Exposing malaria in-host diversity and estimating population diversity by capture-recapture using massively parallel pyrosequencing
Exposing malaria in-host diversity and estimating population diversity by capture-recapture using massively parallel pyrosequencing
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DOI：
10.1073/pnas.1007068107
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发表时间：
2010-11-16
影响因子：
11.1
通讯作者：
Meshnick, Steven R.
中科院分区：
文献类型：
--
作者：
Juliano, Jonathan J.;Porter, Kimberly;Meshnick, Steven R.
疟疾感染通常包含多种不同的遗传变异。数学和动物模型表明，这些变异之间的相互作用对耐药性的出现具有深远的影响。然而，目前用于量化个体感染中寄生虫多样性的方法对低丰度变异不敏感，并且不能定量变异种群规模。为了更完整地描述疟疾感染的宿主复杂性和生态学，我们使用大规模并行焦磷酸测序来表征一组患者感染中的疟原虫多样性。通过对复杂混合物中的单链 DNA 进行单独测序，该技术可以量化混合感染中不常见的变异。这种方法揭示的宿主内多样性远远超过目前推荐的基因分型方法所描述的多样性，每次感染的变异数多达六倍。此外，在配对的治疗前和治疗后样本中，我们显示了复杂的寄生虫环境，包括可能被药物治疗上调和下调选择的变体。与所有多样性调查一样，抽样限制阻碍了充分发现，抽样工作的差异可能会混淆样本、宿主和种群之间的比较。在这里，我们使用物种积累曲线和捕获-再捕获的生态方法来估计我们在种群中未能检测到的变异数量，并表明这些方法可以比较治疗前后以及疟疾种群之间的多样性。生态统计学和大规模并行焦磷酸测序的结合为研究耐药性的演变和疟疾感染的宿主生态学提供了强大的工具。
Malaria infections commonly contain multiple genetically distinct variants. Mathematical and animal models suggest that interactions among these variants have a profound impact on the emergence of drug resistance. However, methods currently used for quantifying parasite diversity in individual infections are insensitive to low-abundance variants and are not quantitative for variant population sizes. To more completely describe the in-host complexity and ecology of malaria infections, we used massively parallel pyrosequencing to characterize malaria parasite diversity in the infections of a group of patients. By individually sequencing single strands of DNA in a complex mixture, this technique can quantify uncommon variants in mixed infections. The in-host diversity revealed by this method far exceeded that described by currently recommended genotyping methods, with as many as sixfold more variants per infection. In addition, in paired pre- and posttreatment samples, we show a complex milieu of parasites, including variants likely up-selected and down-selected by drug therapy. As with all surveys of diversity, sampling limitations prevent full discovery and differences in sampling effort can confound comparisons among samples, hosts, and populations. Here, we used ecological approaches of species accumulation curves and capture-recapture to estimate the number of variants we failed to detect in the population, and show that these methods enable comparisons of diversity before and after treatment, as well as between malaria populations. The combination of ecological statistics and massively parallel pyrosequencing provides a powerful tool for studying the evolution of drug resistance and the in-host ecology of malaria infections.