Multi-Parameter Genomic Evolution of SARS-CoV-2 Across Ten US States During the Vaccination Era, 2020–2025

SARS-CoV-2 has adapted continuously since 2019, yet no multi-parameter framework has characterized this trajectory across geographically distinct United States populations. We present a systematic 13-parameter analysis of 137,059 high-quality genomes from ten diverse US states across three phases: pre-vaccination (January–December 2020), early vaccination (January–June 2021), and sustained immune pressure (July 2021–December 2025), the latter dominated by the high-volume Delta period with an early-Omicron contribution. Approximately 5000 genomes were retrieved per state per phase from GISAID EpiCoV under stringent quality filters, and each phase dataset was partitioned into three subsets, giving 90 subsamples in total, to quantify subsampling stability. Parameters spanned genome-wide mutation rate, dN/dS and Ti/Tv, site-specific selection (HyPhy FUBAR), spike and ACE2 contact-residue profiling, insertion–deletion characterization, furin cleavage site evolution, N-linked glycosylation dynamics, lineage–mutation concordance, amino acid physicochemical shifts, and protein-wise dN/dS ranking and mutation load. Genome-wide mutation rate rose monotonically in all ten states, from 0.050% to 0.149%, and dN/dS rose from 0.611 to 1.103, crossing neutrality in nine of ten states by Phase 3. Amino acid mutation load increased 3.95-fold, from 9.12 to 36.03 substitutions per sequence, while Ti/Tv fell from 3.69 to 2.11. Spike position 142 was diversifying in all ten states in Phase 3, with positions 484, 452, and 478 recurring in 70, 60, and 60 percent of states. ORF8 followed a spike-then-collapse trajectory (dN/dS 3.43, 14.23, 1.01), consistent with progressive loss of function. The reproducibility of these effects across ten geographically distinct state populations establishes a state-level baseline for SARS-CoV-2 immune escape.