Anopheles is a diverse genus of mosquitoes comprising over 500 described species, including all known human malaria vectors. While a limited number of key vector species have been studied in detail, the goal of malaria elimination calls for surveillance of all potential vector species. Here, we develop a multilocus amplicon sequencing approach that targets 62 highly variable loci in the Anopheles genome and two conserved loci in the Plasmodium mitochondrion, simultaneously revealing both the mosquito species and whether that mosquito carries malaria parasites. We also develop a cheap, nondestructive, and high-throughput DNA extraction workflow that provides template DNA from single mosquitoes for the multiplex PCR, which means specimens producing unexpected results can be returned to for morphological examination. Over 1000 individual mosquitoes can be sequenced in a single MiSeq run, and we demonstrate the panel’s power to assign species identity using sequencing data for 40 species from Africa, Southeast Asia, and South America. We also show that the approach can be used to resolve geographic population structure within An. gambiae and An. coluzzii populations, as the population structure determined based on these 62 loci from over 1000 mosquitoes closely mirrors that revealed through whole genome sequencing. The end-to-end approach is quick, inexpensive, robust, and accurate, which makes it a promising technique for very large-scale mosquito genetic surveillance and vector control.

中文翻译：

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目标扩增子测序面板可同时识别整个按蚊属的蚊子种类和疟原虫


按蚊是一个多样化的蚊属，包含超过 500 种描述的物种，包括所有已知的人类疟疾媒介。虽然对有限数量的关键媒介物种进行了详细研究，但消除疟疾的目标要求对所有潜在媒介物种进行监测。在这里，我们开发了一种多位点扩增子测序方法，针对按蚊基因组中的 62 个高度可变的基因座和疟原虫线粒体中的两个保守基因座，同时揭示蚊子种类以及该蚊子是否携带疟疾寄生虫。我们还开发了一种廉价、无损、高通量的 DNA 提取工作流程，为多重 PCR 提供来自单个蚊子的模板 DNA，这意味着产生意外结果的样本可以返回进行形态学检查。单次 MiSeq 运行即可对超过 1000 只蚊子进行测序，我们展示了该面板使用来自非洲、东南亚和南美洲 40 个物种的测序数据来分配物种身份的能力。我们还表明，该方法可用于解决An 内的地理人口结构。冈比亚和安. coluzzii种群，因为根据来自 1000 多个蚊子的 62 个基因座确定的种群结构与全基因组测序揭示的结果非常接近。这种端到端方法快速、廉价、稳健且准确，这使其成为大规模蚊子遗传监测和病媒控制的有前途的技术。