Noninvasive prenatal diagnosis using targeted DNA capture and sequencing.

描述（由申请人提供）：使用靶向DNA捕获和测序的非侵入性产前诊断摘要：非侵入性产前诊断（NIPD）是对母体外周血中循环的胎儿遗传物质的研究，利用该分子诊断在妊娠早期实现，并且对胎儿的健康没有风险。在过去的十年中，已经开发了几种方法来确定胎儿性别的性连锁疾病，胎儿恒河猴D血型状态在恒河猴D阴性的妇女和父系遗传突变的显性疾病。最近，母体血浆DNA的大规模平行测序被应用于检测胎儿染色体非整倍性和单基因病症。然而，少量的无细胞胎儿DNA和由高母体DNA背景引起的干扰极大地限制了DNA变体的准确碱基识别，这取决于感兴趣的基因组区域的足够数量的序列读段（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获得的DNA输入量，并且相对昂贵。在这里，我们建议开发长挂锁探针（LPPs）应用于NIPD，以克服与识别胎儿DNA中的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）的组合允许以高准确性和完整性以及更低的成本和DNA要求对特定疾病相关的基因组区域进行靶向重测序。为了评估碱基识别算法的捕获效率和准确性，例如为了估计和控制新DNA变体的错误发现率（FDR），我们将利用具有已知染色体非整倍性和基因型含量（即21三体、囊性纤维化）的样品序列。我们将建立计算机资源，数据分布和分析软件，用于从考虑序列质量和覆盖率分布的序列数据中调用基因型，并为实验策略提供反馈。我们有针对性的方法允许通过样品索引每次测序运行分析多个样品，并将测序成本降低到约100美元/样品。制定这种综合办法对于在怀孕早期诊断高危家庭和某些人群中普遍存在的遗传疾病或各类疾病具有重要意义。

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.