Single Cell Genomics for Malaria Parasites

描述(由申请人提供)：疟疾(和其他病原体)的多种基因型和多物种感染极为常见。例如，撒哈拉以南非洲地区80%的人恶性疟原虫感染含有多种寄生虫基因。大多数疟疾物种不能在实验室中培养，从而排除了基于培养的单个克隆的隔离。目前，对这类复杂感染的遗传分析要么涉及高变量基因组区域的表征，要么最近涉及感染的深度测序。这两种方法都不够充分，因为不能构建寄生虫单倍型，而且还没有解决复杂单倍型混合物的统计方法。为了解决这个问题，我们开发了强大的疟原虫单细胞基因组学方法，利用流式细胞仪分离单个感染细胞，然后进行全基因组扩增，并进行基因特征和/或深度测序。这些方法为了解复杂疟疾感染的构成、验证寻求从深序列数据重建单倍型的统计方法以及检查非可培养疟疾感染的宿主内部动态提供了新的机会。我们利用这些新的方法：(1)剖析恶性疟原虫在高传播区的感染构成。这些数据将被用来确定存在多少独立的基因类型，感染中各成分单倍型与其相对丰度之间的关系，并对目前采用的间接方法的性能进行批判性评估。(2)我们将解剖泰国患者中复杂的间日疟原虫感染和后续感染。这项工作不可能通过其他方式进行，因为与恶性疟原虫不同，间日疟原虫不可能进行长期培养。我们将阐明初次感染和后续感染之间的关系，以及这种重要的人类病原体的宿主动态。

DESCRIPTION (provided by applicant): Multiple genotype and multi-species infections of malaria (and other pathogens) are extremely common. For example >80% of human P. falciparum infections in sub-Saharan Africa contain multiple parasite genotypes. Most malaria species cannot be cultured in the laboratory, precluding culture based isolation of individual clones. Currently, genetic analysis of such complex infections involves either characterization of hypervariable genome regions or more recently, deep sequencing of infections. Both methods are inadequate because parasite haplotypes cannot be constructed and statistical methods for resolving complex haplotype mixtures are not yet available. To resolve this problem, we have developed robust methods for single cell genomics of Plasmodium, utilizing FACs to isolates single infected cells, followed by whole genome amplification, and genetic characterization and/or deep sequencing. These methods open up new opportunities for understanding the composition of complex malaria infections, for validating statistical methods that seek to reconstruct haplotypes from deep sequence data, and to examine the within host-dynamics of non-culturable malaria infections. We exploit these new methods: (1) to dissect the composition of P. falciparum infections in a region of intense transmission. These data will be used to determine how many independent genotypes are present, the relationships between the component haplotypes and their relative abundance within infections, and to critically evaluate the performance of indirect methods currently employed. (2) We will dissect complex P. vivax infections in Thai patients in primary and subsequent infections. This work is not possible through other means as unlike P. falciparum no long term culture is possible for P. vivax. We will clarify the relationships between primary and subsequent infections, and within host dynamics of this important human pathogen.