Blast and Alignments
Virus genome sequences are compared for identity differences using 2-nucleotide blasts (Needleman-Wunsch Global Align Nucleotide Sequences) and are done in NCBI website using the SARS-COV2 reference genome (NC_045512, Wuhan-Hu-1). This genome has 100% identity with the genome that was collected on 12/01/2019 (MN908947). From the blast result identity differences in nucleotides are noted or counted over the gaps and other artifacts in alignments [Suppl. Table 1 ]. Average nucleotide differences are calculated for each month by using mean differences in nucleotides of all the genome collected in that month. Average nucleotide difference of a month group over the average nucleotide difference of previous month is considered the mutation rate in that month.
Global blast with 300bp flanking sequences of rs35074065 is done in Ensembl website (www.ensembl.org). ACE2 amino acid (aa) homology percentage for each animal with human is obtained from pre-aligned sequences for orthologues groups in Ensembl. Alignments of ACE2 protein sequences from all animals are done using CLUSTALW at https://npsa-prabi.ibcp.fr/.