Metabarcoding mosquitoes: MinION sequencing of bulk samples gives accurate species profiles for vector surveillance (Culicidae)

Mosquitoes (Family: Culicidae) are dominant vectors of pathogens, and their surveillance has been incorporated into major disease control programs worldwide. However, routine, species-level identification of mosquitoes is often a bottleneck for management, and Next Generation Sequencing (NGS) platforms and DNA metabarcoding can revolutionize this process. MinION nanopore technologies promise on-site sequencing and rapid sample processing rates ideal for time-sensitive biosurveillance. Here, we benchmark the results of DNA metabarcoding on the MinION against the Illumina MiSeq platform, which is known for its higher sequencing accuracy. We used metazoan COI mini-barcode primers to carry out DNA metabarcoding of mosquito bulk samples caught during a real vector survey, then compared the mosquito species profiles recovered on each sequencing platform. We also tested the influence of using different trap lures, storage methods, and pooling different specimen body parts on the number of species recovered. We report that mosquito specieslevel identifications were highly congruent between MinION and Illumina (93% overlap). We also find that CO2 gas cylinders outperformed biogenic CO2 sources significantly, by two-fold. Notably, we demonstrated the feasibility of detecting zoonotic reservoirs and pathogen signals from mosquito bulk samples. We present the first use of DNA metabarcoding on the MinION for vector surveillance and discuss future applications.

蚊科（Culicidae）昆虫是多种病原体的主要传播媒介，其监测工作已纳入全球主流疾病防控项目。然而，常规的蚊类物种级鉴定往往是蚊媒防控工作的核心瓶颈，而下一代测序（Next Generation Sequencing, NGS）平台与DNA元条形码技术有望彻底革新这一困境。MinION纳米孔测序技术兼具现场测序能力与快速样本处理效率，完美适配时效性极强的生物监测需求。本研究以测序精度见长的Illumina MiSeq平台为对照，对MinION平台的DNA元条形码测序结果开展基准测试。我们采用后生动物COI迷你条形码引物，对实地媒介生物调查中采集的蚊虫混合样本实施DNA元条形码测序，并比对了两个测序平台所获得的蚊虫物种组成数据。此外，我们还评估了不同诱捕诱饵、样本保存方式以及混合不同标本身体部位对检出物种数量的影响。研究结果显示，MinION与Illumina平台的蚊虫物种级鉴定结果高度一致，物种重合度达93%。同时我们发现，二氧化碳钢瓶的诱捕效果显著优于生物源二氧化碳来源，性能提升达两倍。值得注意的是，本研究证实了从蚊虫混合样本中检测人畜共患病宿主与病原体信号的可行性。本研究首次将MinION平台的DNA元条形码技术应用于媒介生物监测，并对未来的应用场景进行了探讨。