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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一个靶向扩增子测序面板，可同时识别整个按蚊属中的蚊子种类和疟原虫存在

按蚊是一种多样化的蚊子属，包括 500 多种已描述的物种，包括所有已知的人类疟疾媒介。虽然对有限数量的关键病媒物种进行了详细研究，但消除疟疾的目标需要监测所有潜在的病媒物种。在这里，我们开发了一种多位点扩增子测序方法，该方法针对按蚊基因组中的 62 个高度可变位点和疟原虫中的两个保守位点线粒体，同时揭示了蚊子的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、无损和高通量的 DNA 提取工作流程，为多重 PCR 提供来自单个蚊子的模板 DNA，这意味着可以返回产生意外结果的样本进行形态学检查。在一次 MiSeq 运行中可以对 1000 多只蚊子进行测序，我们展示了该小组使用来自非洲、东南亚和南美洲的 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。科卢齐种群，因为基于来自 1000 多只蚊子的这 62 个基因座确定的种群结构密切反映了通过全基因组测序揭示的结果。端到端方法快速、廉价、稳健且准确，这使其成为非常有前景的大规模蚊子遗传监测和媒介控制技术。