in ORF1ab/1b, S and ORF8 proteins (4) . Subsequent analysis of isolates sequenced in India 61 showed higher mutational frequency in ORF1a from Indian samples (n=25) compared to global 62 isolates (n=3932) (5). This prompted us to look for similar dominant mutations, concurrent or 63 unique, present both globally and unique to nations with higher COVID-19 incidence namely 64
Brazil, India, Italy, Spain, UK and USA and compare it to countries where incidence was low, 65
Australia, Germany, Republic of Congo and Saudi Arabia. Here, we provide a comprehensive 66 report on the evolution of local clusters of mutations explored in a large dataset of >25k 67 genomes (whole genome sequences) from multiple geographical locations around the globe 68 and their co-occurrence or exclusivity with common global mutations. We further ascertained 69 effects of these mutations on the structural stability in the corresponding proteins, some of 70 which interact with host proteins, which were shown to be upregulated in patients with severe 71 COVD-19 symptoms. Identification of such mutations may further aid researchers in 72 developing broad spectrum vaccines or understanding variability in drug efficacy. 73 74 Methods 75 76 High quality, full length whole genome sequences of clinical isolates of SARS-CoV-2 were 77 downloaded from Global Initiative on Sharing All Influenza Data (Table 1) (GISAID;  78 https://www.gisaid.org) (23). Glimmer was used for gene prediction (ORFs), and ORF 79 sequences with Ns were masked out and were not considered for protein translation (24). 80
Throughout, site numbering and genome structure are given using Wuhan-Hu-1 81 (NC_045512.2) as reference genome (https://www.ncbi.nlm.nih.gov/nuccore/NC_045512). 82 MAFFT was used for ORF alignment and mutations were computed using BioInception's in-83 house pipeline based on R (https://cran.r-project.org/) and Python. Alignment regions with 84 >25% gaps against the reference genome were ruled out for amino acid mutational changes. 85
The change or mutation ratio for an ORF alignment is computed by the presence of a mutation 86 divided by total number of sequences in the alignment. This ratio is changed into a percentage. 87
Analysis of co-occurring or exclusive mutations (number of times two mutation occur together 88 in a clinical genome) across various ORFs was defined in terms of log-odds ratio (with p-value 89 and standard error) where a positive ratio indicates co-occurring mutations and negative value 90 denotes an exclusive mutation in the significant part of population using statsmodel 91 Here, we report recurrent occurrence of (i) global and (ii) local (region specific) non-100 synonymous mutations present in >25K SARS-CoV-2 clinical isolates (Table 1) . To achieve 101 better signal-to-noise ratio, we have used a double layered protocol to identify statistically 102 significant signature mutations. In general, recurrent or dominant mutations occurring in ≥10% 103 of the functional genome (ORFs), compared to Wuhan_HU-1 (NC_045512), were classified 104 into mutually exclusive or co-occurring (mutations which also have the propensity to exist 105 together with another mutation). These were defined using log-odds ratio (LOR) where a 106 positive or negative log-odds value represents co-occurring and mutually exclusive mutations, 107 respectively. Vibrational entropy changes were predicted for both wild type and mutant 108 proteins for evaluating the impact of mutations on conformation, flexibility and stability of 109 proteins. The SARS-CoV-2 proteome can be classified into ORF1a, ORF1b (or ORF1ab joint), They were also found to be widely accumulated in North American and European populations 119 Whereas in the USA, T265I (ORF1a) and Q57H (ORF3a) were highly concurrent with P314L-137 D614G mutations. The globally dominant P314L, D614G and Q57H were predicted to be 138 stabilizing mutations for nsp12, S and ORF3a proteins, respectively (Table 2) . Similarly, other 139 mutations were predicted to confer mild stabilization or destabilization effects on respective 140 proteins (∆∆G -0.64 to 0.46 kcal.mol -1) ( Scotland. Mutations in England (G251V in ORF3a), Wales (G392D, A876T in ORF1a; S193I 172 in N protein), Scotland (S194L in N protein) and Northern Ireland (P2144L and T3579I in 173 ORF1a) displayed mutual exclusivity (negative LOR) with P314L, D614G and R203K, G204R 174 mutations. Interestingly in England, the G251V mutation was highly concurrent with L3606F 175 mutation of nsp6. Contrarily in Wales, the G392D, A876T and S193I mutations (which have 176 high propensity to co-occur together, showed mutual exclusivity with the L3606F mutation 177 (Supplementary Data Sheet 1). The P2144L and T3579I mutations showed a high propensity 178 to co-occur in Northern Ireland population. Distribution of co-occurring and exclusive 179 mutations in United Kingdom clearly shows G392D, A876T, S193I for Wales and P2144L, 180 T3579I as specific for Northern Ireland, distinct from England. 181 182 Antigenic drift is believed to occur due to immune pressure resulting in selection of viruses 183 that can escape, hence exploring mutations will aid towards vaccine design and development 184 (8) . Virus circulating in humans undergo antigenic drift, thus necessitating periodic updates to 185 the virus strains contained in the seasonal vaccine to maintain a good match with circulating 186 viruses (9) 