key: cord-257403-jujrazsr
authors: Yin, Changchuan
title: Genotyping coronavirus SARS-CoV-2: Methods and implications
date: 2020-04-27
journal: Genomics
DOI: 10.1016/j.ygeno.2020.04.016
sha: 
doc_id: 257403
cord_uid: jujrazsr

Abstract The emerging global infectious COVID-19 disease by novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) presents critical threats to global public health and the economy since it was identified in late December 2019 in China. The virus has gone through various pathways of evolution. To understand the evolution and transmission of SARS-CoV-2, genotyping of virus isolates is of great importance. This study presents an accurate method for effectively genotyping SARS-CoV-2 viruses using complete genomes. The method employs the multiple sequence alignments of the genome isolates with the SARS-CoV-2 reference genome. The single-nucleotide polymorphism (SNP) genotypes are then measured by Jaccard distances to track the relationship of virus isolates. The genotyping analysis of SARS-CoV-2 isolates from the globe reveals that specific multiple mutations are the predominated mutation type during the current epidemic. The proposed method serves an effective tool for monitoring and tracking the epidemic of pathogenic viruses in their global and local genetic variations. The genotyping analysis shows that the genes encoding the S proteins and RNA polymerase, RNA primase, and nucleoprotein, undergo frequent mutations. These mutations are critical for vaccine development in disease control.

through nsp16 in all coronaviruses [8] . Therefore, conserved structure and catalytic sites of 3CLpro may serve as attractive targets for antiviral drugs [9, 10] . Together, nsp3, nsp4, and nsp6

can induce DMV [11] .

SARS-coronavirus RNA replication is unique, involving two RNA-dependent RNA polymerases (RNA pol). The first RNA polymerase is a primer-dependent non-structural protein 12 (nsp12) , and the second RNA polymerase is nsp8. In contrast to nsp12, nsp8 has the primase capacity for de novo replication initiation without primers [12] . Nsp7 and nsp8 are important in the replication and transcription of SARS-CoV-2. The SARS-coronavirus nsp7-nsp8 complex is a multimeric RNA polymerase for both de novo initiation and primer extension [13, 12] . Nsp8 also interacts with ORF6 accessory protein. The nsp9 replicase protein of SARS-coronavirus binds RNA and interacts with nsp8 for its functions [14] .

Furthermore, the SARS-CoV-2 genome encodes four structural proteins. The structural proteins possess much higher immunogenicity for T cell responses than the non-structural proteins [15] . The structural proteins are involved in various viral processes, including virus particle formation. The structural proteins include spike (S), envelope (E), membrane protein (M), and nucleoprotein (N), which are common to all coronaviruses [16, 17] . The spike S protein is a glycoprotein, which has two domains S1 and S2. Spike protein S1 attaches the virion to the cell membrane by interacting with host receptor ACE2, initiating the infection [18, 19] . After the internalization of the virus into the endosomes of the host cells, the S glycoprotein is induced by conformation changes. The S protein is then cleaved by cathepsin CTSL, and the unmasks the fusion peptide of S2, therefore, activating membranes fusion within endosomes. 

Total 558 complete genome sequences of the SARS-CoV-2 strains from the infected individuals are retrieved from the GISAID database [26] as 

The SNP mutations, including nucleotide changes and the corresponding positions in a genome, are called an SNP profile. The SNP profiles of SARS-CoV-2 isolates are retrieved and parsed from the aligned genomes according to the reference genome SARS-CoV-2. The SNP profile of the complete genome of a virus can be considered as the genotype of the virus.

The Jaccard similarity coefficient ( , ) J A B of two sets A and B is defined as the intersectio n size of the two sets divided by the union size of two sets (Equation (1)) [29] .

The Jaccard distance is a metric on the collection of finite sets. The Jaccard distance

of two sets A and B is scored as the difference between 100% and the Jaccard similarity coefficient (Equation (2)).

The genetic relationship of two virus isolates, which are represented by the SNP sets A and B within the two virus genomes, respectively, can be inferred by the Jaccard distance of the SNP sets A and B. The Jaccard distance of SNP variants was adopted in the phylogenetic analysis of human or bacterial genomes [30, 31, 32] . In this study, we use the Jaccard distance of the SNP mutations of SARS-CoV-2 genomes to measure the dissimilarity of virus isolates.

Journal Pre-proof (3)

is negative. In all the descendants of an SNP A , the closest descendant is the one having the minimum

and BA  , then the two viruses are relatives, sharing common SNP mutations. If two SNP sets are ne ither descendant-ancestor nor relatives, the corresponding two viruses are isolated mutants. Hence, the relevance of virus isolates can be identified from the directed Jaccard measure on the SNP genotypes.

Though the source of SARS-CoV-2 varies, we still consider the virus samples were randomly collected for sequencing. If a virus strain among all sequenced viruses has many descendants in the genome set, this strain is conferred with high transmissibility. Therefore, the SNP mutations in this strain are critical for increased transmissibility.

The directed Jaccard distances of the SNP mutations are used to identify the relationships of virus strains, therefore, the genotyping method may determine the virus transmission pattern.

The pipeline for SNP genotyping and analysis is described in Algorithm 1. Combined, these four co-mutations probably can confer increased transmissibility of the virus.

SARS-coronavirus RNA replication is unique, involving two RNA-dependent RNA polymerases (RdRp). The first RNA polymerase is a primer-dependent non-structural protein 12

(nsp12), whereas the second RNA polymerase is nsp8. Nsp8 has the primase capacity for de novo initiation RNA replication without primers [12] . The most abundant SNP mutation in SARS-CoV-2 isolates is (28144T>C) in nsp8 protein, in which amino acid leucine (L) is mutated to serine (S). Our result is consistent with a previous study on 103 SARS-CoV-2 genomes in which SARS-CoV-2 virus is classified as S and L types by the two co-mutations (8782C>T and 28144T>C) [34] .

The third abundant SNP mutation is (26144G>T) in nonstructural protein 3 (nsp3:

G251V). The protein nsp3 works with nsp4 and nsp6 to induce double-membrane vesicles (DMV), membrane complex that acts as a platform for RNA replication and assembly [11] .

The most significant SNP mutation (23403A>G) is located in the gene encoding spike glycoprotein (S protein: D614G). The S protein in the SARS-CoV-2 virus is an important determinant of the host range and pathogenicity. The S protein attaches the virion to the cell membrane by binding the host ACE2 receptor [35] . The mutation D614G is located in the putative S1-S2 junction region near the furin recognition site (R667) for the cleavage of S protein when the viron enters or exists cells [36] . However, the actual functional impact of this high-frequency SNP Especially, the SNP analytics result also shows that the primer independent RNA primase (nsp8) contains more mutations than any other proteins (28144T>C, 28881G>A, 28882G>A, and 28883G>C). The RNA polymerase and primase mutations may confer resistance to mutagenic nucleotide analogs via increased fidelity. The previous study indicated that a single mutation in RNA polymerase can improve the replication fidelity in RNA virus [37] . If a mutation is lethal or reduces the transmission ability, the mutations may not be carried on or get deceased. The SNP profiles demonstrate that the mutations in the envelope glycoprotein and RNA polymerases predominate. Only these mutations in the S protein that have strongly binding capacity to cell ACE2 receptors while escaping from immune system response can have chances to survive.

Therefore, these critical mutations are the results of natural selection in virus evolution.

In the SARS-CoV-2 strains found in the US, the nucleocapsid (N) protein gene has three mutations (28881G>A, 28882G>A, and 28883G>C), The N protein of SARS-CoV is responsible for the formation of the helical nucleocapsid during virion assembly. The N protein may cause an immune response and has potential value in vaccine development [38] . These mutations shall be considered when developing a vaccine using the N protein. 

To spread, a pathogen virus must multiply within the host to ensure transmission, while simultaneously avoiding host morbidity or death. Therefore, during the evolution of a virus, the transmissibility of the virus is usually increased, whereas the pathogenicity becomes reduced [39] .

From the SNP profiles of SARS-CoV-2 strain, high-frequency mutations predominate in the virus isolations, therefore, these high-frequency mutations probably contribute to increased transmissibility. In addition, these high-frequency mutations are associated with different critical proteins. This study analyzes and traces the SNP profiles from 558 SARS-CoV-2 strains which have at least 10 descendants. The result suggests a number of high-frequency mutations that are associated with different critical proteins. The results show that the SNP distribution is not random but is predominated at some positions and then have more descendants. These high-frequency mutations may confer a high transmissibility of the virus (Table 2 ). If we exclude the leader sequence mutation and the synonymous mutations (3037C>T, 8782C>T, 18060C>T), we classify the SNP mutations into four major groups based on the impacted proteins. (1) single mutation in nsp6 (11083G>T) (Fig.2) , (2) single mutation in ORF3a (26144G>T) (Fig.3) in RNA polymerase (nsp8) (8782C>T, 28144T>C) (Fig.4) , and (4) double mutations in S-protein and RNA polymerase: (241C>T, 3037C>T, 14408C>T, 23403A>G ) (Fig.5) ). These strains in one group are derived from the same ancestor stain in that group according to their SNP profiles. 

This study employs the substitutions variants in genotyping for understanding the evolution and transmission of SARS-CoV-2, however, structural variants including insertions, deletions, and copy number variation are critical for virus pathogenicity [40] and human pathology [41, 42] . 

). The tandem or dispersed repeats of copy number variants have not been observed in the SARS-CoV-2 genomes. Whether these structural mutations can spread is unknown given the limited genome data. The phenotype changes of these structural variants need further investigation.

The proposed genotyping method can reveal the SNPs patterns and structural variants of virus isolates during evolution. The method may also be applied to unravel the evolutionary genetics of zoonotic jumps so that infectious diseases can be prevented. Identification and comparison of the SNPs in the Spike protein genes of SARS-CoV-2 and bat-CoVs using the reference genome of a putative ancestral bat-CoV, for example, pangolins-Cov, will provide insights on the genetic mechanisms in the zoonotic jump of SARS-CoV-2.

A few notable limitations in this study due to the nature of the genome data shall be noted.

Because the sample collection dates may not reflect the actual infection date so the transmission path analysis is only an approximation. Caution should be exercised on the genotyping analytics because some countries have not sequenced enough virus samples, the frequencies of the genotype J o u r n a l P r e -p r o o f groups may be unbalanced due to the unavailability of complete genomes in some countries and regions. Whether any of these common SNP mutations will result in biological and clinical differences remains to be determined.

In this study, the complete genomes of SARS-CoV-2 are used for SNP genotype calling.

However, in times of crisis, complete genomes may not be available for SNP genotyping. In this case, the SNP variant calling process may directly use the raw NGS reads [32] . The SNP variants then can be obtained by mapping the NGS reads to the reference genome by BWA alignments [43] , followed by GATK variant calling [44] .

The SARS-CoV-2 pandemic has caused a substantial health emergency and economic stress in the 

The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

WHO, Coronavirus disease 2019 (COVID-19) situation report -63, Coronavirus Disease

Pathogenicity and transmissibility of 2019-nCoV -a quick overview and comparison with other emerging viruses

siRNA targeting the leader sequence of SARS-CoV inhibits virus replication

Emerging coronaviruses: genome structure, replication, and pathogenesis

Nuclear magnetic resonance structure of the nucleic acid-binding domain of severe acute respiratory syndrome coronavirus nonstructural protein 3

Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein

Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs

Broad-spectrum antivirals against 3C or 3C-like proteases of picornaviruses, noroviruses, and coronaviruses

Machine intelligence design of 2019-nCoV drugs, bioRxiv

Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles

The SARS-coronavirus nsp7+ nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension

Identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins

The nsp9 replicase protein of SARS-coronavirus, structure and functional insights

T cell responses to whole SARS coronavirus in humans

The genome sequence of the SARS-associated coronavirus

Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection

Receptor recognition by the novel coronavirus from Wuhan: an analysis based on decade-long structural studies of SARS coronavirus

A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2

The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade

The membrane protein of severe acute respiratory syndrome coronavirus acts as a dominant immunogen revealed by a clustering region of novel functionally and structurally defined cytotoxic T-lymphocyte epitopes

Characterization of protein-protein interactions between the nucleocapsid protein and membrane protein of the SARS coronavirus

Clinical characteristics of coronavirus disease 2019 in China

RNA virus mutations and fitness for surviva l

Viral evolution and the emergence of SARS coronavirus

GISAID: Global initiative on sharing all influenza data-from vision to reality

Clustal Omega, accurate alignment of very large numbers of sequences, in: Multiple sequence alignment methods

A new coronavirus associated with human respiratory disease in China

Distance between sets

Genotyping of genetically monomorphic bacteria: DNA sequencing in mycobacterium tuberculosis highlights the limitations of current methodologies

A novel strategy for clustering major depression individuals using whole-genome sequencing variant data

Whole genome single nucleotide polymorphism genotyping of staphylococcus aureus

Biopython: freely available python tools for computational molecular biology and bioinformatics

Origin and evolution of the 2019 novel coronavirus

The sars-cov s glycoprotein: expression and functional characterization

Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry

A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity

Immune responses against SARS-coronavirus nucleocapsid protein induced by DNA vaccine

Virulence evolution and the trade-off hypothesis: history, current state of affairs and the future

Pathogenicity of severe acute respiratory coronavirus deletion mutants in hACE-2 transgenic mice

Investigation of copy number variation in subjects with major depression based on whole-genome sequencing data

Investigation of short tandem repeats in major depression using whole-genome sequencing data

Aligning sequence reads

The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data

The author sincerely appreciates the researchers worldwide who sequenced and shared the complete genome data of SARS-CoV-2 and other coronaviruses from GISAID (https://www.gisaid.org/). This research is dependent on these precious data. The references of the genomes are in the supplementary material (gisaid_cov2020_acknowledgement_table.csv). The author thanks four anonymous reviewers for their insightful suggestions.

SARS-CoV-2_IDs.csv gisaid_cov2020_acknowledgement_table.csv