OBJECTIVES/GOALS: Whole-genome viral sequencing is vital to inform public health and study evolution. Arboviruses evolve in vectors, reservoir hosts, and humans, and require surveillance at all points. We developed a new rigorous method of sequencing that captures whole viral genomes in field-collected and clinical samples. METHODS/STUDY POPULATION: ClickSeq is a novel method of Next Generation Sequencing (NGS) library synthesis using azido-nucleotides to terminate reverse transcription. The cDNA generated can be ligated to sequencing and indexing primers at room temperature using copper (Cu I) and vitamin C. With this approach, we designed primers located ~250 bp apart along the genomes of the arboviruses Chikungunya 37797, Zika Dakar, Yellow Fever Asibi, Dengue serotype 2, West Nile 385-99, and St. Louis Encephalitis Virus (SLEV) clade II. We tested this method with varying viral titers: lab-infected mosquito pools, field-collected mosquito pools from a Texas West Nile and SLEV outbreak, and patient isolates from a Pakistani CHIKV outbreak. The cDNA was sequenced in the UTMB NGS Core and aligned using bowtie. RESULTS/ANTICIPATED RESULTS: The use of a single protocol to capture whole viral genomes including UTRs for multiple viruses from different sample collection styles is ideal for arboviruses. Primers for multiple viruses were pooled and used to sequence mosquito pools. The Tiled ClickSeq method captured whole viral genomes without the need for host depletion. UTRs were captured even when the viral strain used for primer design differed from the resulting strain. Discreet variants were captured in both the hypervariable nsP3 region and the UTR in the patient isolates from the CHIKV outbreak compared to the 2017 outbreak. Texas WNV and SLEV outbreaks are now defined from the 2020 outbreak and can be further tracked to update public health measures and understand viral evolution. DISCUSSION/SIGNIFICANCE: UTRs impact both human and mosquito fitness, leading to further outbreaks. Tiled ClickSeq aims to capture whole viral genomes with a method and cost that can be implemented by public health researchers to understand disease evolution as it happens to update both public health and basic virology to the effects of evolution on arboviruses.