2.1. Consensus Sequence ORF Generation and Entropy Calculation
A total of 1731 full-length SARS-CoV-2 sequences were downloaded from NCBI (30 April 2020, txid2697049, minimum length = 29,000 bp) and aligned using MAFFT [44]. The alignment was visually inspected and curated using Genbank NC_045512.2 as a coordinate reference [45]. A nucleotide consensus sequence was generated by keeping all nucleotides present in at least 25% of the sequences in the alignment. The amino acid consensus sequence was then created by using NC_045512.2 annotated Open Reading Frames (ORFs) plus additional ORFs described in Finkel et al. [46] using the Biostrings R package. Mixed nucleotide positions were either resolved if they were synonymous or flagged for downstream analysis. Positional entropy was calculated at the amino acid level both as the standard and 22-aminoacid-normalized Shannon entropy for every ORF using Bio3d R package on the alignment [47], and afterward, the mean OLP normalized entropy was calculated.