Single Cell Genomics for Malaria Parasites

疟疾寄生虫的单细胞基因组学

• 批准号: 8975609
• 负责人: Ian Harry Cheeseman
• 金额: $ 45.75万
• 依托单位: TEXAS BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8975609
• 关键词: Address; Africa South of the Sahara; Archives; Biology; Blood Cells; Burmese; Cells; Clinic; Complex; Data; Disease Outcome; Drug resistance; Genetic; Genome; Genomics; Genotype; Haplotypes; Health; Human; Individual; Infection; Laboratories; Life Cycle Stages; Liver; Malaria; Malawi; Methods; Parasites; Patients; Performance; Plasmodium; Plasmodium falciparum; Plasmodium vivax; Population; Population Genetics; Primary Infection; Relapse; Reporting; Research Personnel; Sampling; Severity of illness; Statistical Methods; Thailand; Time; Virulent; Work; base; deep sequencing; genetic analysis; genome sequencing; malaria infection; novel strategies; parasite genome; pathogen; tool; transmission process; whole genome

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.

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非常常见。例如，撒哈拉以南非洲80%的人类恶性疟原虫感染包含多种寄生虫基因型。大多数疟疾物种不能在实验室中培养，这妨碍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到高变基因组区域的特征或最近的感染深度测序。这两种方法都是不够的，因为寄生虫的单倍型无法构建，而且还没有统计方法来解决复杂的单倍型混合物。为了解决这个问题，我们已经开发出了强大的疟原虫单细胞基因组学方法，利用FACs分离单个感染细胞，然后进行全基因组扩增，遗传表征和/或深度测序。这些方法为了解复杂疟疾感染的组成、验证从深度序列数据中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动力学开辟了新的机会。我们利用这些新方法：(1)剖析恶性疟原虫感染在密集传播区域的组成。这些数据将用于确定有多少独立的基因型存在，组成单倍型之间的关系及其在感染中的相对丰度，并批判性地评估目前采用的间接方法的性能。(2)我们将剖析泰国患者原发性和继发性间日疟原虫感染的复杂情况。这项工作是不可能通过其他手段，因为不像恶性疟原虫，间日疟原虫不可能长期培养。我们将阐明原发性和继发性感染之间的关系，以及这种重要人类病原体的宿主动力学。