Analysis of 3.5 million SARS-CoV-2 sequences reveals unique mutational trends with consistent nucleotide and codon frequencies.

BACKGROUND: Since the onset of the SARS-CoV-2 pandemic, bioinformatic analyses have been performed to understand the nucleotide and synonymous codon usage features and mutational patterns of the virus. However, comparatively few have attempted to perform such analyses on a considerably large cohort of viral genomes while organizing the plethora of available sequence data for a month-by-month analysis to observe changes over time. Here, we aimed to perform sequence composition and mutation analysis of SARS-CoV-2, separating sequences by gene, clade, and timepoints, and contrast the mutational profile of SARS-CoV-2 to other comparable RNA viruses. METHODS: Using a cleaned, filtered, and pre-aligned dataset of over 3.5 million sequences downloaded from the GISAID database, we computed nucleotide and codon usage statistics, including calculation of relative synonymous codon usage values. We then calculated codon adaptation index (CAI) changes and a nonsynonymous/synonymous mutation ratio (dN/dS) over time for our dataset. Finally, we compiled information on the types of mutations occurring for SARS-CoV-2 and other comparable RNA viruses, and generated heatmaps showing codon and nucleotide composition at high entropy positions along the Spike sequence. RESULTS: We show that nucleotide and codon usage metrics remain relatively consistent over the 32-month span, though there are significant differences between clades within each gene at various timepoints. CAI and dN/dS values vary substantially between different timepoints and different genes, with Spike gene on average showing both the highest CAI and dN/dS values. Mutational analysis showed that SARS-CoV-2 Spike has a higher proportion of nonsynonymous mutations than analogous genes in other RNA viruses, with nonsynonymous mutations outnumbering synonymous ones by up to 20:1. However, at several specific positions, synonymous mutations were overwhelmingly predominant. CONCLUSIONS: Our multifaceted analysis covering both the composition and mutation signature of SARS-CoV-2 gives valuable insight into the nucleotide frequency and codon usage heterogeneity of SARS-CoV-2 over time, and its unique mutational profile compared to other RNA viruses.

Codon Usage Bias in Autophagy-Related Gene 13 in Eukaryotes: Uncovering the Genetic Divergence by the Interplay Between Nucleotides and Codon Usages.

Synonymous codon usage bias is a universal characteristic of genomes across various organisms. Autophagy-related gene 13 (atg13) is one essential gene for autophagy initiation, yet the evolutionary trends of the atg13 gene at the usages of nucleotide and synonymous codon remains unexplored. According to phylogenetic analyses for the atg13 gene of 226 eukaryotic organisms at the nucleotide and amino acid levels, it is clear that their nucleotide usages exhibit more genetic information than their amino acid usages. Specifically, the overall nucleotide usage bias quantified by information entropy reflected that the usage biases at the first and second codon positions were stronger than those at the third position of the atg13 genes. Furthermore, the bias level of nucleotide 'G' usage is highest, while that of nucleotide 'C' usage is lowest in the atg13 genes. On top of that, genetic features represented by synonymous codon usage exhibits a species-specific pattern on the evolution of the atg13 genes to some extent. Interestingly, the codon usages of atg13 genes in the ancestor animals (Latimeria chalumnae, Petromyzon marinus, and Rhinatrema bivittatum) are strongly influenced by mutation pressure from nucleotide composition constraint. However, the distributions of nucleotide composition at different codon positions in the atg13 gene display that natural selection still dominates atg13 codon usages during organisms' evolution.

The nucleotide usages significantly impact synonymous codon usage in Mycoplasma hyorhinis.

Five annotated genomes of Mycoplasma hyorhinis were analyzed for clarifying evolutionary dynamics driving the overall codon usage pattern. Information entropy used for estimating nucleotide usage pattern at the gene level indicates that multiple evolutionary dynamics participate in forcing nucleotide usage bias at every codon position. Moreover, nucleotide usage bias directly contributes to synonymous codon usage biases with two different extremes. The overrepresented codons tended to have A/T in the third codon position, and the underrepresented codons strongly used G/C in the third position. Furthermore, correspondence analysis and neutrality plot reflect an obvious interplay between mutation pressure and natural selection mediating codon usage in M. hyorhinis genome. Due to significant bias in usages between A/T and G/C at the gene level, different selective forces have been proposed to contribute to codon usage preference in M. hyorhinis genome, including nucleotide composition constraint derived from mutation pressure, translational selection involved in natural selection, and strand-specific mutational bias represented by different nucleotide skew index. The systemic analyses of codon usage for M. hyorhinis can enable us to better understand the mechanisms of evolution in this species.

Significance and roles of synonymous codon usage in the evolutionary process of Proteus.

Proteus spp. bacteria frequently serve as opportunistic pathogens that can infect many animals and show positive survival and existence in various natural environments. The evolutionary pattern of Proteus spp. is an unknown topic, which benefits understanding the different evolutionary dynamics for excellent bacterial adaptation to various environments. Here, the eight whole genomes of different Proteus species were analyzed for the interplay between nucleotide usage and synonymous codon usage. Although the orthologous average nucleotide identity and average nucleotide identity display the genetic diversity of these Proteus species at the genome level, the principal component analysis further shows that these species sustain the specific genetic niche at the aspect of synonymous codon usage patterns. Interestingly, although these Proteus species have A/T rich genes with underrepresented G (guanine) or C (cytosine) at the third codon positions and overrepresented A or T at these positions, some synonymous codons with A or T end are obviously suppressed in usage. The overall codon usage pattern reflected by the effective number of codons (ENC) has a significantly positive correlation with GC3 content (GC content at the third codon position), and ENC has a significantly negative correlation with the adaptation index for these species. These results suggest that the mutation pressure caused by nucleotide composition constraint serves as a dominant evolutionary dynamic driving evolutionary trend of Proteus spp., along with other selections related to natural selection, replication and fine-tune translation, and so on. Taken together, the analyses help to understand the evolutionary interplay between nucleotide and codon usage at the gene level of Proteus.

Analyses of nucleotide, synonymous codon and amino acid usages at gene levels of Brucella melitensis strain QY1.

Brucella melitensis is the causative pathogen of the zoonotic disease brucellosis in China. This work focused on analyses of genetic features represented by nucleotide, synonymous codon and amino acid usages at gene levels of B. melitensis strain QY1 isolated from China. Although nucleotide usage biases at different codon positions all work on synonymous codon usage bias, nucleotide usage biases at the 1st and 3rd positions play more important roles in codon usages. Mutation pressure caused by nucleotide composition constraint influences the formation of over-representative synonymous codons, but neighboring nucleotides surrounding a codon strongly influence synonymous codon usage bias for B. melitensis strain QY1. There is significant correlation between amino acid usage bias and hydropathicity of proteins for B. melitensis strain QY1. Compared with different Brucella species about synonymous codon usage patterns, synonymous codon usages are not obviously influenced by hosts. Due to nucleotide usage bias at the 1st codon position influencing synonymous codon and amino acid usages, good interactions among nucleotide, synonymous codon and amino acid usages exist in the evolutionary process of B. melitensis.

Prevalence and genetic features of hepatitis E virus in swine, in Gansu, China.

The prevalence of hepatitis E virus (HEV) in domestic swine threats the public health, due to the risk of zoonotic transmission. In this study, we investigated the prevalence and genetic features of HEV in swine, in Gansu, which is geographically an important province in China. 377 fecal samples were collected from pig farms in Gansu province of China and tested for HEV RNA by RT-PCR. The prevalence rate is about 23% in pig farms of Gansu province. By RT-nPCR, 5' and 3' RACE methods, a whole genome with 7,284 nt in length, termed as swCH189, was obtained and investigated by nucleotide, codon and amino acid usage analyses. Phylogenetic tree analysis classified swCH189 strain into genotype 4e. Although this subtype has never been reported in the local population, genotype 4 is known as zoonotic and more pathogenic than other genotypes. According to the synonymous codon usage patterns of the three open reading frames (ORFs) of swCH189, compositional constraint mainly influences usage patterns of synonymous codons with A-end, while natural selections dominate in usage patterns of synonymous codons with G, C and U-ends. Genetic diversities of each ORFs, in respect to codon and amino acid usage patterns, are closely related to other members of genotype 4 in general, but with distinct features. Thus, the prevalence rate and the genetic features of HEV determined by this study are important for the prevention of zoonotic transmission of HEV from swine to human in this region as well as in China.

Comparative genomic analysis for nucleotide, codon, and amino acid usage patterns of mycoplasmas.

The evolutionary factors in influencing the genetic characteristics of nucleotide, synonymous codon, and amino acid usage of 18 mycoplasma species were analyzed. The nucleotide usage at the 1st and 2nd codon position which determines amino acid composition of proteins has a significant correlation with the total nucleotide composition of gene population of these mycoplasma species, however, the nucleotide usage at the 3rd codon position which affects synonymous codon usage patterns has a slight correlation with either the total nucleotide composition or the nucleotide usage at the 1st and 2nd codon position. Other evolutionary factors join in the evolutionary process of mycoplasma apart from mutation pressure caused by nucleotide usage constraint based on the relationships between effective number of codons/codon adaptation index and nucleotide usage at the 3rd codon position. Although nucleotide usage of gene population in mycoplasma dominates in forming the overall codon usage trends, the relative abundance of codon with nucleotide context and amino acid usage pattern show that translation selection involved in translation accuracy and efficiency play an important role in synonymous codon usage patterns. In addition, synonymous codon usage patterns of gene population have a bigger power to represent genetic diversity among different species than amino acid usage. These results suggest that although the mycoplasmas reduce its genome size during the evolutionary process and shape the form, which is opposite to their hosts, of AT usages at high levels, this kind organism still depends on nucleotide usage at the 1st and 2nd codon positions to control syntheses of the requested proteins for surviving in their hosts and nucleotide usage at the 3rd codon position to develop genetic diversity of different mycoplasma species. This systemic analysis with 18 mycoplasma species may provide useful clues for further in vivo genetic studies on the related species.

Unique features of nucleotide and codon usage patterns in mycoplasmas revealed by information entropy.

Currently, the comparison between GC usage pattern at the 3rd codon position and codon usage index is commonly used to estimate the roles of evolutionary forces in shaping synonymous codon usages, however, this kind of analysis often losses the information about the role of A/T usage bias in shaping synonymous codon usage bias. To overcome this limitation and better understand the interplay between nucleotide and codon usages for the evolution of bacteria at gene levels, in this study, we employed the information entropy method with some modification to estimate roles of nucleotide compositions in the overall codon usage bias for 18 mycoplasma species in combination with Davies-Bouldin index. At gene levels, the overall nucleotide usage bias represents A content as the highest, followed by T, G and C for mycoplasmas, resulting in a low GC content. This feature is universal across these species derived from different hosts, suggesting that the hosts have the limited impact on nucleotide usage bias of mycoplasmas. Information entropy and Davies-Bouldin index can better reveal that the nucleotide usage bias at the 3rd codon position is essential in shaping the overall nucleotide bias for all given mycoplasmas except M. pneumoniae M129. Davies-Bouldin index revealed that the 1st and 2nd codon position play more important role in synonymous codon usage bias than that of the 3rd position at gene levels. To our knowledge, this is the first comprehensive investigation into cooperation between nucleotide and codon usages for mycoplasma and extends our knowledge of the mechanisms that contribute to codon usage and evolution of this microorganism.

Transcription-associated mutational pressure in the Parvovirus B19 genome: Reactivated genomes contribute to the variability of viral populations.

In this study we used non-overlapping parts of the two long open reading frames coding for nonstructural (NS) and capsid (VP) proteins of all available sequences of the Parvovirus B19 subgenotype 1a genome and found out that the rates of A to G, C to T and A to T mutations are higher in the first long reading frame (NS) of the virus than in the second one (VP). This difference in mutational pressure directions for two parts of the same viral genome can be explained by the fact of transcription of just the first long reading frame during the lifelong latency in nonerythroid cells. Adenine deamination (producing A to G and A to T mutations) and cytosine deamination (producing C to T mutations) occur more frequently in transcriptional bubbles formed by DNA "plus" strand of the first open reading frame. These mutations can be inherited only in case of reactivation of the infectious virus due to the help of Adenovirus that allows latent Parvovirus B19 to start transcription of the second reading frame and then to replicate its genome by the rolling circle mechanism using the specific origin. Results of this study provide evidence that the genomes reactivated from latency make significant contributions to the variability of Parvovirus B19.

Opposite nucleotide usage biases in different parts of the Corynebacterium diphtheriae spaC gene.

In this work we described a bacterial open reading frame with two different directions of nucleotide usage biases in its two parts. The level of GC-content in third codon positions (3GC) is equal to 40.17 ± 0.22% during the most of the length of Corynebacterium diphtheriae spaC gene. However, in the 3'-end of the same gene (from codon #1600 to codon #1873) 3GC level is equal to 64.61 ± 0.91%. Using original methodology ('VVTAK Sliding window' and 'VVTAK VarInvar') we approved that there is an ongoing mutational AT-pressure during the most of the length of spaC gene (up to codon #1599), and there is an ongoing mutational G-pressure in the 3′-end of spaC. Intragenic promoters predicted by three different methods may be the cause of the differences in preferable types of nucleotide mutations in spaC parts because of their autonomous transcription.