Noninvasive prenatal diagnosis using targeted DNA capture and sequencing.

摘要：无创产前诊断（Noninvasive prenatal diagnosis， NIPD）是对母体外周血中循环的胎儿遗传物质进行研究，在妊娠早期实现分子诊断，且对胎儿健康无风险。在过去十年中，已经开发了几种方法来确定性别连锁疾病的胎儿性别，恒河D阴性妇女的胎儿恒河D血型状况以及显性疾病的父系遗传突变。最近，大量平行测序的母体血浆DNA被用于检测胎儿染色体非整倍体和单基因条件。然而，胎儿无细胞DNA的数量少以及母体DNA高背景所造成的干扰极大地限制了DNA变异的准确碱基召唤，这取决于感兴趣的基因组区域的足够数量的序列读取（覆盖）。多重DNA序列捕获提供了一种新的策略，但目前的方法，如基于杂交的靶富集，需要的DNA输入量超过从母体血浆样品中获得的DNA输入量，并且相对昂贵。在这里，我们建议开发用于NIPD的长挂锁探针（LPPs），以克服识别胎儿DNA遗传和结构变异相关的困难。基于lpp的捕获与下一代测序（例如Illumine HiSeq）相结合，允许以高精度和完整性对特定疾病相关的基因组区域进行靶向重测序，并且成本和DNA要求相对较低。为了评估碱基调用算法的捕获效率和准确性，例如估计和控制新DNA变异的错误发现率（FDR），我们将利用已知染色体非整倍体和基因型含量（即21三体，囊性纤维化）的样本序列。我们将建立计算机资源、数据分布和分析软件，从序列数据中调用基因型，考虑序列质量和覆盖分布，并为实验策略提供反馈。我们的目标方法允许通过样品索引分析每个测序运行的多个样品，并将测序成本降低到约100美元/样品。发展这种综合方法对于在妊娠早期诊断高危家庭和某些人群中普遍存在的遗传疾病或疾病群体具有重要意义。

DESCRIPTION (provided by applicant): Noninvasive prenatal diagnosis using targeted DNA capture and sequencing Abstract: Noninvasive Prenatal Diagnosis (NIPD) is the study of fetal genetic material circulating in maternal peripheral blood, with which molecular diagnosis is achieved earlier in pregnancy and without risk to the health of the fetus. During the last decade, several approaches have been developed to determine fetal sex for sex-linked disorders, fetal rhesus D blood group status in rhesus D negative women and paternally inherited mutations for dominant diseases. More recently, massively parallel sequencing of maternal plasma DNA was applied to detect fetal chromosomal aneuploidies and a monogenic condition. However, the small amount of cell-free fetal DNA and the interference caused by the high maternal DNA background greatly limits the accurate base calling of DNA variants, which depends on a sufficient number of sequence reads (coverage) of the genomic regions of interest. Multiplex DNA sequence capture provides a new strategy but the current methods such as hybridization-based target enrichment require DNA input amounts beyond those obtained from a maternal plasma sample and are relatively expensive. Here we propose to develop long padlock probes (LPPs) for applications in NIPD in order to overcome the difficulties associated with identifying genetic and structural variants in fetal DNA. LPP-based capture combined with next- generation sequencing (e.g. Illumine HiSeq) allows for targeted resequencing of specific disease- related genomic regions at high accuracy and completeness and at comparably lower costs and DNA requirement. To assess capturing efficiency and accuracy of the base calling algorithms, such as to estimate and control the false discovery rate (FDR) for novel DNA variants, we will utilize sample sequences with known chromosomal aneuploidies and genotype content (i.e. Trisomy 21, Cystic fibrosis). We will establish computer resources, data distribution and analysis software for calling genotypes from sequence data that take into account sequence quality and coverage distributions and provide feedback to the experimental strategy. Our targeted approach allows multiple samples to be analyzed per sequencing run through sample indexing and reduces the sequencing costs to approximately $100/sample. Developing this integrated approach has important implications for the diagnosis, in early pregnancy, of genetic diseases or groups of disorders that are prevalent in high- risk families and in certain populations.