Improved species assignments across the entire Anopheles genus using targeted sequencing

Accurate species identification of the mosquitoes in the genus Anopheles is of crucial importance to implement malaria control measures and monitor their effectiveness. We use a previously developed amplicon panel (ANOSPP) that retrieves sequence data from multiple short nuclear loci for any species in the genus. Species assignment is based on comparison of samples to a reference index using k-mer distance. Here, we provide a protocol to generate version controlled updates of the reference index and present its latest release, NNv2, which contains 91 species, compared to 56 species represented in its predecessor NNv1. With the updated reference index, we are able to assign samples to species level that previously could not be assigned. We discuss what happens if a species is not represented in the reference index and how this can be addressed in a future update. To demonstrate the increased power of NNv2, we showcase the assignments of 1789 wild-caught mosquitoes from Madagascar and demonstrate that we can detect within species population structure from the amplicon sequencing data

Improved species assignments across the entire Anopheles genus using targeted sequencing.

Peer reviewed: TrueAcknowledgements: In tribute to our colleague and co-author Tatamo, who died conducting field work. We thank Edel Sheerin for general logistic support for ANOSPP. We thank Petra Korlević for valuable suggestions and Nil Rahola for providing information on morphological keys used in taxonomic information. We thank the Medical Entomology Research Unit from the Institut Pasteur de Madagascar, VectorLink and the National Program for the Fight Against Malaria (PNLP) for sample collection and identification in this country. We also thank the Vector Biology and Control Section from the Department of Entomology at the Armed Forces Research Institute of Medical Sciences for sample collection and identification in Thailand. We thank the teams of the Entomology, Malaria and Laboratory departments of the Shoklo Malaria Research Unit for their help with sample collection, processing and management. For field sampling in Uganda we would like to thank Target Malaria Uganda. For sample collection in Ghana we would like to thank Richardson Kwesi Egyirifa, Christopher Dorcoo and Sampson Otoo. We thank Sanger’s Scientific Operation Teams for carrying out all PCRs, library generation, and sequencing on data presented here and Catherine McCarthy for her support in ensuring all samples are compliant with Access and Benefit Sharing of sequence data as laid out by the Nagoya Protocol. ThermaStop was provided to the Wellcome Sanger Institute by ThermaGenix Inc. (Natick Massachusetts, USA) free of charge.Accurate species identification of the mosquitoes in the genus Anopheles is of crucial importance to implement malaria control measures and monitor their effectiveness. We use a previously developed amplicon panel (ANOSPP) that retrieves sequence data from multiple short nuclear loci for any species in the genus. Species assignment is based on comparison of samples to a reference index using k-mer distance. Here, we provide a protocol to generate version controlled updates of the reference index and present its latest release, NNv2, which contains 91 species, compared to 56 species represented in its predecessor NNv1. With the updated reference index, we are able to assign samples to species level that previously could not be assigned. We discuss what happens if a species is not represented in the reference index and how this can be addressed in a future update. To demonstrate the increased power of NNv2, we showcase the assignments of 1789 wild-caught mosquitoes from Madagascar and demonstrate that we can detect within species population structure from the amplicon sequencing data