Tracking arboviruses, their transmission vectors and potential hosts by nanopore sequencing of mosquitoes
The risk to human health from  mosquito-borne viruses such as dengue, chikungunya and yellow fever is increasing due to increased human expansion, deforestation and climate change. To anticipate and predict the spread and transmission of mosquito-borne viruses, a better understanding of the transmission cycle in mosquito populations is needed. We present a pathogen-agnostic combined sequencing protocol for identifying vectors, viral pathogens and their hosts or reservoirs using portable Oxford Nanopore sequencing. Using mosquitoes collected in São Paulo, Brazil, we extracted RNA for virus identification, and DNA for blood meal and mosquito identification. Mosquitoes and blood meals are identified using cytochrome c oxidase I sequencing (COI) against a curated barcode of life database (BOLD). Viruses were identified using the SMART-9N protocol which allows  amplified cDNA to be prepared with native barcoding for nanopore sequencing. Kraken2 was employed to detect viral pathogens, and Minimap2 and BOLD identified the contents of the blood meal. Due to the high similarity of some species, mosquito identification was conducted using BLAST after generation of consensus COI sequences using RACON polishing. This protocol can simultaneously uncover viral diversity, mosquito species, and mosquito feeding habits. It also has the potential to increase understanding of mosquito genetic diversity and transmission dynamics of zoonotic mosquito-borne viruses.