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

 描述（由申请人提供）：遗传缺陷，包括单基因孟德尔疾病和非整倍性是流产和先天性出生障碍的主要原因之一。非侵入性产前检测（NIPT）目前用于检测非整倍性，但是考虑到下一代测序（NGS）的成本下降、分子/生物化学方法的日益复杂以及计算能力的不断增加，胎儿基因组的完整确定（即，在SNV和大非整倍性水平上的基因型和单倍型）似乎是可以达到的。在这方面，母体血浆中的无细胞DNA（cfDNA）已被靶向用于胎儿遗传缺陷的非侵入性检测和诊断，因为cfDNA含有来源于母亲和胎儿两者的遗传物质的混合物。但是，由于源自胎儿的cfDNA的分数很小（约10-15%），并且由高度片段化的DNA组成，因此将胎儿基因型确定为单核苷酸变体（SNV）水平仍然具有挑战性，并且目前涉及cfDNA的过度昂贵的深度测序（高达70倍）。除了用于诊断单基因孟德尔疾病的基因型知识之外，胎儿单倍型的非侵入性解卷积对于评估复杂的多基因疾病的风险可能是必要的。已经做出努力来确定具有亲本单倍型的胎儿基因组，但是目前对亲本进行单倍型分析的方法通常具有成本过高、方法和仪器复杂性和/或依赖于难以或不可能获得的遗传物质的缺点;并且它们仅提供部分单倍型 信息（短单倍型块和不完全定相的变体），阻碍了他们的效用， 具有成本效益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方法通过根据已建立的HiC方案经由邻近连接和NGS优先回收同源染色体上的物理连接的DNA变体来保留单倍型信息。HaploSeq实现了真正的跨染色体单倍型，以高准确度（~ 99%）解析人类基因组中的绝大多数等位基因（> 93%），从而构成了第一个可扩展的、具有成本效益的组装完整人类单倍型的方法。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染色体的亲本单倍型。此外，HaploSeq的全基因组定相能力还有助于利用来自母体血浆的非常低深度的cfDNA测序来进行完整的胎儿基因型和单倍型测定。总之，通过利用我们专有的单倍型分型技术来告知新的cfDNA测序分析算法，一个HaploSeq-Ft血液测试将使父母能够以不危及怀孕的成本效益的方式了解其胎儿的完整基因型和单倍型。

 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.