem repeats (hundreds to thousands of nucleotides) have been implicated in over 40 genetic disorders to date, which are collectively known as repeat expansion diseases. Although repeat expansion diseases represent a diverse group of genetic disorders, they typically involve the nervous system and are marked by neurological, neurodegenerative, neuromuscular, and/or developmental abnormalities. Current approaches for discovering the genetic basis of repeat expansion disorders are challenging in multiple respects and are often limiting to their study: they rely on the availability of large families with multiple affected individuals, and employ low-throughput and laborious molecular biology methods. Genetic investigations for many diseases have now shifted over to using “next-generation” sequencing methods which are capable of examining variants on a genome-wide scale. However, because next-generation sequencing reads are typically quite short (100-250 nucleotides) compared to the length of expanded repeat tracts (several hundred to several thousand nucleotides), there are technical barriers to identifying and accurately typing expanded tandem repeats using this approach. Here we propose a novel, next-generation sequencing approach which will enable expanded short tandem repeats to be effectively enriched and typed using short next-generation sequencing reads. In our approach, artificial genetic diversity is introduced into monotonous repeat sequences through the incorporation of synthetic hybridization probes containing unique, random DNA tags (unique molecular identifiers). After hybridization of these probes to target DNA, covalent linkage, and PCR amplification of converted repeat tract DNA, standard protocols for DNA fragmentation and next-generation sequencing library preparation are applied. Repeat tracts, now containing integrated molecular tags, are then subjected to massively parallel sequencing. During analysis, reads spanning across adjacent molecular tags are computationally assembled together into a larger contiguous sequence, enabling reconstruction of the converted tract on the basis of these synthetic, high diversity regions. After computational removal of the molecular tags, the composition of the original tract can be accurately resolved. In our first Aim, we will develop and optimize this technology to enable accurate sizing of expanded repeats across several representative repeat expansion diseases involving tracts of different lengths and composition. In our second Aim, we will adapt our methods in order to develop a multiplexed panel of probes targeting all short tandem repeats in the human genome, enabling inexpensive, high-throughput, whole genome screening for both known and previously unknown expanded repeat diseases. The availability of robust, cost-effective, quantitative, and generally applicable tools for the detection and characterization of expanded repeat disorders will provide enhanced, transformative capabilities in the diagnosis and genetic investigation of these disorders. Consequently, these methods will greatly facilitate genetic discovery and study of repeat expansion diseases, and will have application to typing other repetitive elements in the genome, including centromeres and telomeres.