Noninvasive prenatal diagnosis using targeted DNA capture and sequencing.

描述(申请人提供)：利用靶向DNA捕获和测序进行非侵入性产前诊断摘要：非侵入性产前诊断(NIPD)是对孕妇外周血中循环的胎儿遗传物质的研究，通过这种遗传物质，可以在怀孕早期实现分子诊断，而不会对胎儿健康造成风险。在过去的十年里，已经开发了几种方法来确定性别连锁障碍的胎儿性别，D阴性恒河猴的胎儿D血型状态，以及显性疾病的父系遗传突变。最近，母亲血浆DNA的大规模平行测序被应用于检测胎儿染色体非整倍体和单基因情况。然而，少量的无细胞胎儿DNA和高母体DNA背景造成的干扰极大地限制了DNA变体的准确碱基调用，这取决于对感兴趣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，如基于杂交的靶浓缩，需要的DNA输入量超过从母体血浆样本获得的DNA输入量，而且相对昂贵。在这里，我们建议开发用于NIPD的长挂锁探针(LPP)，以克服与识别胎儿DNA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)相结合，允许以高精度和完整性以及相对较低的成本和DNA需求对特定疾病相关基因组区域进行有针对性的重新测序。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变体的错误发现率(FDR)，我们将使用具有已知染色体非整倍体和基因型含量的样本序列(即21三体，囊性纤维化)。我们将建立计算机资源、数据分配和分析软件，从考虑序列质量和覆盖率分布的序列数据中调用基因类型，并为实验策略提供反馈。我们的定向方法允许在每次测序运行中通过样本索引分析多个样本，并将测序成本降低到大约100美元/样本。开发这一综合方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病或疾病组具有重要意义。 公共卫生相关性：使用有针对性的DNA捕获和测序进行非侵入性产前诊断相关性：非侵入性产前诊断(NIPD)是对孕妇外周血中循环的胎儿遗传物质的研究。建立一种灵敏且经济有效的方法，准确检测妊娠早期少量母体血浆中无细胞胎儿DNA中的染色体变化和单基因紊乱，对包括胎儿筛查和妊娠管理在内的临床医学具有重要意义。

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. PUBLIC HEALTH RELEVANCE: Noninvasive prenatal diagnosis using targeted DNA capture and sequencing Relevance: Noninvasive Prenatal Diagnosis (NIPD) is the study of fetal genetic material circulating in maternal peripheral blood. Developing a sensitive and cost-effective assay to accurately detect chromosomal changes and monogenic disorders in cell-free fetal DNA from small amounts of maternal plasma in early pregnancy has important implications for clinical medicine including fetal screening and pregnancy management.