A multiplex assay to detect mosquito species, bloodmeal host source and <scp> <i>Plasmodium</i> </scp> in malaria vectors using Nanopore amplicon sequencing

Multiple species of Anopheles mosquitoes transmit malaria-causing Plasmodium around the world. Molecular methods are often employed to confirm vector species, detect parasites and determine bloodmeal host sources; these assays are often performed separately and can be time-consuming and expensive. However, in this study, we show that the Oxford Nanopore Technologies (ONT) MinION Sequencer offers a cost-effective and efficient alternative to accurately identify mosquito species, host bloodmeal sources and detect parasites simultaneously in malaria vectors. We sequenced 150 insectary-reared mosquitoes representing nine species and 150 blood-fed mosquitoes with one of five vertebrate blood sources. We also analysed the presence of Plasmodium falciparum (Welch, 1897) in 40 infected mosquito samples. A final combined assay integrated all three previously optimized assays into a single sequencing run, demonstrating the high-throughput capability of the Nanopore sequencing platform. This run included 32 samples for each targeted amplicon, totalling 96 samples. For comparison, we sequenced all samples using a standard Sanger sequencing protocol. Our results showed that the MinION sequencing platform accurately identified all nine mosquito species, five different bloodmeal hosts from the blood-fed mosquitoes up to 48 h post blood feed, and detected P. falciparum in the 40 positive controls. The sequencing results obtained using the MinION platform exhibited high concordance with those from standard Sanger sequencing, as demonstrated by comparable similarity scores and correct mosquito species identification. This demonstrates that our MinION sequencing and analysis protocol offers a novel, highly precise, cost-effective solution for combined mosquito species identification, bloodmeal analysis and parasite detection.