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Responding to the ongoing and severe public health threat of viruses of the family Flaviviridae , including dengue, hepatitis C, West Nile, yellow fever, and Zika, demands a greater understanding of how these viruses emerge and spread. Updated phylogenies are central to this understanding. Most cladograms of Flaviviridae focus on specific lineages and ignore outgroups, hampering the efficacy of the analysis to test ingroup monophyly and relationships. This is due to the lack of annotated Flaviviridae genomes, which has gene content variation among genera. This variation makes analysis without partitioning difficult. Therefore, we developed an annotation pipeline for the genera of Flaviviridae ( Flavirirus , Hepacivirus , Pegivirus , and Pestivirus , named "Fast Loci Annotation of Viruses" (FLAVi; http://flavi-web.com/), that combines ab initio and homology-based strategies. FLAVi recovered 100% of the genes in Flavivirus and Hepacivirus genomes. In Pegivirus and Pestivirus , annotation efficiency was 100% except for one partition each. There were no false positives. The combined phylogenetic analysis of multiple genes made possible by annotation has clear impacts over the tree topology compared to phylogenies that we inferred without outgroups or data partitioning. The final tree is largely congruent with previous hypotheses and adds evidence supporting the close phylogenetic relationship between dengue and Zika.

FLAVi: An Enhanced Annotator for Viral Genomes of Flaviviridae