key: cord-0911591-13tc3loo authors: Wang, Rui; Hozumi, Yuta; Yin, Changchuan; Wei, Guo-Wei title: Decoding SARS-CoV-2 Transmission and Evolution and Ramifications for COVID-19 Diagnosis, Vaccine, and Medicine date: 2020-06-12 journal: J Chem Inf Model DOI: 10.1021/acs.jcim.0c00501 sha: 9cbdf53fc5faaec953ff31e069cccbb1a0558e64 doc_id: 911591 cord_uid: 13tc3loo [Image: see text] Tremendous effort has been given to the development of diagnostic tests, preventive vaccines, and therapeutic medicines for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much of this development has been based on the reference genome collected on January 5, 2020. Based on the genotyping of 15 140 genome samples collected up to June 1, 2020, we report that SARS-CoV-2 has undergone 8309 single mutations which can be clustered into six subtypes. We introduce mutation ratio and mutation h-index to characterize the protein conservativeness and unveil that SARS-CoV-2 envelope protein, main protease, and endoribonuclease protein are relatively conservative, while SARS-CoV-2 nucleocapsid protein, spike protein, and papain-like protease are relatively nonconservative. In particular, we have identified mutations on 40% of nucleotides in the nucleocapsid gene in the population level, signaling potential impacts on the ongoing development of COVID-19 diagnosis, vaccines, and antibody and small-molecular drugs. The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed crucial threats to public health and the world economy since it was detected in Wuhan, China, in December 2019. 15 As of June 1, 2020, 6 057 853 cases of COVID-19 have been reported in more than 200 countries and territories, resulting in more than 371 166 deaths. 27 However, there have been no signs of slowing down nor relief at this monument partially due to the fact there are no specific anti-SARS-CoV-2 drugs and effective vaccines. SARS-CoV-2 is a positive-strand RNA virus that belongs to the beta coronavirus genus. The genomic information underpins the development of antiviral medical interventions, prophylactic vaccines, and viral diagnostic tests. The first SARS-CoV-2 genome was reported on January 5, 2020. 28 It has a genome size of 29.99 kb, which encodes for multiple nonstructural and structural proteins. The leader sequence and ORF1ab encode nonstructural proteins for RNA replication and transcription. Among various nonstructural proteins, viral papain-like (PL) proteinase, main protease (or 3CL protease), RNA polymerase, and endoribonuclease are the common targets in antiviral drug discovery. Yet, it typically takes more than ten years to put an average drug to the market. The downstream regions of the genome encode structural proteins, including spike (S) protein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein. Notably, S-protein uses one of its two subunits to bind directly to the host receptor angiotensin-converting enzyme 2 (ACE2), enabling virus entry into host cells. 29 The N protein, one of the most abundant viral proteins, can bind to the RNA genome and is involved in replication, assembly, and host cellular response during viral infection. 13 As a virulence factor, the E protein is a small integral membrane protein that regulates cell stress response and apoptosis and promotes inflammation. 4 The structural protein, especially, the S protein, is the candidate antigen for vaccine and antibody drug development. Developing safe and effective vaccines is urgently needed to prevent the spread of SARS-CoV-2. However, it typically takes over one year to design and test a new vaccine. Furthermore, the replication in RNA viruses, such as Influenza A, is subject to errors, 14 except nidoviruses. Coronaviruses, a kind of nidoviruses, have the ability to proofread their genomes during their genetic replication and recombination. 6 Therefore, SARS-CoV-2 might not mutate as fast as Influenza A viruses do, but still has heterogeneous and dynamic populations. The SARS-CoV-2 genome undergoes rapid mutations that are partially stimulated as a response to the challenging immunological environments arising from its transmission to the COVID-19 patients of different races, ages, and medical conditions. The vaccine developed at one time may not be effective for mitigating the infection by new mutated virus isolates. An alarming fact is that many of these mutations may devastate the ongoing effort in the development of effective medicines, preventive vaccines, and diagnostic tests. Accurate identification of the antigens and their mutations represents the most important roadblock in developing effective vaccines against COVID-19. For example, different vaccines are needed for various geographic locations due to predominant mutations in the corresponding regions. In COVID-19 diagnosis, the diagnostic kits are designed using two major methods: serological tests and molecular tests. Serological tests are to detect specific neutralizing antibodies from COVID-19 infections. Molecular diagnoses look for specific COVID-19 pathogenic genes, which usually rely on the polymerase chain reaction (PCR). Because of the fast mutations of the SARS-CoV-2 genome, genotyping analysis of SARS-CoV-2 may optimize the PCR primer design to detect SARS-CoV safely and to reduce the risk of false-negatives caused by genome sequence variations. In addition, the genotyping analysis may also reveal those highly conserved regions with very few mutations, which can be selected as a target sequence for clinical diagnosis and effective drug therapy. The evolution pattern through the highly frequent mutations of SARS-CoV-2 can be observable on short time scales. In the early infection period (i.e., February 2020), the SARS-CoV-2 variants were clustered as S and L types. 23 Recent genotyping analysis reveals a large number of mutations in various essential genes encoding the S protein, the N protein, and the RNA polymerase in the SARS-CoV-2 population. 30 Monitoring the evolutionary patterns and spread dynamics of SARS-CoV-2 is of great importance for COVID-19 control and prevention. Mutations occur in many different ways. Some mutations occur randomly. Other mutations are enforced by the host immune system surveillance, which induces viral responses. The most preserved mutations and viral evolution can be regarded as the result of the dynamic equilibrium between the random perturbation, host cell defense, and viral fitness. Therefore, the faster and wider the SARS-CoV-2 spread, the more frequent and diverse the mutations will be. The tracking and analysis of COVID-19 dynamics, transmission, and spread are of paramount importance for winning the ongoing battle against COVID-19. Genetic identification and characterization of the geographic distribution, intercontinental evolution, and global trends of SARS-CoV-2 are the most effective approaches for studying COVID-19 genomic epidemiology and offer the molecular foundation for region-specific SARS-CoV-2 vaccine design, drug discovery, and diagnostic development. 16 For example, different vaccines for the shell can be designed according to predominant mutations. This work provides the most comprehensive genotyping to reveal the transmission trajectory and spread dynamics of COVID-19 to date. Based on genotyping 15 140 SARS-CoV-2 genomes from the world as of June 1, 2020, we trace the COVID-19 transmission pathways and analyze the distribution of the subtypes of SARS-CoV-2 across the world. We use Kmeans methods to cluster SARS-CoV-2 mutations, which provides updated molecular information for the region-specific design of vaccines, drugs, and diagnoses. Our clustering results show that, globally, there are at least six distinct subtypes of SARS-CoV-2 genomes. While, in the U.S., there are four significant SARS-CoV-2 genotypes. We introduce mutation hindex and mutation ratio to characterize conservative and nonconservative proteins and genes. We unveil the unexpected nonconservative genes and proteins, rendering a warning for the current development of diagnostic tests, preventive vaccines, and therapeutic medicines. 2.1. COVID-19 Evolution and Clustering. Tracking the SARS-CoV-2 transmission pathways and analyzing the spread dynamics are critical to the study of genomic epidemiology. Temporospatially clustering the genotypes of SARS-CoV-2 in the transmission provides insights into diagnostic testing and vaccine development in disease control. In this work, we retrieve and genotype 15 140 SARS-CoV-2 isolates from the world as of June 1, 2020. There are 8309 single mutations in 15 140 SARS-CoV-2 isolates. Based on these mutations, we classify and track the geographical distributions of 15 140 genotype isolates by K-means clustering. The SARS-CoV-2 genotypes, represented as single nucleotide polymorphism (SNP) variants, are clustered as six groups in the world, including the U.S.. In particular, the genotypes in the U.S. are further clustered into four groups. Table 1 lists the comutations with the highest number of descendants in different clusters in the world. Optimal clustering groups are established using the Elbow method in the K-means clustering algorithm (Supporting Information). The detailed distribution of the SNP variants from the world for each cluster is provided in the Supporting Information. The SNP variant clusters from 76 countries that have a high number of the COVID-19 cases are listed in Table 2 . The pie chart plot on the world map is described in Figure 1 which was created by Highcharts (https://www.highcharts.com/maps/demo). The light blue, dark blue, green, red, purple, and yellow represent the Cluster I, II, III, IV, V, and VI, respectively. The color of the dominated cluster decides the base color of each country. The geographic distribution of the SNP variant clusters reflects the approximate transmission pathways and spread dynamics across the world. Several findings can be made from Furthermore, we analyze the statistics of SNP variants located in the United States. In Table 3 Table 4 lists the co-mutations with the highest number of descendants in different clusters in the United States. Notably, several findings on the genotypes of clusters in the US are as follows: 1. The subtypes of SARS-CoV-2 in all of the clusters are spreading out among the west coast states. Especially, the state of Washington is dominated by cluster B. 2. East coast states are dominated by subtypes from clusters A and C, especially in New York. 3. The subtypes of SARS-CoV-2 in cluster A are spread throughout the United States. Figure 2 is the pie chart plot of the four distinct clusters in the US, which was also created by Highcharts. The colors, blue, red, yellow, and green represent clusters A, B, C, and D, respectively. The base color of each state corresponds to its dominant cluster. We note that cluster D in the U.S. is derived from cluster V in the world, with an additional mutation at the leader sequence 241. The high spread in New York is consistent with the high transmission of SARS-CoV-2 in European countries, where the subtype in cluster V is predominant. 2.2. Ramifications for COVID-19 Diagnosis, Vaccine, and Medicine. 2.2.1. Protein-Specific Mutation Analysis. Figures 3 and 4 depict the distribution and frequencies of SNP mutations of SARS-CoV-2 isolates from 15 140 genome samples in the world with respect to the reference genome of January 5, 2020. The statistics of single mutations on various SARS-CoV-2 proteins that occurred in the recorded genomes between January 5, 2020, and June 1, 2020, are listed in Table 5 . The spike protein has the highest number of mutations on gene of 1004, while the envelope protein has the lowest number of mutations of 52. Since the sizes of proteins vary dramatically from 1273 for the spike protein to 75 for the envelope protein, it is useful to consider the mutation ratio, i.e., the number of mutations per residue. In this category, the RNA-dependent RNA polymerase has the lowest score of 0.217, whereas the nucleocapsid protein has the highest score of 0.400, i.e, 503 mutations on its 1257 nucleotides (419 residues). Note that main protease has the second-lowest mutation ratio of 0.221, indicating its conservative nature. Another relatively conservative protein judged by the mutations ratio in terms of gene is the envelope protein, the MR Gene = 0.231. Counting the number of single mutations and mutation ratio does not reflect the fact that some mutations occur numerous times over genome samples while other mutations may happen only on a few genome samples. To account for the frequency effect of mutations, we introduce a mutation h-index to measure both the number of mutations and the frequency of mutations of a given protein or genetic section. It is defined as the maximum value of h such that the given protein genetic section has h single mutations that have each occurred at least h times. It is very interesting to note from Table 5 that the mutation h-index correlates very well with the number of mutations on gene; the Pearson correlation coefficient is 0.711. Specifically, N protein has both the highest MR Gene of 0.400 and the highest h-index of 33, suggesting that it is the most nonconservative protein in SARS-CoV-2 genomes. In contrast, the envelope protein has the third-lowest number of mutations per residues of 0.231 and the lowest h-index of 9, indicating its relatively conservative nature. By combining the number of mutations per residue and the mutation h-index, we report that the most conservative SARS-CoV-2 proteins is the envelope. It is found that the most nonconservative SARS-CoV-2 proteins are (1) the nucleocapsid protein, (2) the spike protein, and (3) the papain-like protease. The number of mutations in terms of gene (NM Gene ) and the number of mutations in terms of protein (NM Pro ) we reported are accumulated numbers that from all of the 15 140 genome isolates. If we focus on the single genome isolate, the maximum number of mutations on the whole genome sequence is 24. 2.2.2. Diagnosis. Real-time RT-PCR (rRT-PCR) is routinely used in the qualitative detection of nucleic acid from SARS-CoV-2 for diagnostic testing COVID-19. 3,24 The primers used in the rRT-PCR are critical for the precise diagnosis of COVID-19 and the discovery of new strains. The primer sequences are specially designed for amplifying the conserved regions across the different existing strains for high specificity and sensitivity and also are subject to genotype changes as the SARS-CoV-2 coronavirus evolves. In diagnostic testing COVID-19, many rRT-PCR primers are designed to detect for three perceived conservative SARS-CoV-2 regions: (1) RNA-dependent RNA polymerase (RdRP) gene in ORF1ab region, (2) the E protein gene, and (3) the N protein gene. 3 Our genotyping statistics given in Table 5 indicate that the nucleocapsid protein is the worst choice. Among the four structural proteins of SARS-CoV-2, the spike surface glycoprotein (S) of 1273 amino acid residues, nucleocapsid protein (N) of 419 amino acid residues, membrane protein (M) of 222 amino acid residues, and envelope protein (E protein) of 75 amino acid residues, the S Journal of Chemical Information and Modeling pubs.acs.org/jcim Article protein is the most divergent with 1004 unique mutations among the 15 140 SARS-CoV-2 genomes. The N protein has 503 unique mutations, and the envelope (E) protein has 52 mutations. Considering the lengths of the proteins, all the four structural proteins undergo many mutations. The RdRP gene, which is often used in diagnostic testing COVID-19, also has 607 mutations. Therefore, all three regions in the routine rRT-PCR target, namely RdRP, the N protein gene, and the E protein genes, have significant mutations. Precise and robust diagnosis tools must be re-established according to the conserved regions and predominated mutations in the SARS-CoV-2 genomes detailed in the Supporting Information. 2.2.3. Vaccine Development. Vaccines are mostly associated with the S protein. Compared to SARS-CoV, SARS-CoV-2 has a unique furin cleavage site, where four amino acid residues (PRRA) are inserted into the S1−S2 junction region 681−684 of the S protein. 25 The furin cleavage site is crucial for zoonotic transmission of SARS-CoV-2. 7 This study reveals crucial mutations near the S1−S2 junction region in the S protein, including 23403A>G-(D614G), 23422C>T-(V620V), 23575C>T-(C671C), 23586A>G-(Q675R), 23611G>A-(R683R), 23707C>T-(P715P), 23731C>T-(T723T), 23849T>C-(L763L), and 23929C>T-(Y789Y). Moreover, these mutations of the S protein SARS-CoV-2 are located at the epitope region, corresponding to the regions 469−882 and 599−620 in SARS-CoV. 19 Additionally, many mutated amino acids are on the receptorbinding domain (RBD) of the S protein, as shown in Figure 5 . Unfortunately, the S protein is the second most nonconservative protein in the genome based on the number of mutations per residue and mutation h-index. In fact, about half of the receptor-binding domain residues of the S proteins have had mutations in the past few months as shown in Figure 6 . Because the surface accessibility of epitope is also important for the interaction of antibody and antigen, these mutations are critical for the antigenicity of the S protein. Convalescent COVID-19 patients show a neutralizing antibody response after infection, which is directed mostly against the S protein. 18 The neutralizing antibody responses against SARS-CoV-2 could give some defense against SARS-CoV-2 infection, thus having implications for preventing SARS-CoV-2 outbreaks. The divergence of S proteins and the nonconserved regions of the S proteins might contribute to the antigenicity. The highly frequent mutations identified in the S protein may reduce the durability of the SARS-CoV-2 vaccine's immunity or undermine the current development of vaccines. The existing mutations must be considered when designing a new vaccine. Additionally, a cocktail of multiple vaccines has a better chance of preventing COVID-19 infections. 2.2.4. Drug Discovery. Unfortunately, there is no specific effective drug for SARS-CoV-2 at this point. Potential drugs include small-molecular drugs and antibody drugs. Much of the effort in small-molecular drug discovery focuses on SARS-CoV-2 nonstructural proteins. Among the major nonstructural proteins of SARS-CoV-2, the main protease of 306 amino acids has 78 mutations with 0.255 mutations per residue and the mutation h-index of 16, RNA polymerase of 932 amino acids has 228 mutations with 0.245 mutations per residue and the mutation h-index of 21, and papain-like protease of 945 amino acids has 105 mutations with 0.333 mutations per residue and the mutation h-index of 10. In fact, the main protease is the most popular drug target because there are no similar known genes in the human genome, which implies that SARS-CoV-2 main protease inhibitors will likely be less toxic. 10 The present study suggests that the main protease is the second most conservative protein. Therefore, it remains the most attractive target for drug discovery. Therapeutic antibodies got started from cancer treatments and now applies to infectious diseases by targeting pathogens. 1 Antibody drugs are highly specificity and versatile in the treatment of infectious diseases. Their working principle involves the host immune system. The time used to develop antibody therapeutics are usually considerably shorter than that used to develop a vaccine. Many SARS-CoV-2 antibody drugs are isolated from patient blood and target the S proteins. Although there many binding sites on the S protein that antibodies can target, the ones that are most effective in neutralizing SARS-CoV-2 block the receptor-binding domain (RBD) of the host cell angiotensin-converting enzyme 2 (ACE2) receptor. The RBD is a dongle-shape protein at the Journal of Chemical Information and Modeling pubs.acs.org/jcim Article end of the virus's spikes. As mentioned above, there are many mutations on the S proteins. The RBD is also prone to mutations. Some mutations that break hydrogen bonds and/or salt bridges in antibody−antigen interactions will have a large impact. However, silent mutations, such as those that replace hydrophobic residues with other hydrophobic residues, will typically have little effect. To avoid the failure of one specific antibody, the cocktail treatments that include several different antibodies might be required to treat SARS-CoV-2 that undergoes antigenic mutations. The SARS-CoV-2 spike glycoprotein, or S protein, comprised of two subunits, S1 and S2, of very different properties; 25 see Figure 5 . Among them, the S1 subunit, as shown in Figure 5 , contains the receptor-binding domain (RBD) responsible for binding to the host cell receptor angiotensin-converting enzyme 2 (ACE2). The RBD is also the common binding domain for antibodies. The S2 subunit offers the structural support of the S protein and mediates fusion between the viral and host cell membranes. After the fusion, the virus releases the viral genome into the host cell. The S1 RBD protein plays key parts in the induction of neutralizing-antibody and T-cell responses, as well as protective immunity. However, S2 and extracellular domain (ECD) of spike protein and their combination are commonly used in recombinant proteins in SARS-CoV-2 antibody development. As shown in Table 5 , the S protein is the most heterogeneous structural protein with a significant number of mutations as shown in Figures 5 and 6 and Table 6 . The divergence of the spike protein, the nonconserved regions of the spike protein might contribute to the antigenicity difference in SARS-CoV-2 isolates. We found that most of the high frequent mutations of the S protein are located in the S1 subunit. Figure 6 indicates that near half of the amino acid residues have had mutations since January 5, 2020. One of the important mutations at S1 is 23010T>C (V483A) within the RBD for ACE2 binding, and the total frequency of 23010T>C (V483A) is 23. The structural study revealed that the amino acids 442−487 in the S1 subunit may impact viral binding to human ACE2. 9, 26 The mutations identified in this study imply the change in ACE2 binding affinity and the transmissibility of SARS-CoV-2 as well as negative impacts in preventive vaccine and diagnostic test development. 2.2.5.2. Main Protease. SARS-CoV-2 main protease, or 3CL protease, is essential for cleaving the polyproteins that are translated from the viral RNA. 10 It operates at multiple cleavage sites on the large polyprotein through the proteolytic processing of replicase polyproteins and plays a pivotal role in viral gene expression and replication. SARS-CoV-2 main protease is one of the most attractive targets for anti-CoV drug design because its inhibition would block viral replication and it is unlikely to be toxic due to no known similar human proteases. Another reason for the focused drug discovery efforts in developing SARS-CoV-2 main protease inhibitors is that this protein is relatively conservative as shown in Table 5 . Figure 7 illustrates the main protease mutation patterns. Figure 8 further highlights the inhibitor binding domain (BD). I cluster II cluster III cluster IV cluster V cluster VI top1 23403A>G D614G 10969 2333 2609 70 2965 2991 1 top2 23731C>T T723T 228 24 0 1 203 0 0 top3 23929C>T Y789Y 228 2 0 225 1 0 0 top4 24368G>T D936Y 110 37 0 1 2 70 0 top5 21575C>T L5F 98 22 9 28 15 14 10 top6 24862A>G T1100T 90 14 58 0 18 0 0 top7 24390G>C S943T 56 20 7 28 1 0 0 top8 24389A>C S943R 56 20 7 28 1 0 0 top9 24933G>T G1124V 47 15 0 21 7 1 3 top10 23707C>T P715P 44 1 0 Indeed, the main protease is relatively conservative compared to the spike protein. Table 7 lists top 10 mutations and their frequency in our data set. It is interesting to see that many mutations, such as D176D, R298R, N151N, are degenerate ones. One possible explanation is that nondegenerates may be nonsilent and likely cause unsurvivable disruption to the virus. Note that mutation G15S mostly occurs in cluster IV. Mutation R298R is restricted to cluster IV. Some other mutations, such as D248E, A266V, N151N, and T45I are specific to certain clusters. Nonetheless, some mutations at the BD shown in Figure 8 are worth noting. They can undermine the ongoing drug discovery effort. Protease. SARS-CoV-2 papain-like protease (PLPro) is a cysteine cleavage protein located within the nonstructural protein 3 (NSP3) section of the viral genome. 17 Like the main protease, PLPro activity is required to cleave the viral polyprotein into functional, mature subunits and, thereby, contributes to the biogenesis of the virus replication. Additionally, PLPro possesses a deubiquitinating activity. The SARS PLPro is also a major therapeutic and diagnostic target. As shown in Table 5 , the SARS PLPro is prone to mutations. Figure 9 shows that mutations are all over the places in PLPro. Table 8 lists the top 10 mutations in PLPro. Three of these mutations are degenerate ones. Note that only two of the top mutations occurred in cluster II. In contrast, cluster I has many different mutations. 2.2.5.4. RNA Polymerase. SARS RNA-dependent RNA polymerase (RdRP) is an enzyme that catalyzes the synthesis of the SARS RNA strand complementarily to the SARS-CoV-2 RNA template and is thus essential to the replication of SARS-CoV-2 RNA. 8 As one of the nonstructural proteins, RdRPs are located in the early part of ORF1b section. Like most other RNA viruses, SARS-CoV-2 RdRPs are considered to be highly conserved to maintain viral functions and thus targeted in antiviral drug development as well as diagnostic tests. On the other hand, the SARS-CoV-2 RNA polymerase lacks proofreading capability and thus its mutations are deemed to happen as shown in Table 5 . Figure 10 illustrates the SARS-CoV-2 RdRP mutations since January 5, 2020. Surprisingly, there are many mutations in SARS-CoV-2 RdRP. Table 9 describes the top 10 mutations. As in other cases, five of these mutations are degenerate ones. 2.2.5.5. Endoribo-nuclease. Endoribo-nuclease (NendoU) protein is a nidoviral RNAuridylate-specific enzyme that cleaves RNA. 11 It contains a C-terminal catalytic domain belonging to the NendoU family RNA processing. The NendoU protein is presented among coronaviruses, arteriviruses, and toroviruses. The many aspects of the detailed function and activity of SARS-CoV-2 NendoU protein are yet to be revealed. Figure 11 depicts SARS-CoV-2 NendoU protein mutations. As in most other SARS-CoV-2 proteins, mutations have occurred over different parts. Table 5 shows that NendoU is relatively conservative. Table 10 lists the top 10 high-frequency mutations of the SARS-CoV-2 NendoU protein that occurred in the past few months. Four of these mutations are degenerate ones. The frequencies of these mutations range from 153 to 15. Note that Cluster VI only has one of these mutations. 2.2.5.6. Envelope Protein. The SARS-CoV-2 envelope (E) protein is one of SARS-CoV's four structural proteins. As a transmembrane protein, it involves in ion channel activity and thus facilitates viral assembly, budding, envelope formation, pathogenesis, and release of the virus. 22 The E protein may not be essential for viral replication, but it is for pathogenesis. Figure 12 illustrates E protein as a very small pentamer with a few mutations. Table 11 shows its top 10 mutations. Note that the first four mutations are degenerate ones. All other mutations have relatively low frequencies. As shown in Table 5 , the SARS-CoV-2 E protein is very conservative. 2.2.5.7. Nucleocapsid Protein. SARS-CoV-2 nucleocapsid (N) protein 2 is another structural protein. Its primary function is to encapsidate the viral genome. To do so, it is heavily phosphorylated (or charged) and, thereby, can bind with RNA. Additionally, SARS-CoV-2 N protein confirms the viral genome to replicase-transcriptase complex (RTC) and plays I cluster II cluster III cluster IV cluster V cluster VI top1 10097G>A G15S 224 23 0 1 200 0 0 top2 10323A>G K90R 95 8 71 13 1 1 1 top3 10798C>A D248E 88 44 44 0 0 0 0 top4 10851C>T A266V 86 25 0 0 0 61 0 top5 10582C>T D176D 53 20 1 1 0 31 0 top6 10319C>T L89F 50 28 1 4 0 17 0 top7 10948A>G R298R 33 0 0 0 33 0 0 top8 10507C>T N151N 32 3 12 17 0 0 0 top9 10265G>A G71S 31 3 0 0 28 0 0 top10 10188C>T T45I 27 23 0 1 0 3 0 Figure 9 . Illustration of SARS-CoV-2 papain-like protease mutations using 6W9C as a template. a crucial role in viral genome encapsulation. Therefore, it may function completely differently at different stages of the viral life cycle. SARS-CoV-2 N protein is considered to be one of the most conservative SARS-CoV-2 proteins in the literature and is a popular target for diagnosis of vaccine development. 3 The present works shown in Table 5 indicate that the SARS-CoV-2 N protein is the worst target of any drug, vaccine, and diagnostic development. Figure 13 is the illustration of SARS-CoV-2 nucleocapsid phosphoprotein mutations using 6VYO as a template. Table 12 presents the top 10 mutations of the SARS-CoV-2 N protein since January 5, 2020. Note that only 2 out of the top 10 mutations are degenerate ones, which is a significantly lower ratio than that of other proteins. The frequency of 10th mutation is 78, which suggests there are many mutations associated with these mediate-sized proteins. Most top mutations occurred to clusters I, III, and IV. Clusters V and VI have almost none of the top 10 mutations. 2.2.5.8. Membrane Protein. SARS-CoV-2 membrane (M) protein is another structural protein and plays a central role in viral assembly and viral particle formation. It exists as a dimer in the virion and has certain geometric shapes to enable certain membrane curvature and binding to nucleocapsid proteins. Similar to other SARS-CoV proteins, M protein is also a popular target for viral diagnosis and vaccines. Table 5 gives SARS-CoV-2 M protein the middle ranking for its conservation. Table 13 details the top 10 mutations in SARS-CoV-2 M protein that occurred in the past few months. Eight of these mutations are degenerate. Clusters I and V have relatively a few of these mutations. I cluster II cluster III cluster IV cluster V cluster VI top1 5142C>T T808I 41 0 0 41 0 0 0 top2 5730C>T T1004I 22 3 0 4 9 2 4 top3 5784C>T T1022I 19 0 0 0 2 0 17 top4 5062G>T L781F 15 1 0 14 0 0 0 top5 5467C>T Y916Y 15 10 0 5 0 0 0 top6 5183C>T P822S 15 2 1 3 2 7 0 top7 5230G>T K837N 12 7 5 0 0 0 0 top8 5572G>T M951I 11 0 0 9 0 0 2 top9 5812C>T D1031D 10 1 0 5 3 1 0 top10 5284C>T N855N 10 8 0 1 1 0 0 Figure 10 . Illustration of SARS-CoV-2 RNA-polymerase mutations using 6M71 as a template. top1 14408C>T P323L 10925 2309 2602 68 2955 2991 0 top2 14805C>T Y455Y 1242 9 0 1202 30 0 1 top3 15324C>T N628N 405 128 253 18 5 1 0 top4 13730C>T A97V 263 11 20 232 0 0 0 top5 13536C>T Y32Y 121 23 0 1 92 5 0 top6 13862C>T T141I 118 61 53 2 0 2 0 top7 14786C>T A449V 98 53 14 3 22 6 0 top8 15540C>T V700V 39 1 0 37 1 0 0 top9 13627G>T D63Y 36 0 1 35 0 0 0 top10 14877C>T Y479Y 34 2 0 2 1 0 29 Figure 11 . Illustration of SARS-CoV-2 Endoribo-nuclease protein mutations using 6VWW as a template. 3. MATERIAL AND METHODS 3.1. Data Collection and Preprocessing. On January 5, 2020, the complete genome sequence of SARS-CoV-2 was first released on GenBank (access number: NC_045512.2) by Zhang's group at Fudan University. 28 Since then, there has been a rapid accumulation of SARS-CoV-2 genome sequences. In this work, 15 140 complete genome sequences with high coverage of SARS-CoV-2 strains from the infected individuals in the world have been downloaded from the GISAID database 20 (https://www.gisaid.org/) as of June 1, 2020. All the records in GISAID without the exact submission date were not taken into considerations. To rearrange the 15 140 complete genome sequences according to the reference SARS-CoV-2 genome, multiple sequence alignment (MSA) was carried out by using Clustal Omega 21 with default parameters. 3.2. SNP Genotyping. SNP genotyping measures the genetic variations between different members of a species. Establishing the SNP genotyping method for the investigation of the genotype changes during the transmission and evolution of SARS-CoV-2 is of great importance. By analyzing the rearranged genome sequences, SNP profiles which record all of the SNP positions in teams of the nucleotide changes and its corresponding positions can be constructed. The SNP profiles of a given genome of a COVID-19 patient capture all the differences from a complete reference genome sequence and can be considered as the genotype of the individual SARS-CoV-2. 3.3. Distance of SNP Variants. The Jaccard distance measures dissimilarity between sample sets. The Jaccard distance of SNP variants is widely employed in the phylogenetic analysis of human or bacterial genomes. 30 In this work, we utilize the Jaccard distance to compare the I cluster II cluster III cluster IV cluster V cluster VI top1 19839T>C N73N 153 7 0 0 146 0 0 top2 19684G>T V22L 63 2 0 57 4 0 0 top3 20578G>T V320L 59 42 16 1 0 0 0 top4 20134G>T V172L 39 1 0 25 10 3 0 top5 20148C>T F176F 31 3 1 20 5 0 2 top6 19999G>T V127F 30 14 0 0 1 15 0 top7 20316C>T F232F 25 0 0 25 0 0 0 top8 20270C>T A217V 22 3 0 19 0 0 0 top9 20275G>A D219N 20 1 17 1 0 1 0 top10 20031C>A A137A 15 1 A32A 16 0 2 14 0 0 0 top2 26256C>T F4F 12 2 6 0 4 0 0 top3 26319A>T V25V 10 1 0 8 0 0 1 top4 26319A>G V25V 8 1 0 7 0 0 0 top5 26270C>T T9I 7 4 1 0 2 0 0 top6 26416G>T V58F 5 1 0 1 3 0 0 top7 26326C>T L28L 5 difference between the SNP variant profiles of SARS-CoV-2 genomes. The Jaccard similarity coefficient, also known as the Jaccard index, is defined as the intersection size divided by the union of two sets A, B: 12 The Jaccard distance of two sets A, B is scored as the difference between one and the Jaccard similarity coefficient and is a metric on the collection of all finite sets: Therefore, the genetic distance of two genomes corresponds to the Jaccard distance of their SNP variants. If A ∩ B ≠ ⌀, A ⊂ B, and B ⊂ A, then we say these two SNP variants are relatives. If A ⊂ B, then A is the ancestor of B and B is the descendant of A. In principle, the Jaccard distance measure of SNP variants takes account of the ordering of SNP positions, i.e., transmission trajectory, when an appropriate reference sample is selected. However, one may fail to identify the infection pathways from the mutual Jaccard distances of multiple samples. In this case, the dates of the sample collections offer useful information. Additionally, clustering techniques, such as k-means described below, enable us to characterize the spread of COVID-19 onto the communities. 3.4. K-Means Clustering. K-means clustering is one of the fundamental unsupervised algorithms in machine learning which aims at partitioning a given data set X = {x 1 , x 2 , ..., x n , ..., x N }, x n ∈  d into k clusters {C 1 , C 2 , ..., C k }, k ≤ N such that the specific clustering criteria are optimized. More specifically, the standard K-means clustering algorithm starts to pick k points as cluster centers randomly and then allocates each data to its nearest cluster. The cluster centers will be updated iteratively by minimizing the within-cluster sum of squares (WCSS) which is defined by where μ k is the mean of points located in the kth cluster C k and n k is the number of points in C k . Here, ∥•∥ 2 denotes the L 2 distance. The algorithm above only provides a way to obtain the optimal partition for a fixed number of clusters. However, we are interested in finding the best number of clusters for the SNP variants. Therefore, the Elbow method is applied. By varying the number of clusters k, a set of WCSS can be calculated in the K-means clustering process, and then the plot of WCSS according to the number of clusters k can be carried out. The location of the elbow in this plot will be considered as the optimal number of clusters. To be noticed, the WCSS measures the variability of the points within each cluster which is influenced by the number of points N. Therefore, as the number of total points of N increases, the value of WCSS becomes larger. Additionally, the performance of k-means clustering depends on the selection of the specific distance. In this work, we propose to implement K-means clustering with the Elbow method for analyzing the optimal number of the subtypes of SARS-CoV-2 SNP variants. The Jaccard distance-based and location-based representations are considered as the input features for the K-means clustering method. 3.4.1. Jaccard Distance-Based Representation. Suppose we have a total of N SNP variants concerning a reference genome in a SARS-CoV-2 sample. The location of the mutation sites for each SNP variant will be saved in the set S i , i = 1, 2, ..., N. The Jaccard distance between two different sets (or samples) S i , S j is denoted as d J (S i , S j ). Therefore, the N × N Jaccard distance-based representation will be I cluster II cluster III cluster IV cluster V cluster VI top1 28881G>A R203K 3083 100 1 17 2963 1 1 top2 28882G>A R203K 3076 96 0 14 2966 0 0 top3 28883G>C G204R 3077 96 1 14 2966 0 0 top4 28311C>T P13L 323 1 3 317 1 0 1 top5 28657C>T D128D 191 1 2 183 3 0 2 top6 28688T>C L139L 163 1 1 161 0 0 0 top7 28836C>T S188L 120 64 53 1 0 2 0 top8 28878G>A S202N 91 0 0 91 0 0 0 top9 28580G>T D103Y 79 1 1 3 74 0 0 top10 29148T>C I292T 78 3 1 1 73 0 0 (5) Therefore, an N × M location-based representation will be = L i j v ( , ) i j (6) 3.4.3. Principal Component Analysis (PCA). Hundreds of complete genome sequences are deposited to GISAID every day, which results in an ever-growing massive quantity of high dimensional data representations for the K-means clustering. For example, if the data set of an organism involves 10 000 SNPs, the initial representation will be a 10 000-dimensional vector for each sample, which can be computationally difficult for a simple K-means clustering algorithm. Therefore, a dimensionality reduction method is used to preprocess the data. The essential idea of PCA-based K-means clustering is to invoke the PCA to obtain a reduced-dimensional representation of each sample before performing the K-means clustering. In practice, one can select a few lowest dimensional principal components as the K-means input for each sample. In ref 5, the authors proved that the principal components are the continuous solution of the cluster indicators in the K-means clustering method, which provides us a rigorous mathematical tool to embed our high-dimensional data into a lowdimensional PCA subspace. The rapid global transmission of coronavirus disease 2019 (COVID-19) has offered some of the most heterogeneous, diverse, and challenging mutagenic environments to stimulate dramatic genetic evolution and response from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This work provides the most comprehensive genotyping of SARS-CoV-2 transmission and evolution up to date based on 15 140 genome samples and reveals six clusters of the COVID-19 genomes and associated mutations on eight different SARS-CoV-2 proteins. We introduce mutation h-index and mutation ratio to qualify individual protein's degree of nonconservativeness. We unveil that SARS-CoV-2 envelope protein, main protease, and endoribonuclease protein are relatively the most conservative, whereas SARS-CoV-2 nucleocapsid protein, spike protein, and papain-like protease are relatively the most nonconservative. We report that all of the SARS-CoV-2 proteins have undergone intensive mutations since January 5, 2020, and some of these mutations might seriously undermine ongoing efforts on COVID-19 diagnostic testing, vaccine development, antibody therapeutics, and small-molecular drug discovery. The nucleotide sequences of the SARS-CoV-2 genomes used in this analysis are available, upon free registration, from the GISAID database (https://www.gisaid.org/). Eighteen tables are provided in the Supporting Information for SNP variants of 15 140 SARS-CoV-2 samples across the world, SNP variants of 4587 SARS-CoV-2 samples in the US, SNP variants in six global clusters, SNP variants in four US clusters, and mutation records for eight SARS-CoV-2 proteins. The acknowledgments of the SARS-COV-2 genomes are also given in the Supporting Information. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jcim.0c00501. 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