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Generated in collaboration by the Loman, Andersen, and Grubaugh labs. For general use of the protocol and primer design, please cite: Quick, J. et al. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nature Protocols 12 (6), 1261-1276 (2017)https://www.nature.com/nprot/journal/v12/n6/abs/nprot.2017.066.html For measuring intrahost virus genetic diversity and calling variants using iVar, please cite: Grubaugh, ND. et al. An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar. Genome Biology 20,8 (2019) https://genomebiology.biomedcentral.com/articles/10.1186/s13059-018-1618-7 The general approach to this protocol is to amplify the virus genome in small (~400 bp) overlapping fragments using two highly multiplexed PCR reactions (where the overlapping segments are in separate reactions). The amplicons are combined after PCR and are the correct size for library preparation and paired-end 250 nt sequencing using the Illumina MiSeq. Version 4 notes: This protocol has been updated to include considerations for measuring intra-batch contamination through the introduction of sample-specific barcoded spike-ins. All of the primers are now listed in a separate spreadsheet. We currently have 400 bp amplicon schemes for Zika virus, West Nile virus (North America lineage I genotype), Usutu virus, and chikungunya virus (ECSA genotype). More will be made available soon. You can build your own primer sets by using Primal Scheme. Overview of tiled virus amplicon sequencing protocol