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Variant analysis of SARS-CoV-2 genomes

Abstract Objective To analyse genome variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Methods Between 1 February and 1 May 2020, we downloaded 10 022 SARS CoV-2 genomes from four databases. The genomes were from infected patients in 68 countries. We identified variants by extracting pairwise alignment to the reference genome NC_045512, using the EMBOSS needle. Nucleotide variants in the coding regions were converted to corresponding encoded amino acid residues. For clade analysis, we used the open source software Bayesian evolutionary analysis by sampling trees, version 2.5. Findings We identified 5775 distinct genome variants, including 2969 missense mutations, 1965 synonymous mutations, 484 mutations in the non-coding regions, 142 non-coding deletions, 100 in-frame deletions, 66 non-coding insertions, 36 stop-gained variants, 11 frameshift deletions and two in-frame insertions. The most common variants were the synonymous 3037C > T (6334 samples), P4715L in the open reading frame 1ab (6319 samples) and D614G in the spike protein (6294 samples). We identified six major clades, (that is, basal, D614G, L84S, L3606F, D448del and G392D) and 14 subclades. Regarding the base changes, the C > T mutation was the most common with 1670 distinct variants. Conclusion We found that several variants of the SARS-CoV-2 genome exist and that the D614G clade has become the most common variant since December 2019. The evolutionary analysis indicated structured transmission, with the possibility of multiple introductions into the population.


Introduction
In late 2019, several people in Wuhan, China, were presenting with severe pneumonia at the hospitals. As the number of patients rapidly increased, the Chinese government decided on 23 January 2020 to lock down the city to contain the virus. Unfortunately, the virus had already spread across China and throughout the world. The World Health Organization (WHO) officially declared the outbreak a pandemic on March 11, 2020. As of 23 May 2020, over 5 million cases worldwide had been reported to WHO and the death toll has exceeded 330 000. 1 Researchers isolated the virus causing the pneumonia in December 2019 and found it to be a strain of β-coronavirus (CoV). The virus showed a high nucleotide sequence homology with two severe acute respiratory syndrome (SARS)-like bat coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21 (88% homology) and with SARS-CoV (79.5% homology), while only 50% homology with the Middle East respiratory syndrome coronavirus (MERS) CoV. 2,3 The virus, now named SARS-CoV-2, contains a single positive stranded RNA (ribonucleic acid) of 30 kilobases, which encodes for 10 genes. 4 Researchers have shown that the virus can enter cells by binding the angiotensin-converting enzyme 2 (ACE2), through its receptor binding domain in the spike protein. 5 The virus causes the coronavirus disease 2019 (CO-VID-19), with common symptoms such as fever, cough, shortness of breath and fatigue. 6,7 Early data indicated that about 20% of patients develop severe COVID-19 requiring hospitalization, including 5% who are admitted to the intensive care unit. 8 Initial estimates of the case fatality rates were from 3.4% to 6.6% which is lower than that of SARS or MERS, 9.6% and 34.3% respectively. [9][10][11] The mortality from COVID-19 is higher in people older than 65 years and in people with underlying comorbidities, such as chronic lung disease, serious heart conditions, high blood pressure, obesity and diabetes. [12][13][14] Community transmission of the virus, as well as anti-viral treatments, can engender novel mutations in the virus, potentially resulting in more virulent strains with higher mortality rates or emergence of strains resistant to treatment. 15 Therefore, systematic tracking of demographic and clinical patient information, as well as strain information is indispensable to effectively combat COVID-19.
Here we analysed the SARS-CoV-2 genome from 10 022 samples to understand the variability in the viral genome landscape and to identify emerging clades.

Methods
In total, we downloaded 15 755 genome sequences from the following databases: the Chinese National Microbiology Data Center on 1 February 2020; the Chinese National Genomics Data Center Genome Warehouse on 4 February 2020; GISAID 16 on 1 May 2020 and GenBank on 1 May 2020. We removed redundant sequences with the China National Center for Bioinformation annotations. To reduce the number of false positive variants, we removed sequences with more than 50 ambiguous bases.
For this study, we used the sequence of established SARS-CoV-2 reference genome, NC_045512. 17 This genome was sequenced in December 2019. Each sample was first aligned to the reference genome in a pairwise manner using EMBOSS needle (Hinxton, Cambridge, England), with a default gap penalty of 10 and extension penalty of 0.5. 18 Then, we developed a custom script in Python (Python Software Foundation, Wilmington, United States of America) to extract the differences between the genome variants and the reference genome. Nucleotide variants in the coding regions were converted to corresponding encoded amino acid residues. For the open reading frame 1 (ORF1), we used the protein coordinates from YP_009724389.1 19 for translation. Finally, we carefully Objective To analyse genome variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Methods Between 1 February and 1 May 2020, we downloaded 10 022 SARS CoV-2 genomes from four databases. The genomes were from infected patients in 68 countries. We identified variants by extracting pairwise alignment to the reference genome NC_045512, using the EMBOSS needle. Nucleotide variants in the coding regions were converted to corresponding encoded amino acid residues. For clade analysis, we used the open source software Bayesian evolutionary analysis by sampling trees, version 2.5. Findings We identified 5775 distinct genome variants, including 2969 missense mutations, 1965 synonymous mutations, 484 mutations in the non-coding regions, 142 non-coding deletions, 100 in-frame deletions, 66 non-coding insertions, 36 stop-gained variants, 11 frameshift deletions and two in-frame insertions. The most common variants were the synonymous 3037C > T (6334 samples), P4715L in the open reading frame 1ab (6319 samples) and D614G in the spike protein (6294 samples). We identified six major clades, (that is, basal, D614G, L84S, L3606F, D448del and G392D) and 14 subclades. Regarding the base changes, the C > T mutation was the most common with 1670 distinct variants. Conclusion We found that several variants of the SARS-CoV-2 genome exist and that the D614G clade has become the most common variant since December 2019. The evolutionary analysis indicated structured transmission, with the possibility of multiple introductions into the population.  20 Using the identified recurrent variants, we performed hierarchical clustering in SciPy library, Python, to identify clades. First, a binary matrix of samples and distinct variants was created. Then, we did hierarchical clustering using the Ward's method 21 in SciPy library. 22 We investigated the mutation patterns of SARS-CoV-2 to find potential causes of mutations, by looking at the changes in bases. Since coronavirus genomes are positive sense, single stranded RNA, we did not combine C > T with G > A mutations.
The spike protein is a key protein for SARS-CoV-2 viral entry and a target for vaccine development. We, therefore, wanted to find amino acid conservation between other coronavirus sequences in the spike protein. We used the basic local alignment search tool BLAST (National Center for Biotechnology Information [NCBI], Bethesda, United States) 23 followed by the constraint-based multiple alignment tool COBALT (NCBI, Bethesda, United States). 24 We carefully investigated mutations within the receptor binding domain and predicted B-cell epitopes. 25,26 The mutations were further analysed to identify cross species conservation and to understand the nature of amino acid changes. We visualized the aligned sequence using the open source software alv. 27 For the phylogenetic analysis, we used the open source software Bayesian evolutionary analysis by sampling trees (BEAST), version 2.5. 28 BEAST uses a Bayesian Monte-Carlo algorithm generating a distribution of likely phylogenies given a set of priors, based on the probabilities of those tree configurations determined from the viral genomes. This analysis presents a different view than the variant analysis described above and is an independent test of the structure that individual haplogroup markers identify. First, we aligned sequences to NC_045512, using the multiple sequence alignment software, MAFFT. 29 Subsequently, we adjusted for length and sequencing errors, by truncating the bases in the 5'-UTR and 3'-UTR, without losing key sites. We excluded sequences showing a variability higher than 30 bases. For an optimal output of the phylogenetic tree, we randomly selected a subset of 2000 samples by using a random number generator in Python. We ran BEAST using sample collection dates with the Hasegawa-Kishino-Yano mutation model, 30 with the strict clock mode. Finally, we estimated the mutation rate and median tree height from the resulting BEAST trees.

Results
In total, we analysed 10 022 SARS CoV-2 genomes (sequences are available from the data repository) 20  Of the 2969 missense variants, 1905 variants are found in ORF1ab, which is the longest ORF occupying two thirds of the entire genome. ORF1ab is transcribed into a multiprotein and subsequently cleaved into 16 nonstructural proteins (NSPs). Of these proteins, NSP3 has the largest number of missense variants among ORF1ab proteins. Of the NSP3 missense variants, A58T was the most common (159 samples) followed by P153L (101 samples; Table 2). We also detected mutations in the nonstructural protein RNA-dependent RNA polymerase (RdRp), such as P323L (6319 samples). Deletions are also common in 3'-5' exonuclease (11 deletions) including those resulting in frameshifts. A comprehensive list of variants is available in data repository. 20 Variants with recurrence over 100 samples are shown in Table 3. The most common variants were the synonymous variant 3037C > T (6334 samples), OR-F1ab P4715L (RdRp P323L; 6319 samples) and SD614G (6294 samples). They occur simultaneously in over 3000 samples, mainly from Europe and the United States. Other variants including ORF3a Q57H (2893 samples), ORF1ab T265I (NSP3 T85I; 2442 samples), ORF8 L84S (1669 samples), N203_204delinsKR (1573 samples), ORF1ab L3606F (NSP6 L37F; 1070 samples) were the key variants for identifying clades.
We identified six major clades with 14 subclades ( Fig. 1 and Table 4 (80 samples) and Iceland (76 samples). However, the basal clade now accounts only for a small fraction of genomes (670 samples mainly from China). The remaining two clades D448del and G392D are small and they are without any significant subclades at this point.
All non-coding deletions are either located within 3'-UTR, 5'-UTR or intergenic regions. Of the in-frame deletions, ORF1 D448del stands out with 250 samples. In contrast, we only detected two distinct in-frame insertions in our data set. We also detected 11 frameshift deletions and 36 stop-gained variants. The recurrent stop-gained variant Y4379* (NSP10 Y126*) is found in 51 samples in the D614G clade. NSP10 Y126* is located only 13 residues upstream of the stop codon; therefore, a truncation may not significantly affect function of the protein. Most of frameshift variants in ORF1ab do not recur except for S135fs (three samples) and L3606fs (two samples). Although frameshift variants are considered deleterious, for instance, S135fs (more precisely S135Rfs*9) In-frame deletion Non     The most common base change is C > T (Fig. 2). As expected, 31 we observed a strong bias in transition versus transversion ratio (7:3). C > T transitions might be intervened by cytosine deaminases. Surprisingly, G > T transversions, likely introduced by oxo-guanine from reactive oxygen species, 32 were also frequently observed.
Assessing variants in the spike protein revealed 427 distinct non-synonymous variants with many variants located within the receptor binding domain and B-cell epitopes (Fig. 3). Among the variants in the receptor binding domain, V483A (26 samples), G476S (9 samples) and V367F (12 samples) are highly recurrent. Fig. 4 shows the consensus tree from the phylogenetic analysis. The tree has a coalescence centre with exponential expansion identified by haplotype markers. The colour mapped phylogenies largely support the 14 identified subclades. We note that substantial numbers of samples from the United States show affinity with European lineages rather than those directly derived from East Asia. Except for the earliest cases, European clades dominate even in samples from western states in the United States. Further, European samples tend to associate with lineages that expanded through Australia.

Discussion
Here we show the evolution of the SARS-Co-2 genome as it has spread across the world. Although, our methods do not allow us to investigate whether the mutations observed led to a loss or gain of function, we can speculate on the implications of viral function of these mutations.
14 other variants besides D614G. Almost all strains with D614G mutation also have a mutation in the protein responsible for replication (ORF1ab P4715L; RdRp P323L), which might affect replication speed of the virus. This protein is the target of the anti-viral drugs, remdesivir and favipiravir, and the susceptibility for mutations suggests that treatment resistive strains may emerge quickly. Mutations in the receptor binding domain of the spike protein suggest that these variants are unlikely to reduce binding affinity with ACE2, since that would decrease the fitness of the virus. V483A and G476S are primarily observed in samples from the United States, whereas V367F is found in samples from China, Hong Kong Special Administrative Region, France and the Netherlands. The V367F and D364Y variants have been reported to enhance the structural stability of the spike protein facilitating more efficient binding to the ACE2 receptor. 34 In summary, structural and functional changes concomitant with spike protein mutations should be meticulously studied during therapy design and development.
We detected several non-recurring frameshift variants, which can be se-quencing artefacts. The frameshift at Y3 in ORF10, although only detected in one sample, might not be essential for survival of the new coronavirus, since ORF10, a short 38-residue peptide, is not homologous with other proteins in the NCBI repository.
The phylogenetic analysis suggest population structuring in the evolution of SARS-CoV-2. The analysis provides an independent test of the major clades we identified, as well as the geographic expansions of the variants. While the earliest samples from the United Stated appear to be derived from China, belonging either to basal or L84S clades, the European clades, such as D614G/ Q57H, tend to associate with most of the subsequent increase in infected people in the United States. D614G was first observed in late January in China and became the largest clade in three months. The mutation rate of 1.12 × 10 −3 mutations per site-year is similar to 0.80 × 10 −3 to 2.38 × 10 −3 mutations per site-year reported for SARS-CoV-1. 35 The rapid increase of infected people will provide more genome samples that could offer further insights to the viral dissemination, particularly the possibility of at least two zoonotic transmissions of SARS-CoV-2 into the human population. An understanding of the biological reservoirs carrying coronaviruses and the modalities of contact with human population through trade, travel or recreation will be important to understand future risks for novel infections. Further, populations may be infected or even re-infected via multiple travel routes.
The number of people with confirmed COVID-19 has rapidly increased over the last five months with no sign of decline in the near future. The fight against COVID-19 will be long, until vaccines and other effective therapies are developed. To facilitate rapid therapeutic development, clinicopathological, genomic and other societal information must be shared with researchers, physicians and public health officials. Given the evolving nature of the SARS-CoV-2 genome, drug and vaccine developers should continue to be vigilant for emergence of new variants or sub-strains of the virus. ■ Notes: Each sample is coloured with corresponding subclade. We used the Bayesian evolutionary analysis by sampling trees software. 28 Research Severe acute respiratory syndrome coronavirus 2 genomes Takahiko Koyama et al.
Flu Database, GenBank, and NGDC Genome Warehouse, and the National Microbiology Data Center on which this research is based. The list of genomes is available from the data repository. 20 We also thank Jane Snowdon and Dilhan Weeraratne.

Research
Severe acute respiratory syndrome coronavirus 2 genomes Takahiko Koyama et al.