Non-invasive determination of complete fetal genomes from cfDNA using HaploSeq

 描述(申请人提供)：遗传缺陷，包括单基因孟德尔病和非整倍体，是导致流产和先天出生障碍的主要原因。非侵入性产前检测(NIPT)目前被用于检测非整倍体，但鉴于下一代测序(NGS)成本的下降、分子/生化方法的日益复杂以及计算能力的不断增强，完全确定胎儿基因组(即SNV和大型非整倍体水平的基因类型和单倍型)似乎是遥不可及的。在这方面，孕妇血浆中的无细胞DNA(CfDNA)已成为非侵入性检测和诊断胎儿遗传缺陷的目标，因为cfDNA含有来自母亲和胎儿的遗传物质的混合物。但是，由于来自胎儿的cfDNA的比例很小(~10-15%)，并且由高度碎片化的DNA组成，因此确定单核苷酸变异(SNV)水平的胎儿基因型仍然具有挑战性，目前涉及成本过高的cfDNA深度测序(高达70X)。除了了解用于诊断单基因孟德尔疾病的基因类型外，对胎儿单倍型的非侵入性去卷积对于评估复杂的多基因疾病的风险可能是必要的。人们已经努力用父母的单倍型来确定胎儿基因组，但目前的单倍型父母的方法普遍存在成本过高、方法和仪器复杂、和/或对难以或不可能获得的遗传物质的依赖；而且它们只提供部分单倍型。 信息(较短的单倍型块和变异的不完全阶段)，阻碍了它们在 性价比高的完整胎儿基因组测定。我们的团队之前开发了一种创新的方法，HaploSeq，可以解决这个问题。HaploSeq方法根据已建立的HIC协议，通过邻近连接和NGS优先恢复同源染色体上物理连接的DNA变体，从而保留单倍型信息。HaploSeq实现了真正的跨越染色体的单倍型，以高精度(~99%)解析人类基因组中的绝大多数等位基因(&gt；93%)，从而构成了第一个可扩展的、具有成本效益的完整人类单倍型组装方法。在这里，我们提出了一种创新的方法，HaploSeq-Ft，用于非侵入性地确定完整的胎儿基因型和单倍型，使用HaploSeq从血液样本中生成跨越染色体的父母单倍型。此外，HaploSeq的全基因组分期能力还有助于从母体血浆中进行非常低深度的cfDNA测序，以确定完整的胎儿基因和单倍型。总而言之，通过利用我们专有的单倍型技术为新的cfDNA测序分析算法提供信息，一项HaploSeq-Ft血液测试将使父母能够以经济高效的方式了解其胎儿的完整基因型和单倍型，而不会危及怀孕。

 DESCRIPTION (provided by applicant): Genetic defects including single-gene Mendelian disorders and aneuploidies are among the leading causes of miscarriages and congenital birth disorders. Non-invasive prenatal testing (NIPT) is currently used to detect aneuploidies, but given the falling cost of next-generation sequencing (NGS), growing sophistication in molecular/biochemical methods, and ever increasing computational power, complete determination of the fetal genome (i.e., genotypes and haplotypes at the level of both SNVs and large aneuploidies) seems within reach. In this regard, cell-free DNA (cfDNA) in the maternal plasma has been targeted for non-invasive detection and diagnosis of fetal genetic defects, as cfDNA contains a mixture of genetic material derived from both the mother and the fetus. But because the fraction of cfDNA derived from the fetus is small (~10-15%), and consists of DNA that is highly fragmented, determination of fetal genotypes to the level of single-nucleotide variants (SNV) remains challenging, and presently involves excessively costly deep sequencing of cfDNA (up to 70X). In addition to knowledge of genotypes for diagnosing single gene Mendelian disorders, non-invasive deconvolution of fetal haplotypes is likely necessary for assessing the risk for complex multi-genic disorders. Efforts have been made to determine fetal genome with parental haplotypes, but the current methods to haplotype parents generally suffer from excessive costs, methodological and instrumentation complexity, and/or reliance on genetic material that is difficult or impossible to obtain; and they provide only partial haplotype information (short haplotype blocks and incomplete phasing of variants), hindering their utility in cost-effective complete fetal genome determination. Our team previously developed an innovative approach, HaploSeq, that can solve this problem. The HaploSeq method preserves haplotype information by preferentially recovering physically linked DNA variants on a homologous chromosome via proximity-ligation and NGS as per the established HiC protocol. HaploSeq achieves truly chromosome-spanning haplotypes, resolving the vast majority of alleles (>93%) at high accuracy (~99%) in human genomes, thus constituting the first scalable, cost-effective method for assembling complete human haplotypes. Here, we propose an innovative approach, HaploSeq-Ft, for non-invasive determination of complete fetal genotype and haplotype, using HaploSeq to generate chromosome-spanning parental haplotypes from blood samples. In addition, HaploSeq's whole-genome phasing capabilities also facilitate utilization of very low-depth cfDNA sequencing from maternal plasma for complete fetal genotype and haplotype determination. Taken together, by leveraging our proprietary haplotyping technology to inform novel cfDNA sequencing analysis algorithms, one HaploSeq- Ft blood test will enable parents to know the complete genotype and haplotype of their fetus in a cost-effective manner that does not endanger the pregnancy.