MosquitoID: Rapid metagenomic sequencing for offline mosquito surveillance.

Mosquitoes transmit numerous infectious diseases, with climate change expanding their global distribution through warmer environments. Next-generation sequencing offers significant advantages for mosquito genomic surveillance and potential early warning systems. In this study, a portable metagenomic sequencing approach using Oxford Nanopore Technologies (ONT) for field-based mosquito analysis (MosquitoID protocol) was developed, enabling species and host feeding patterns identification, and pathogen detection, all coming from a single amplification-free workflow. DNA was extracted from 62 mosquito samples (Aedes albopictus, Aedes cretinus, Culex pipiens, Culiseta longiareolata) from Greece and Spain, either single-species pools (1-10 specimens) or mixed-species pools, with reverse purification method or archived samples. Additionally, 30 pooled Aedes aegypti samples from Bangladesh underwent cDNA reverse purification. All samples were sequenced using ONT rapid barcoding kits. Offline bioinformatics analysis via Geneious screened custom BLAST databases for species, host, and virus identification. MosquitoID accurately identified mosquito species in 89% of samples overall, with main discrepancies in Aedes cretinus. Virus screening detected Phasi Charoen-like virus in cDNA samples. Host DNA sequences identified multiple species including horses, cattle, and ducks. This study demonstrates metagenomic ONT sequencing's effectiveness for rapid host, species, and virus identification. After further benchmarking, the approach shows potential for real-time disease monitoring and enhanced surveillance systems. Integrating portable next-generation sequencing with offline bioinformatics tools could significantly strengthen mosquito-borne disease prevention strategies, particularly for non-bioinformaticians and in resource-limited settings.