Single Cell Genomics for Malaria Parasites

描述（由申请人提供）：疟疾（和其他病原体）的多基因型和多物种感染极为常见。例如，撒哈拉以南非洲>80%的人类恶性疟原虫感染含有多种寄生虫基因型。大多数疟疾物种不能在实验室中培养，排除了基于培养的单个克隆的分离。目前，这种复杂感染的遗传分析涉及高变基因组区域的表征或最近的感染深度测序。这两种方法都是不够的，因为寄生虫单倍型不能构建和统计方法解决复杂的单倍型混合物还没有。为了解决这个问题，我们已经开发了用于疟原虫单细胞基因组学的稳健方法，利用FACs分离单个感染细胞，然后进行全基因组扩增，以及遗传表征和/或深度测序。这些方法为理解复杂疟疾感染的组成，验证试图从深层序列数据重建单倍型的统计方法，以及检查不可培养疟疾感染的宿主内动力学提供了新的机会。我们利用这些新的方法：（1）解剖恶性疟原虫感染的组成在一个地区的强烈传播。这些数据将用于确定有多少独立的基因型存在，感染内的组分单倍型和它们的相对丰度之间的关系，并严格评估目前采用的间接方法的性能。(2)我们将解剖泰国患者原发性和继发性感染中的复杂间日疟原虫感染。这项工作不可能通过其他手段，因为与恶性疟原虫不同，间日疟原虫不可能长期培养。我们将阐明原发感染和继发感染之间的关系，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.