Codon Usage and Phenotypic Divergences of SARS-CoV-2 Genes

Figure A1. Phylogenetic tree from GISAID.

Figure A2. RSCU vectors of coronavirus. Patterns of RSCU vectors for 306 patients with SARS-CoV-2 from different countries (data downloaded from GISAID).

Figure A3. Unrooted ML-based tree of the 30 CoV genomic sequences. The four distinct color-coded clades correspond to the respective genera of CoVs. The SARS-CoV-2 sequence is indicated by a star. The branch lengths depict evolutionary distance. Bootstrap values higher than 50 are shown at the nodes. The scale bar at the lower left denotes the length of nucleotide substitutions per position.

Figure A4. Similarity index (SiD) of SARS-CoV-2, using different host organisms as references. On the horizontal axis, the 13 eukaryotic species that were considered in the comparisons are shown. The host species are ranked in ascending order. CAI values for the bat, rat, hamster, snake, pangolin, and human are higher compared to the other species.

Figure A5. Heatmaps of RSCU vectors for genes RdRP (upper panel) and S (bottom panel).

Figure 1. Clustering of the relative synonymous codon usage (RSCU) vectors associated with the 30 coronaviruses. Human coronaviruses are shown in red. The newly identified SARS-CoV-2 coronavirus is closer to HCoV-229E and SARSr-CoV in terms of codon usage, as measured by their RSCU vectors. Heatmap was drawn with the CIMminer software [20], which uses Euclidean distances and the average linkage algorithm.

Figure 2. Z-score values. Z-score is calculated for two codon bias indeces: effective number of codons (ENC) and the competition adaptation index (CAI). CAI values are calculated by considering the hosts specified in Table 3 by Woo et al. [4]. Regarding SARS-CoV-2, we considered a human host. In red, we show the human coronaviruses. Several coronaviruses have a codon usage preference values higher than the average value of the family (|Z-score| > 3). The statistically significant differences are marked with asterisks. In particular, SARS-CoV-2 genes have average values of CAI and ENC that are higher than the average of all coronaviruses. (*): |Z-score| > 3.

Figure 3. CAI values of SARS-CoV-2 for different hosts. On the horizontal axis, the 12 eukaryotic species are shown that were considered in the comparisons. The host species are ranked in ascending order. CAI values for snake and human hosts are higher than those for other hosts.

Figure 4. RSCU vectors of three different coronavirus genes. Heatmaps confirm that the RSCU patterns of the newly identified coronavirus SARS-CoV-2 sequence are more related to those of SARSr-CoV and SARSr-Rh-BatCoV HKU3 for genes E, M and N.

Figure 5. ENC-plots of genes M, N, S, E and RdRP. In these plots, each point corresponds to a single gene. The black-dotted lines in all panels are plots of Wright’s theoretical curve corresponding to codon usage biases (CUBs) that occur merely due to mutational bias (no selective pressure). Red dots represent SARS-CoV-2 genes.

Figure 6. Violin plots of the distances of genes M, N, S, E and RdRP from Wright’s theoretical curve.

Figure 7. Neutrality plot of genes M, N, S, E and RdRP. In these plots, each point corresponds to a single gene in a virus. The solid black lines in all panels are the bisectors corresponding to those CUBs occurring merely due to mutational bias (no selective pressure). The black-dotted lines are the linear regressions. Red dots represent SARS-CoV-2 genes.