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Prediction of species composition ratios in pooled specimens of the Anopheles Hyrcanus group using quantitative sequencing
Malaria Journal ( IF 3 ) Pub Date : 2021-08-06 , DOI: 10.1186/s12936-021-03868-y
Do Eun Lee 1 , Heung-Chul Kim 2 , Sung-Tae Chong 2 , Terry A Klein 2 , Ju Hyeon Kim 3 , Si Hyeock Lee 1, 3
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Plasmodium vivax is transmitted by members of the Anopheles Hyrcanus Group that includes six species in the Republic of Korea: Anopheles sinensis sensu stricto (s.s.), Anopheles pullus, Anopheles kleini, Anopheles belenrae, Anopheles lesteri, and Anopheles sineroides. Individual Anopheles species within the Hyrcanus Group demonstrate differences in their geographical distributions, vector competence and insecticide resistance, making it crucial for accurate species identification. Conventional species identification conducted using individual genotyping (or barcoding) based on species-specific molecular markers requires extensive time commitment and financial resources. A population-based quantitative sequencing (QS) protocol developed in this study provided a rapid estimate of species composition ratios among pooled mosquitoes as a cost-effective alternative to individual genotyping. This can be accomplished by using species- or group-specific nucleotide sequences of the mitochondrial cytochrome C oxidase subunit I (COI) and the ribosomal RNA internal transcribed spacer 2 (ITS2) region as species identification alleles in a two-step prediction protocol. Standard genomic DNA fragments of COI and ITS2 genes were amplified from each Anopheles species using group-specific universal primer sets. Following sequencing of the COI or ITS2 amplicons generated from sets of standard DNA mixtures, equations were generated via linear regression to predict species-specific nucleotide sequence frequencies at different positions. Species composition ratios between An. sineroides, An. pullus and An. lesteri were estimated from QS of the COI amplicons based on the mC.260A, mC.122C and mC.525C alleles at the first step, followed by the prediction of species composition ratios between An. sinensis, An. kleini and An. belenrae based on QS of the ITS2 amplicons using the rI.370G and rI.389T alleles. The COI copy number was not significantly different between species, suggesting the reliability of COI-based prediction. In contrast, ITS2 showed a slightly but significantly higher copy number in An. belenrae, requiring an adjustment of its predicted composition ratio. A blind test proved that predicted species composition ratios either from pooled DNA specimens or pooled mosquito specimens were not statistically different from the actual values, demonstrating that the QS-based prediction is accurate and reliable. This two-step prediction protocol will facilitate rapid estimation of the species composition ratios in field-collected Anopheles Hyrcanus Group populations and is particularly useful for studying the vector ecology of Anopheles population and epidemiology of malaria.

中文翻译:

使用定量测序预测 Anopheles Hyrcanus 组汇集标本中的物种组成比

间日疟原虫由大韩民国的六种按蚊群成员传播:中华按蚊(ss)、普鲁斯按蚊、克莱尼按蚊、贝伦按蚊、莱斯特按蚊和黑按蚊。Hyrcanus 组内的单个按蚊物种在地理分布、媒介能力和杀虫剂抗性方面表现出差异,这对于准确识别物种至关重要。使用基于物种特异性分子标记的个体基因分型(或条形码)进行的常规物种鉴定需要大量的时间投入和财政资源。本研究中开发的基于种群的定量测序 (QS) 协议提供了对混合蚊子物种组成比率的快速估计,作为个体基因分型的一种具有成本效益的替代方案。这可以通过使用线粒体细胞色素 C 氧化酶亚基 I (COI) 和核糖体 RNA 内部转录间隔区 2 (ITS2) 区域的物种或组特异性核苷酸序列作为两步预测协议中的物种识别等位基因来实现。使用组特异性通用引物组从每个按蚊物种中扩增 COI 和 ITS2 基因的标准基因组 DNA 片段。在对由标准 DNA 混合物组生成的 COI 或 ITS2 扩增子进行测序后,通过线性回归生成方程以预测不同位置的物种特异性核苷酸序列频率。An之间的物种组成比。西内里德斯,安。普鲁斯和安。在第一步,根据 mC.260A、mC.122C 和 mC.525C 等位基因,从 COI 扩增子的 QS 估计 l​​esteri,然后预测 An 之间的物种组成比。中华,安。克莱尼和安。belenrae 基于使用 rI.370G 和 rI.389T 等位基因的 ITS2 扩增子的 QS。COI 拷贝数在物种之间没有显着差异,表明基于 COI 的预测的可靠性。相比之下,ITS2 在 An 中显示出略高但显着更高的拷贝数。belenrae,需要调整其预测的组成比例。盲测证明,混合 DNA 样本或混合蚊子样本的预测物种组成比与实际值没有统计学差异,表明基于 QS 的预测是准确可靠的。这种两步预测协议将有助于快速估计现场采集的按蚊群种群中的物种组成比,特别适用于研究按蚊种群的载体生态学和疟疾流行病学。
更新日期:2021-08-07
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