Role of next-generation sequencing in diagnosing, tracking and vaccine development of severe acute respiratory syndrome coronavirus 2

Nagamani Kammili; Amrithesh Kumar Arun; Winnie Thomas; Madhavi Latha Manolla

doi:10.4103/jacm.jacm_18_22

REVIEW ARTICLE

Year : 2022 | Volume : 24 | Issue : 3 | Page : 25-31

Role of next-generation sequencing in diagnosing, tracking and vaccine development of severe acute respiratory syndrome coronavirus 2

Nagamani Kammili¹, Amrithesh Kumar Arun², Winnie Thomas³, Madhavi Latha Manolla³
¹ Department of Multi-Disciplinary Research Unit, Department of Microbiology, Gandhi Medical College and Hospital, Secunderabad, Telangana, India
² Department of Microbiology, Gandhi Medical College and Hospital, Secunderabad, Telangana, India
³ Department of Multi-Disciplinary Research Unit, Gandhi Medical College and Hospital, Secunderabad, Telangana, India

Date of Submission	26-Sep-2022
Date of Acceptance	29-Sep-2022
Date of Web Publication	11-Nov-2022

Correspondence Address:
Madhavi Latha Manolla
Department of MDRU, Gandhi Medical College and Hospital, Secunderabad - 500 003, Telangana
India

Source of Support: None, Conflict of Interest: None

Check

DOI: 10.4103/jacm.jacm_18_22

Abstract

An objective method of detecting infections without the requirement for clinical hypotheses is provided using next-generation sequencing (NGS) technology for the diagnosis of infectious disorders. In order to inform nations and the general public about any potential changes that may be required to respond to the variant and stop its spread, the World Health Organization and its international networks of experts have been continuously monitoring changes to the virus based on NGS data throughout the current pandemic. When tracking ongoing outbreaks, monitoring for novel pathogens or spotting potentially harmful variations of well-known diseases, NGS offers substantial advantages. Due to the technology's quick creation of high-resolution sequence data, researchers and research teams can access it easily and share it with one another. Numerous candidate vaccines have been created on several platforms quickly. Many of them have worked in crisis circumstances in numerous nations worldwide. The breakthrough infections could only be tracked by the use of NGS technology. In this review, we discussed in silico analysis using current bioinformatics approaches, and sequencing reveals unique emerging severe acute respiratory syndrome coronavirus 2 variations which have the potential to cause novel illnesses in the future.

Keywords: Coronavirus disease-2019 pandemic, next-generation sequencing, Variants of Concern, Variants of Interest

How to cite this article:
Kammili N, Arun AK, Thomas W, Manolla ML. Role of next-generation sequencing in diagnosing, tracking and vaccine development of severe acute respiratory syndrome coronavirus 2. J Acad Clin Microbiol 2022;24, Suppl S1:25-31

How to cite this URL:
Kammili N, Arun AK, Thomas W, Manolla ML. Role of next-generation sequencing in diagnosing, tracking and vaccine development of severe acute respiratory syndrome coronavirus 2. J Acad Clin Microbiol [serial online] 2022 [cited 2023 Jun 3];24, Suppl S1:25-31. Available from: https://www.jacmjournal.org/text.asp?2022/24/3/25/360980

Next-generation Sequencing and Coronavirus Disease-2019 Diagnostics

In late December 2019, patients presenting with viral pneumonia due to an unknown microbial agent were reported in Wuhan, China. A novel coronavirus was then identified as the causative pathogen, provisionally named 2019 novel coronavirus (2019-nCoV).^[1] In the year 2020, the World Health Organization (WHO) has designated the emergent novel CoV as coronavirus disease-2019 (COVID-19), while the International Committee on the Taxonomy of Viruses has designated it as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).^[2]

As of May 2022, approximately 517,126,859 cases of 2019-nCoV infections have been confirmed, and SARS-CoV-2 accumulated mutations and changed throughout the pandemic (WHO Health Emergency Dashboard). Most variations have little to no impact on the virus' properties. However, a few of these changes may affect the viral characteristics, such as ease of its spread, the associated disease severity, the performance of vaccines, therapeutic medicines, diagnostic tools or influence the performance of other public health and social measures.^[3] Therefore, rapid and cost-efficient whole-genome sequencing of SARS-CoV-2 has become critical for understanding viral transmission dynamics and managing the outbreak.^[4]

The WHO, in collaboration with associates, skill networks, national authorities, institutions and researchers, has been intensively monitoring, assessing and evaluating the evolution of SARS-CoV-2 since January 2020.^[5] In late 2020, the appearance of variants posed an increased risk to global public health provoked the characterisation of specific Variants of Interests (VOIs) and Variants of Concerns (VOCs) to prioritise the ongoing response to the COVID-19 pandemic.^[6]

Next-generation sequencing (NGS) technology provides an objective method of detecting infections without the need for an initial clinical hypothesis or culturing.^[7] During the current pandemic, the WHO and its international networks of experts have been constantly monitoring changes to the virus on the basis of NGS data, scrutinising and identifying any significant amino acid substitutions, in order to provide information and alert countries and the public about any changes that may be needed to respond to the variant and prevent its spread.^[8] Globally, NGS systems and pipelines have been established and are being strengthened to detect 'signals' of potential VOIs or VOCs and assess these based on the risk posed to global public health.^[9]

This review aims to serve as a reference guide to understand the background, utilisation and implementation of NGS technology in the setting of infectious disease outbreaks for pathogen diagnosis, variant identification and vaccine development, with the SARS-CoV-2 pandemic serving as a backdrop.^[10] First, we reviewed the current diagnostic methods for COVID-19, followed by an introduction to NGS technology and its bioinformatic processes. Next, we reviewed the studies implementing NGS early in the SARS-CoV-2 pandemic in an effort to highlight its multiple uses/applications in different identification, phylogenetic monitoring and scientific inquiry. We concluded this review by discussing how NGS technology is implemented in vaccine development.^[11],[12] NGS offers significant advantages in tracking ongoing outbreaks, surveilling for new pathogens or identifying potentially dangerous variants of known pathogens. The technology allows the rapid generation of sequence information at a high resolution, allowing great accessibility and shareability amongst investigators/research groups. On a fast-track basis, various candidate vaccines have been developed on several platforms. Many of them have been employed in emergencies in various countries worldwide. Inactivated virus, nucleic acid, adenovirus-based vectors and recombinant components were employed in the development of these COVID-19 vaccines^[13] The mechanisms used by different manufacturers of NGS technology to generate raw data vary extensively, and a comprehensive review of the step-by-step process of read generation used by each company is outside the scope of this article.

Next-generation Sequencing

NGS technologies have redefined the modus operandi in both human and microbial genetics research, allowing the generation of substantial sequencing datasets on a short time scale and at inexpensive costs.^[14] Bulk molecular testing for COVID-19 by high throughput NGS-based technology has shown great potential to be a highly scalable workflow.

NGS technology has reformed the field of genomics by providing a novel and effective method for screening samples and detecting pathogens. This technology, in conjunction with bioinformatics tools, is changing the way research and diagnostic centres respond to infectious disease outbreaks.^[15] This progress is paving the way for new approaches and improving our understanding of disease origin, occurrence and transmission through a large-scale genomic approach.^[16] In the event of a pandemic, mass testing in the population is required to prevent infection spread and reduce mortality rates. In response to this need, researchers created an NGS-based diagnostic test for the COVID-19 virus that has 96% accuracy in paired analyses with the current gold standard real-time polymerase chain reaction (RT-qPCR).^[17] This new technology expands global diagnostic capacity, allowing for rapid treatment initiation and isolation of infected individuals, all of which contribute to the control of COVID-19 outbreaks.

Coronavirus Sequencing Current Methods

Amplicon sequencing

By sequencing specific regions of the viral genome, we can detect the presence of the SARS-CoV-2 coronavirus. The ultra-deep sequencing of polymerase chain reaction amplicons is used to analyse genomic regions of interest.^[18]

Target enrichment sequencing

Identify and characterise coronaviruses, flu viruses and other pathogenic respiratory tract organisms, as well as the antimicrobial resistance alleles associated with them. These findings can assist researchers in monitoring respiratory infections and optimising infection control strategies.^[19] This method uses hybridisation to target-specific probes to capture genomic regions of interest.

Shotgun metagenomics

Comprehensively sequence all organisms in each sample and identify novel pathogens such as coronaviruses. This NGS method can help accelerate outbreak investigations and support the development of new laboratory tests.^[20]

Application of next-generation sequencing technology

Track the transmission routes of the virus globally
Detect mutations quickly to prevent the spread of new strain types
Identify viral mutations that can affect vaccine potency or avoid detection by established molecular diagnostic assays
Screen targets for possible COVID-19 therapeutics
Identify and characterise respiratory co-infections and antimicrobial resistance alleles.

NGS is unravelling the complex dynamics of viral evolution and host responses against these viruses, thus contributing substantially to the likelihood of successful vaccine development.

Next-Generation Sequencing and Coronavirus Disease-2019 Variants

As evidenced by the efforts in controlling the COVID-19 pandemic, NGS has now become a crucial technology in real-time management of the worldwide pandemic: To track transmission paths globally, detection of new strains, detect mutations that can interfere with vaccination or diagnosis, spot possible targets for therapeutics, identify co-infections and antimicrobial resistance alleles. Necessarily, the integration of pathogen genomics into public health strategies is vital to prevent and manage communicable diseases.

The causative agent of the currently ongoing pandemic-SARS-CoV-2 virus was first identified by sequence data of isolates from the patients at Wuhan in 2019.^[20],[21] Although the initial sequences were found to be largely similar to the bat coronaviruses, there were regions sharing similarities with the severe acute respiratory syndrome virus, specifically the sequences of the Spike protein (S) and the receptor-binding domain (S1). Phylogenetic analysis of the sequence data allotted SARS-CoV-2 as a Sarbecovirus subtype of the Beta coronavirus genera, one amongst seven coronaviruses that is able to infect human populations.^[11] NGS technology has significantly impacted efforts to trace the origins of the novel coronavirus and subsequently has been applied to great effect in detecting new mutations and identifying new variants/subtypes [Table 1].

Table 1: Different variants of severe acute respiratory syndrome coronavirus 2 based on spike mutations

Click here to view

Worldwide efforts to identify variants via sequencing began in early 2020, where a small study in Italy yielded sequences of high similarity ~ 99% to the original Wuhan-Hu-1 reference strain barring SNP mutations within the N protein.^[22] Eventually, larger studies with numerous sample sizes elucidated some of the mutational characteristics of the SARS-CoV-2 genome,^[23] showing non-conservative nature of the viral N-gene and S-gene. One similar analysis of the available public data between January and April 2020 presented an insight into the mutation patterns of the viral genome over time across geographical regions; identifying five main groups from the worldwide data with recurrent mutations through k-clustering.^[24] The first two clusters identified in the study were predominant in Asiatic regions, given the geographical origin of the virus, and more mutations were accumulated during the subsequent spread of the virus around the world [Table 2]. A point of interest in the following three clusters was the mutation of 23403A > G, resulting in an amino acid substitution in the Spike protein (D614G) that increased the viral pathogenicity leading to high infection rates all throughout Europe and the United States.^[25] The data from the US were divided into three clusters, Cluster A (widespread across the US but in small numbers), Cluster B (predominantly affecting the west coast of the country) and Cluster C (widespread through the US east coast via Europe, originating from cluster 3).

Table 2: Severe acute respiratory syndrome coronavirus 2 genomic clusters as reported by Wang et al. Worldwide data

Click here to view

As the pandemic has progressed since the outbreak in 2019, the eventual increase in the adoption of NGS and the overall availability of sequences from different regions of the world enabled a better understanding of the virus. Genomic regions were assessed for the proneness to gather mutations and protein alterations within, and the h-index was assigned accordingly. The envelope protein (E), protease and endoribonuclease were generally the most conserved regions within the SARS CoV 2 genome, and the nucleocapsid protein (N), spike protein (S) and Papain like protease were the least conserved and likely to accumulate mutations over time. [Table 3].

Table 3: Wild type variants as noted several have mutations (i.e. differences from the Wuhan-Hu reference variant)

Click here to view

In the early phases of the pandemic, certain mutations such as the Spike protein D614G (nt 23403) and the P323 L (nt 14408) in the RdRP enzyme (RNA-dependent RNA polymerase) gained prominence, especially the D614G that resulted in higher transmissibility of the virus and also a potential target for vaccination strategy. The P323 L, since first identified in the RdRP protein, has been correlated with a higher number of overall mutations gathered within the genome, suggesting an effect on proofreading during viral replication.

SARS-CoV-2 has been observed to accumulate mutations rapidly and evolve at a high rate. A vast number of these mutations rarely impact the viral characteristics, a few, such as those mentioned above, give rise to novel variants that pose a higher risk. The WHO classifies these novel strains as VOIs (mutants with modified receptor binding, resistance to treatment or neutralisation by antibodies, etc.,) and VOCs (strongly evidenced increase in transmissibility, severity, resistance to treatment, reduction in neutralisation by antibodies and overall higher risk).

The past 2 years during the pandemic have witnessed the emergence of new variants (Alpha, Beta, Gamma, Delta and Omicron) of the SARS-CoV-2 virus associated with the increased ability of transmission, reinfection and reduced efficacy of vaccination. Apart from these, there are many more subtypes that share mutations and characteristics with these, especially features that increase and maintain infectivity despite rising population immunity. Variants are classified based on their transmissibility (reproduction rate over other circulating types), the severity of infection (based on the rate of hospitalisation and mortality) and their resistance to host immune response.

The Indian SARS-CoV-2 Consortium on Genomics or Indian SARS-CoV-2 Genetics Consortium (INSACOG) is the forum set up under the Ministry of Health and Family Welfare by the Government of India on 30 December 2020 to study and monitor genome sequencing and virus variation of circulating strains of COVID-19 in India. Contact tracing of the case in which the mutation is detected. Epidemiological aspects of the mutant detected with respect to the number of cases, deaths in the community, etc., clinical spectrum of the positive case to detect any change in the severity or mortality. All family members in whom a variant has been found, as well as any of their contacts, needs to have samples collected and delivered to the designated INSACOG Genome Sequencing Laboratories for whole-genome sequencing (IGSL), the procedure of which is briefly illustrated in [Figure 1]. Gandhi Medical College and Hospital is one of the mapped IGSL centres of Telangana State. This activity should be overseen by the rapid response team. Take necessary containment measures in the area in conjunction with the district administration and provide daily status reports.

Figure 1: Illustrative View of COVID-19 NGS basis analysis. COVID-19: Coronavirus disease-2019, NGS: Next generation sequencing

Click here to view

Next-Generation Sequencing and Coronavirus Disease-2019 Vaccine Development

Vaccines have come a long way since the time of Louis Pasteur. It is today known as vaccinology, a discipline that encompasses not only vaccine manufacture, delivery strategies and influence on the clinical course of the disease and the immune system response of the host but also regulatory, ethical, economic and ecological elements of their use.^[25] A century after Pasteur developed the first vaccine, another significant scientific advance in this sector occurred, namely Sanger sequencing. The genomic era has changed the vaccine landscape even more by the modernisation of molecular biology techniques. The introduction of high-throughput sequencings, such as NGS, has revolutionary potential in the field of vaccine design and development due to its myriad applications, which include sequencing of host and pathogen genomes^[26] and transcriptomes,^[27],[28] as well as studies of the diversity of host immune responses in T- and B-cells.^{[29],[30],[31]}

Coronaviruses (CoVs) are a group of viruses that have the potential to trigger an epidemic due to their ability to overcome species barriers and propagate quickly in a new host species. There are various coronavirus vaccines for animals (particularly against IBV caused by gamma coronaviruses), but no vaccinations for humans existed until recently, despite the serious public health hazard posed by CoV-related disorders.^[32] One of the first stages in dealing with the COVID-19 epidemic was to define the virus and determine how quickly it was spreading. Early in the outbreak, NGS was utilised to decode the first novel SARS-CoV-2 sequence, and this sequence served as the foundation for several techniques to halt the rapid progression of the pandemic. First, the initial sequence supplied enough information to conduct preliminary research into successful virus detection techniques. Later, with the worsening COVID-19 scenario, it was imperative to protect human life, and numerous scientific institutions began developing vaccinations targeted at reducing the disease burden. The adoption of NGS technology in the COVID-19 pandemic allowed for an earlier start to the vaccine development process than was possible with traditional assays in previous disease outbreaks.

On 1 January and 2 January 2021, the National Regulatory Authority of India accorded restricted emergency use authorisation (EUA) to the viral vector vaccine developed by Oxford–AstraZeneca (Covishield, manufactured in India) and inactivated vaccine BBV152 (Covaxin), respectively.^[33] Sputnik V, a vaccine produced by the Gamaleya Research Institute of Epidemiology and Microbiology in Russia, is made up of two components: recombinant adenovirus type 5 and recombinant adenovirus type 26 vectors, both of which contain the gene for SARS-CoV-2 spike glycoprotein.^[34] In India, Sputnik received EUA on 13 April 2021. In other parts of the world, Johnson and Johnson, USA, created a similar but single adenovirus-based vaccination. Two firms, Pfizer-BioNTech, the collaboration between German and American companies, have commercialised a vaccine named Comirnaty, which is based on messenger RNA (mRNA) technology and the first COVID-19 vaccine licenced for use in the European Union. Moderna, USA, has also designed an mRNA-based COVID-19 vaccine, which after following a satisfactory examination by the European Medicines Agency, got approval for the mRNA-1273 vaccine. The German CureVac business is developing a vaccine based on a similar concept of mRNA. All the available COVID-19 vaccines in use are extremely successful, with efficacy ranging from 70% to 95%, depending on the dose. In India, the national COVID-19 vaccination programme was launched on 16 January 2021. Up to 10 May 2022, 1,00,63,06,450 had received one dose, 86,96,09,528 individuals had received two doses and 2,85,52,357 individuals received a booster/precaution dose.^[35] However, despite vaccination, there were breakthrough infections observed in the initial days of the vaccinations raising uncertainty about how effective these vaccinations will be against the newly emerging SARS-CoV-2 strains. Individuals who tested positive for SARS-CoV-2 by rRT-PCR or rapid antigen test within 14 days of receiving one dose of either of the authorised COVID-19 vaccinations were considered to have a breakthrough COVID-19 infection. On 09 April 2021, Israel was the first to report breakthrough COVID-19 infections in people who had received the Pfizer vaccination.^[36] From March 2021, we had a second increase of COVID-19 cases in India, which was followed by a disastrous second wave. The neutralization potential of Covishield/AstraZeneca vaccined sera against the B.1.1.7 variation was found to be lowered compared to the ancestral strain, in invitro experiments reported in April 2021^[37] The only method for tracking breakthrough infections was the use of NGS technology. Sequencing reveals unique, emerging SARS COV2 variations, which can be further analysed in silico using bioinformatic tools to reveal their potential for novel infections in the future.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	Alteri C, Cento V, Piralla A, Costabile V, Tallarita M, Colagrossi L, et al. Genomic epidemiology of SARS-CoV-2 reveals multiple lineages and early spread of SARS-CoV-2 infections in Lombardy, Italy. Nat Commun 2021;12:434.
2.	Shen L, Niu J, Wang C, Huang B, Wang W, Zhu N, et al. High-Throughput screening and identification of potent broad-spectrum inhibitors of coronaviruses. J Virol 2019;93:e00023-19.
3.	Chiara M, D'Erchia AM, Gissi C, Manzari C, Parisi A, Resta N, et al. Next generation sequencing of SARS-CoV-2 genomes: Challenges, applications and opportunities. Brief Bioinform 2021;22:616-30.
4.	de Mello Malta F, Amgarten D, Val FC, Cervato MC, de Azevedo BM, de Souza Basqueira M, et al. Mass molecular testing for COVID19 using NGS-based technology and a highly scalable workflow. Sci Rep 2021;11:7122.
5.	Dilliott AA, Farhan SMK, Ghani M, Sato C, Liang E, Zhang M, et al. Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease. J Vis Exp 2018;(134):57266. doi: 10.3791/57266.
6.	Forbes JD, Knox NC, Ronholm J, Pagotto F, Reimer A. Metagenomics: The next culture-independent game changer. Front Microbiol 2017;8:1069.
7.	Freed NE, Vlková M, Faisal MB, Silander OK. Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding. Biol Methods Protoc 2020;5:bpaa014.
8.	Knighton AJ, Ranade-Kharkar P, Brunisholz KD, Wolfe D, Allen L, Belnap TW, et al. Rapid implementation of a complex, multimodal technology response to COVID-19 at an integrated community-based health care system. Appl Clin Inform 2020;11:825-38.
9.	La Rosa G, Brandtner D, Mancini P, Veneri C, Bonanno Ferraro G, Bonadonna L, Lucentini L, Suffredini E. Key SARS-CoV-2 Mutations of Alpha, Gamma, and Eta Variants Detected in Urban Wastewaters in Italy by Long-Read Amplicon Sequencing Based on Nanopore Technology. Water 2021;13:2503. https://doi.org/10.3390/w13182503.
10.	Li N, Cai Q, Miao Q, Song Z, Fang Y, Hu B. High-Throughput metagenomics for identification of pathogens in the clinical settings. Small Methods 2021;5:2000792.
11.	Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: Implications for virus origins and receptor binding. Lancet 2020;395:565-74.
12.	Luciani F, Bull RA, Lloyd AR. Next generation deep sequencing and vaccine design: Today and tomorrow. Trends Biotechnol 2012;30:443-52.
13.	Jin Y, Yang H, Ji W, Wu W, Chen S, Zhang W, et al. Virology, epidemiology, pathogenesis, and control of COVID-19. Viruses 2020;12:E372.
14.	World Health Organization. Regional Office for the Western Pacific. . Inaugural Meeting of the Western Pacific Region Emerging Molecular Pathogen Characterization Technologies (EMPaCT) Surveillance Network, Virtual meeting, 21-22 September 2021: meeting report. WHO Regional Office for the Western Pacific 2021. https://apps.who.int/iris/handle/10665/352822.
15.	Pandey, Anuj & Mishra, Sidharth & Wadgave, Yogesh & Mudgil, Nidhi & Gawande, Sonal & Dhange, Vijay. The COVID-19 variants: an overview. International Journal Of Community Medicine And Public Health 2021;8:5148. 10.18203/2394-6040.ijcmph20213826.
16.	Pattabiraman C, Prasad P, George AK, Sreenivas D, Rasheed R, Reddy NV, et al. Importation, circulation, and emergence of variants of SARS-CoV-2 in the South Indian state of Karnataka. Wellcome Open Res 2021;6:110.
17.	Safiabadi Tali SH, LeBlanc JJ, Sadiq Z, Oyewunmi OD, Camargo C, Nikpour B, et al. Tools and techniques for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/COVID-19 Detection. Clin Microbiol Rev 2021;34:e00228-20.
18.	Hurwitz AM, Huang W, Estes MK, Atmar RL, Palzkill T. Deep sequencing of phage-displayed peptide libraries reveals sequence motif that detects norovirus. Protein Eng Des Sel 2017;30:129-39.
19.	Yan Y, Wu K, Chen J, Liu H, Huang Y, Zhang Y, et al. Rapid acquisition of high-quality SARS-CoV-2 genome via amplicon-oxford nanopore sequencing. Virol Sin 2021;36:901-12.
20.	Zumla A, Al-Tawfiq JA, Enne VI, Kidd M, Drosten C, Breuer J, et al. Rapid point of care diagnostic tests for viral and bacterial respiratory tract infections – Needs, advances, and future prospects. Lancet Infect Dis 2014;14:1123-35.
21.	Li X, Song Y, Wong G, Cui J. Bat origin of a new human coronavirus: There and back again. Sci China Life Sci 2020;63:461-2.
22.	Dallavilla T, Bertelli M, Morresi A, Bushati V, Stuppia L, Beccari T, et al. Bioinformatic analysis indicates that SARS-CoV-2 is unrelated to known artificial coronaviruses. Eur Rev Med Pharmacol Sci 2020;24:4558-64.
23.	Decaro N, Lorusso A. Novel human coronavirus (SARS-CoV-2): A lesson from animal coronaviruses. Vet Microbiol 2020;244:108693.
24.	Wang R, Hozumi Y, Yin C, Wei GW. Decoding SARS-CoV-2 transmission and evolution and ramifications for COVID-19 diagnosis, vaccine, and medicine. Journal of chemical information and modeling 2020;60:5853-65.
25.	Li X, Wang L, Yan S, Yang F, Xiang L, Zhu J, et al. Clinical characteristics of 25 death cases with COVID-19: A retrospective review of medical records in a single medical center, Wuhan, China. Int J Infect Dis 2020;94:128-32.
26.	Pasik K, Domańska-Blicharz K. High-throughput sequencing in vaccine research. J Vet Res 2021;65:131-7.
27.	Ley TJ, Mardis ER, Ding L, Fulton B, McLellan MD, Chen K, et al. DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature 2008;456:66-72.
28.	Margeridon-Thermet S, Shulman NS, Ahmed A, Shahriar R, Liu T, Wang C, et al. Ultra-deep pyrosequencing of hepatitis B virus quasispecies from nucleoside and nucleotide reverse-transcriptase inhibitor (NRTI)-treated patients and NRTI-naive patients. J Infect Dis 2009;199:1275-85.
29.	Ozsolak F, Milos PM. RNA sequencing: Advances, challenges and opportunities. Nat Rev Genet 2011;12:87-98.
30.	Birzele F, Fauti T, Stahl H, Lenter MC, Simon E, Knebel D, et al. Next-generation insights into regulatory T cells: Expression profiling and FoxP3 occupancy in Human. Nucleic Acids Res 2011;39:7946-60.
31.	Burwitz BJ, Ende Z, Sudolcan B, Reynolds MR, Greene JM, Bimber BN, et al. Simian immunodeficiency virus SIVmac239Deltanef vaccination elicits different Tat28-35SL8-specific CD8+T-cell clonotypes compared to a DNA prime/adenovirus type 5 boost regimen in rhesus macaques. J Virol 2011;85:3683-9.
32.	Wu X, Zhou T, Zhu J, Zhang B, Georgiev I, Wang C, et al. Focused evolution of HIV-1 neutralizing antibodies revealed by structures and deep sequencing. Science 2011;333:1593-602.
33.	Gupta N, Kaur H, Yadav PD, Mukhopadhyay L, Sahay RR, Kumar A, et al. Clinical characterization and genomic analysis of samples from COVID-19 breakthrough infections during the second wave among the various states of India. Viruses 2021;13:1782.
34.	Logunov DY, Dolzhikova IV, Zubkova OV, Tukhvatullin AI, Shcheblyakov DV, Dzharullaeva AS, et al. Safety and immunogenicity of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine in two formulations: Two open, non-randomised phase 1/2 studies from Russia. Lancet 2020;396:887-97.
35.	CoWIN Dashboard. Available from: https://dashboard.cowin.gov.in/(22). [Last accessed on 10 May 20].
36.	Goldberg Y, Mandel M, Bar-On YM, Bodenheimer O, Freedman L, Haas EJ, et al. Waning immunity after the BNT162b2 vaccine in Israel. New England Journal of Medicine 2021;385:e85.
37.	Emary KR, Golubchik T, Aley PK, Ariani CV, Angus B, Bibi S, et al. Efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine against SARS-CoV-2 variant of concern 202012/01 (B. 1.1. 7): an exploratory analysis of a randomised controlled trial. The Lancet 2021;397:1351-62.