https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366528/
MEGA software version 7.0.1116 was used for the multiple alignments of the sequences retrieved in this study and the sequences from the Global Initiative on Sharing All Influenza Data (GISAID) database (https://www.gisaid.org/) (Supplementary Table (available from http://www.ijmr.org.in/articles/2020/151/2/images/IndianJMedRes_2020_151_2_244_282559_sm7.pdf)). A neighbour-joining tree was generated using the best substitution model (Kimura 2-parameter model) with a bootstrap of 1000 replicates. As per Tang et al17, the circulating SARS-CoV-2 can be grouped into two types (S and L type) based on the two different single-nucleotide polymorphisms (SNPs) at positions 8782 and 28144 in the genome. The S type possesses TC SNPs while the L type possesses CT SNPs at positions 8782 and 28144, respectively. In the present study, it was observed that two sequences from clinical samples (nCoV-763 and nCoV-770) had TT SNPs, while the other sequences had CT as the SNP (L type) (Table II). The TT SNPs have been observed in few of the GISAID sequences, including one of the Kerala genome sequences (nCoV-19/India/31 January 2020) submitted by us earlier. All the isolates of the clinical samples were of L type. Specific amino acid mutations in the nsp3 region, spike protein and ORF8, in general, lead to the formation of V, G and S genetic variants/clades, respectively, as per the recent classification followed by GISAID. It was observed that the clinical samples, as well as the isolates, had the mutation D614G in the spike protein, classifying the study samples and isolates into the G clade (Table II and Fig. 4). No specific substitutions were observed in any of the isolate sequences with respect to the corresponding clinical sample sequences, as these were sequences from a low passage. The sequences of the clinical samples and the isolate from the contact of the infected Delhi-based individual, who returned from Italy, further showed two mutations, R203K and G204R in the nucleocapsid protein (N). Although all strains demonstrated 99.6 per cent identity with the original Wuhan Hu-1 sequence, the role of unique SNPs and mutations in identifying the source of infection needs to be explored.
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