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Clinical Research in Infectious Diseases

Surveillance of Emerging SARSCoV-2 Variants by Nanopore Technology-based Genome Sequencing

Research Article | Open Access

  • 1. Department of Virology, Medical Research Institute, P.O.Box: 527, Baseline Road, Colombo 08, Sri Lanka
  • 2. Department of Virology, Medical Research Institute, P.O.Box: 527, Baseline Road, Colombo 08, Sri Lanka
  • 3. Department of Virology, Medical Research Institute, P.O.Box: 527, Baseline Road, Colombo 08, Sri Lanka
  • 4. Department of Pathology, Stanford University School of Medicine, Palo Alto, California, USA
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Corresponding Authors
JI Abeynayake, Department of Virology, Medical Research Institute, P.O. Box: No. 527, Baseline Road, Colombo 08, Sri Lanka, E-mail: janakiiabeynayake@yahoo.com
Abstract

Background:

Detection of emerging variants of severe acute respiratory syndrome coronavirus-2, genome sequencing in all countries at least 1% of their infections is recommended. Nanopore technology platform was set-up at the Reference laboratory during pandemic, sequencing is continued to understand the circulated variants in the country.

Objectives:

This study was to describe the surveillance of emerging variants by nanopore technology-based genome sequencing in different COVID-19 waves in Sri Lanka and to demonstrate the association with the sample characteristics, and vaccination status.

Methodology:

The study analyzed 207 RNA positive swab samples received to sequence laboratory during different waves. The N gene cut-off threshold < 30 considered as the major inclusion criteria. Viral RNA was extracted, elutes were subjected to nanopore sequencing according to the manufacturer’s instructions using the SQK-RBK110.96 rapid barcoding kit. All the sequencing data were uploaded in the publicly accessible database, GISAID.

Results:

Analysis revealed variants distributed throughout the period were 58% Omicron, 22% Delta, 4% Alpha, and only less than 1% of Kappa variant. 16% study samples were remained unassigned. Omicron variant was circulated among all age groups and in all the provinces. Ct value and variants assigned percentage was 100% in Ct values 10-15 while only 45% assigned Ct value over 25.

Conclusion:

The present study declared the emergence, prevalence, and distribution of SARS-CoV-2 variants locally and summarized the establishment of Nanopore Technology by enabling whole genome sequencing in a low resource setting country

Keywords

Emerging SARS-Cov-2 Variants, Laboratory Surveillance, Nanopore Technology, Genome Sequencing, Bioinformatics Analysis And Phylogeny, Sociodemographic And Sample Cut off (Ct) Threshold, Global Sharing Of Genomic Data/GISAID

citation

Abeynayake JI , Chathuranga GP, Fernando MAY, Sahoo MK (2023) Surveillance of Emerging SARS-CoV-2 Variants by Nanopore Technologybased Genome Sequencing. Clin Res Infect Dis 7(1): 1058.

INTRODUCTIO

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve giving rise to many variants with higher transmissibility and immune evasion abilities which continue to drive the pandemic [1]. To detect emerging of such possible new variants, the World Health Organization has recommended to carry out genomic sequencing in all countries for at least 1% of their infections [2]. New variants of SARS-CoV-2 virus reinforce the critical role of whole genomic sequencing since it is important to find useful information about the viral lineages, Variants of Interests (VOI) and Variants of Concern (VOC) [3].

SARS-CoV2 is a virus with a single stranded RNA. The genome is about 30 kb and consists of genes encoding multiple nonstructural, structural, and accessory proteins. The non-structural proteins include NSP1 to NSP16 which are necessary for virus transcription and replication [4]. The structural proteins include Spike (S), Envelope (E), Membrane (M) and Nucleoprotein(N).The virus attachment, entry, and infectivity are mediated by structural proteins [3,4]

During virus replication, mutations can occur altering its protein functions leading to little to no impact on the virus’s properties, causes response to on vaccines, therapeutic medicines, diagnostic tools, and other public health and social measures [5]. Factors such as possible transmission between humans and other mammals, have contributed to the rapid increase in the number of mutations [3]. During the pandemic different variants have been identified and broadly categorized as VOC, VOI, and Variants Under Monitoring (VUM). Identification and monitoring their diversified transmission routes in a community in all countries is of key importance.

Nowadays Next-Generation Sequencing (NGS) is an effective method to identify different mutations and new variants of epidemiological and clinical importance. Even though Capillary sequencing technology was the first sequencing technology designed it has been progressively displaced by high-throughput“NGS” technologies. There are several NGS platforms such as Illumina dye sequencing, pyrosequencing, and single molecule real-time sequencing [6,7]. Nanopore technology is another technology that has been emerged and its read length is on average much longer, being especially valuable in Whole-Genome Sequencing (WGS) applications. Further, Nanopore technologies provides fast SARS-CoV-2 sequencing, at low cost, in a portable sequencing platform, with requirement of relatively minimal laboratory infrastructure [8].

Therefore, this Nanopore technology platform was setup in the state sector laboratories during the pandemic and sequencing was carried out during different waves and post COVID (Coronavirus disease) period with positive SARS-CoV-2 samples for better understanding of the circulating SARS-CoV-2 variants, and its mutations in the country.

OBJECTIVES

The objective of this study was to describe the laboratory surveillance of emerging SARS-CoV-2 variants by Nanopore technology-based genome sequencing in different COVID waves and post- COVID period in Sri Lanka and to demonstrate the association with the sample characteristics, clinical profiles, and the vaccination status.

MATERIAL AND METHODOLOGY

Clinical Samples

The study retrospectively analyzed Nasopharyngeal/ Oropharyngeal (NP/OP) samples received for WGS at the sequencing laboratory of the National Virus Reference Laboratory (NVRL), in the country during different COVID waves, using the Nanopore technology-based genome sequencing. All samples included had previously tested positive for SARSCoV-2 RNA using rRT-PCR (Real-Time Reverse-TranscriptasePolymerase Chain Reaction) with targets in the nucleocapsid and envelope genes or lateral flow testing with Panbio COVID-19 Ag rapid test of Abbott manufactures. Following primary detection of COVID-19 positivity samples were matched with pre-defined criteria developed by the Ministry of Health [9] and selected for genomic sequencing.

Sequencing laboratory at NVRL received NP/OP specimens in viral transport media maintaining the cold chain, from different provinces of the country through health care institutions of both state and private sector in Sri Lanka. Upon arrival samples were aliquoted, deidentified, and stored at -80?C until nucleic acid extraction and the test run was initiated. The N gene Cut off threshold value (Ct value) less than 30 was considered as the major inclusion criteria during selection of samples [9,10]. The other criteria intensified were positive samples from overseas returnees/foreign travelers, critically ill patients in ICU, moderate to severe symptomatic inward patients despite having the full course of COVID-19 vaccination, positive patients with initial recovery and discharged, community clusters with high complications, and samples from COVID-19 deaths

RNA extraction and Genomic sequencing

A total of 207 samples were re-extracted with a single freezethaw cycle. QIAamp® Viral RNA Mini kit was used following the manufacturers instruction to extract the viral RNA [11]. The workflow of the nanopore sequencing procedure is as Figure 1.

Workflow of the Nanopore Sequencing.

Figure 1: Workflow of the Nanopore Sequencing.

The extracted samples were subjected to nanopore sequencing according to the manufacturer’s instructions using the SQKRBK110.96 rapid barcoding kit (ONT, Oxford, UK). The extracted RNA was converted to cDNA using Luna Script RT Super Mix followed by sample amplification in two reactions with Midnight primer pool A & B and Q5 HS Master Mix designed for whole genome amplification.

1200 bp tiled PCR amplicons were generated with midnight primers as described in Freed et al., 2020 [12]. All the thermal cycling steps were carried out in the ABI 7500 Real Time PCR instrument (Applied biosystems, USA). Barcodes were attached to resulting DNA amplicons with Rapid Barcode Kit and pooled together before the clean-up step. Subsequently, DNA library was loaded into R9 version of the Oxford Nanopore MinION Spot-ON flowcell (FLO- MIN106D) and sequenced on the Oxford Nanopore Minion Mk1C. The run was terminated once the desired number of reads, a minimum of 20,000 reads per sample was achieved.

During the sequence run streaming the raw data generated were converted to FASTQ files through a process called base calling. FASTQ text files containing sequence data for each read were then uploaded to the EPI2ME Agent software which is a cloud-based data analysis platform that appraises the putative variant of SARS-CoV-2. Furthermore, the resulting consensus sequences (FASTA files) were analyzed through several web-based software like Nextclade, Pangolin COVID-19 lineage assigner, and Stanford University coronavirus antiviral & resistance database. Finally, all the sequencing data were uploaded and published in the publicly accessible database, GISAID platform which is a global initiative on sharing of genomic data [13]. In addition, distribution of variants during different COVID-19 waves, Cut off threshold (Ct value) of the tested samples, vaccination status, clinical profile and sociodemographic data gathered from the accompanied test requests were scrutinized with sequencing results.

Statistical Analysis

Excel software was used to calculation of means, and percentages. Mode was calculated using the SPSS version 25. Descriptive statistics were used to show the characteristics of the study sample.

RESULTS

Here we present genome sequenced results of samples during October 2021 through January 2023 with sample characteristics and variants distribution across the variables

Sample Characteristics

A total of 207 SARS-CoV-2 positive patients’ swab-based samples were sequenced. Sample characteristics such as age range, gender, geography, vaccination status, and clinical profile are depicted in Table 1.

Table 1: Sample characteristics.

Variable Frequency (percentage)
Age range, Mean, Mode, (n = 207) 4 months - 80 years, 37 years, 28 years
< 1 year 2 (0.97%)
1-15 years 22 (10.63%)
16-50 years 134 (64.73%)
52-65 years 40 (19.32%)
> 65 years 9 (4.35%)
Gender (n=207)
Female 76 (36.71%)
Male 131 (63.29%)
Geographical distribution of cases (n=207)
Western 162 (78.26%)
Northwestern 32 (15.46%)
Eastern 2 (0.97%)
Sabaragamuwa 1 (0.48%)
Vaccination status (n = 207)
Vaccinated 84 (40.58%)
Not vaccinated 7 (3.38%)
Unknown 116 (56.04%)
Clinical Profile (n = 207)
Symptomatic 59 (28.50%)
Asymptomatic 16 (7.73%)
Unknown 132 (63.77%)
N Gene Ct value (n = 207)
15-Oct 23 (11.11%)
15-25 139 (67.15%)
> 25 31 (14.98%)
Antigen Positive cases 6 (2.90%)
Unknown 8 (3.86%)

Of these, majority were nasopharyngeal samples. Among the sequenced, males were 63%, and females only 37%. Age range was four months to 80 years and mean age was 37 years. Of the total, highest numbers subjected to sequencing were from Western province and rest represented North-western, Southern, Eastern and Sabaragamuwa provinces. Vaccination status was provided only in 44% whereas status was unknown in others. 59% of the samples were from symptomatic patients, while 17% were from asymptomatic and the remainder was not documented. Samples that were subjected to sequencing covered a scale of 10-30 in the N gene Cut-off threshold (Ct) with 139 samples within the Ct of 15-25.

Bioinformatics analysis of sequencing data generated a SARS CoV-2 phylogenetic tree depicted in Figure 2,

Nextclade based Phylogenetic Tree created using Sequences  data during the study period in Sri Lanka.

Figure 2: Nextclade based Phylogenetic Tree created using Sequences data during the study period in Sri Lanka.

which illustrated the different variants according to WHO nomenclature during the period. The circles represent the Sri Lanka sequences in comparison with published sequences from all over the world. Here, Nextclade lineages are clustered according to the indicated color code. Figure 2 discloses the details of evolutions such as nucleotide and amino acid changes from root, divergence, and clade leading to understand the virus molecular epidemic profile of the country at a given specific time point. Retrospective analysis revealed variants distributed throughout the period were 58% Omicron, 22% Delta, 4% Alpha, and only less than 1% of Kappa variant. 16% study samples were remained unassigned Table 2.

Table 2: Variants distribution during the period.

Variant Frequency (n = 207)
Omicron (B.1.1.529) 119 (57.49%)
Delta (B.1.617.2) 46 (22.22%)
Alpha (B.1.1.7) 8 (3.87%)
Kappa (B.1.617.1) 1 (0.48%)
Unassigned 33 (15.94%)

Data in the Table 3

Table 3: Distribution of Variants/Sub variants during the period.

Month Variant/Frequency Sub Variant/Frequency
December,2021 (n = 53) Delta 42 (79.2%) AY.104 28 (66.7%)
    AY.28 6 (14.3%)
    AY.39 4 (9.5%)
    AY.67 2 (4.8%)
    AY.95 1 (2.4%)
    AY.101 1 (2.4%)
  Omicron 3 (5.7%) BA.1 3 (100%)
January,2022 (n = 95) Omicron 66 (69.47%) BA.1 66 (100%)
  Alpha 8 (8.42%) B.1.1.7 8 (100%)
  Delta 4 (4.21%) AY.104 3 (100%)
  Kappa 1 (1.05%) -  
July,2022 (n = 36) Omicron 34 (94.4%) BA.5 27 (79.4%)
    BA.2 6 (17.6%)
    BE.1 1 (2.9%)
January,2023 (n = 23) Omicron 16 (69.57%) BA.5 4 (25.00%)
    BA.2 4 (25.00%)
    XBB.1 4 (25.00%)
    BF.15 1 (6.25%)
    BF.28 1 (6.25%)
    XBB.3 1 (6.25%)
    CH.1.1 1 (6.25%)

displayed different sub-variants circulated throughout the study period. It was evident leading variants circulated in the months of December 2021 and January 2022 were Delta (79%), Omicron (6%) and Omicron (69%), Delta (4%) respectively. Different variants were circulated during the period, and highest numbers of sub variants were associated with Omicron variant. Association between Ct value and variant assignation is demonstrated in Figure 3.

Association of Ct value and Variant Assigned %.

Figure 3: Association of Ct value and Variant Assigned %.

It was 100% in samples with Ct values 10-15 while only 45% demonstration in samples with a Ct value over 25.

Sample variables and variant distribution are displayed in the Table 4

Table 4: Distribution of Variants with the sample Variables.

Variable Alpha (n = 8) Delta (n = 46) Omicron (n = 119)
Age
< 1 - - 2 (0.97%)
15-Jan 6 (2.90%) 4 (1.93%) 13 (6.28%)
16-50 2 (0.97%) 26 (12.56%) 80 (38.65%)
51-65 - 12 (5.80%) 18 (8.70%)
>65 - 4 (1.93%) 6 (2.90%)
Province
Western 6 (2.90%) 25 (12.08%) 105 (50.72%)
Northwestern - 21 (10.14%) 6 (2.90%)
Southern 2 (0.97%) - 7 (3.38%)
Eastern - - 2 (0.97%)
Sabaragamuwa - - 1 (0.48%)

and Figure 4. Omicron variant was circulated among all

Distribution of Variants with the sample Variables.

Figure 4: Distribution of Variants with the sample Variables.

age groups and in all the provinces. Delta variant was detected in all age groups except in less than one year old patient, but it was detected only in two provinces. Both genders were affected with Omicron (58%), Delta (22%) and Alpha (4%). 30% of the vaccinated population was affected with the Omicron variant. Symptomatic clinical profile was detected in higher percentages than asymptomatic profiles associated with both Delta and Omicron variants.

DISCUSSION

Genomic surveillance is a crucial weapon in the public health fight against infectious diseases, providing rapid identification and complete characterization of infectious disease pathogens. Surveillance related to the SARS-CoV-2 is highly recommended by WHO since RNA viruses are often characterized with high mutation rates [14,15]. Moreover, identification of mutations is critical for not only understanding the infectious mechanism but also for tracking the evolution and transmission routes of the virus [4,16-18]. The manuscript describes the nanopore based gene sequencing, criteria for sample selection and appraises the analysis of putative variants during the period which covers different COVID waves and post COVID period. Criteria considered for selection of samples, filtered the viral load, tracked the imported variants, vaccine escaped mutants, variants in critically ill patients and in deaths as well as in reinfected patients.

We have setup and utilize the nanopore technology for generating and analyzing sequencing data due to its several advantages of this technique which include, single molecule sequencing, enable rapid generation of sequencing data and real-time analysis [19]. Further we experienced, that it requires a comparatively simple procedures for library preparation, offer flexibility in sample throughput by accommodating low to high numbers of specimens per flow-cell, and most importantly its low capital and recurrent cost which is well suited for any low resource setting country. Inbuilt quality control produces standard plots such as distributions of read lengths and quality scores, the number of reads generated per barcode, and the total yield of bases over time are some of the benefits added while using this technology. Along with plots it simplifies optimization of laboratory procedures in sequencing, through rapid diagnosis of common issues like bubbles introduced during library loading, and the presence of contaminants during sequencing, as which also explained well in several studies [20,21].

In this study, 207 positive SARS-CoV-2 genomes were subjected to genomic sequencing. The study analyzed combine residual samples received at the reference laboratory as well as the samples aliquoted and stored at -80? in the reference laboratory during the 1st and 2nd waves. In addition, samplespositive following COVID-19 Ag testing too counted for genome sequence in the current study. The study comprised samples from four months to 80 years of age whereas clinical profile varies from asymptomatic infection to symptomatic, deaths and with some unknown clinical profiles. Majority of the samples were from males and the rest represented the female population. The study subjects mixed with vaccinated, unvaccinated, and unknown status. Samples from different provinces accommodated to sequence study, announced highest numbers are from Western province.

Genomic sequencing data demonstrated majority of samples to be assigned with variants while some samples were unassigned and is most likely due to greater RNA instability or may be due to inadequate original RNA load. Interestingly, almost 70% of Ag positive samples subjected to sequencing were assigned with variants which is an encouraging sign for sequencing during the post COIVD-19 period. The overall incidence of Omicron variant, heavily mutated variant was symbolic indicating Omicron activity in the period of October 2021 to January 2023, confirming the current study period was basically after the 1st and 2nd COVID-19 waves which the 1st and 2nd waves went on from January 2020 to October 2021. However, Delta, Alpha and Kappa variants were also identified for a lesser extent at the same time as the study analyzed a few stored samples at the reference center. Meanwhile, monthly distribution of Omicron variant shown marked increase from 5.7% to 69.47% while Delta variant shown marked decrease from 79.2% to 4.21% from December 2021 to January 2022, representing the trends in some other counties [22]. Beside this feature, same data demarcated the 2nd and the 3rd COVID-19 waves, in Sri Lanka. Apart from those signals, data demonstrated the significant surge of the 3rd wave around January 2022, which may coincide with the emergence of the specific Omicron variant. Alternately, data spelled how Delta variant was replaced by the Omicron variant during the months of January 2022 to July 2022. This shift in the country explained the findings that have been observed globally owing to increased mutations of the Omicron variant [22].

Interestingly, bioinformatics analysis of sequencing data generated the SARS-CoV-2 family tree which showed off branching into many limbs and illustrated the phylogeny of the variants detected in Sri Lanka during the targeted period. This powerful genetic tool makes sense of genome sequence diversity leading to emergence of viruses, sub variants and trace how they are related through sub-sampling on different geographics and different time periods as of its core fundamentals. It conveys the vital information of subsequent mutation rates with time where it was low in Delta variant and considerably high in Omicron which led to massive changes in the Omicron overtime. The rapid mutations acquired by the virus have significantly contributed to the 3rd COVID-19 wave in the pandemic

The sequence data exhibited Delta variant dominance during the December 2021 with AY sub- variants. Alpha variant or sub variant B.1.1.7 detection was relatively low revealing that it was more prevalent in 1st and 2nd waves across the world [22].Although, different variants were circulated during the study period, highest number of sub variants were associated with the Omicron. Emergence of Omicron was noted in January 2022 drastically and it continued to predominate over time with significant number of sub-variants namely BA.1, BA.5, BA.2, BE.1, XBB.1, XBB.3, BF.15, BF.28 and CH.1.1 during the rest of the study period parallel to the other countries [23]. Sri Lanka too has series of recent mutants XBB and BF although BF.7 and XBB.1.5 which are more virulent and transmissible [23,24] not identified within the current data. Emergence of variants and sub variants justified the high mutation rate of the SARS-CoV-2 RNA virus. Furthermore, bioinformatics analysis of sequencing data emphasizes the interval of emergence of BF and XBB. These significant mutations attributed to gradual disappearance of BA.5 sub variant which is comparable to the global scenario [23,24,25].

As previously mentioned in the text, 201 representative samples were selected with a Ct value spanning from10 to 30, and six others were from Ag positive samples tested during the post COVID-19 period due to the limited availability of rRT-PCR positive samples. The analysis disclosed lower Ct values to be more productive for genome analysis and the context assures a 100% or near detection with Ct value less than 25 among the study samples. Findings also persuades to sequence Ag positive samples instead of obliging Ct values over 25. This fact is justifiable since antigen positivity showed up in early stage of the infection with a relatively significant viral load. This further reveals a correlation between the ability to analyze SARS-CoV-2 viral genome with nanopore technology in the presence of higher viral load. However, the nasopharyngeal swabs predominated the type of samples in this study with a low number of different other types of samples which limited the possibility of detecting the more superior sample for sequencing..

Data indicated Omicron variant infectivity occurred irrespective of the age and this observation held across the provinces dispatched samples to this study. Delta variant activity remains almost consistent with that of Omicron in relation to the age. However, mix circulation with Alpha, Delta and Omicron was evident in the Western province during the period may be due to high population density in the province. Surprisingly, even though Omicron infectivity was high despite the individual’s positive vaccination status, the current study unable to compare the outcome of Omicron activity within unvaccinated population. Despite the variant circulated, individuals with symptomatic clinical profile were leading among the study population which is comparable to some other countries [4]. Even with the percentage difference of Delta and Omicron, available data is inadequate to convince the association of variants and deaths during the pandemic. Although, the percentage of Omicron activity is greater among many variables contrast to the other variants, this study partially demonstrates the impact of other variants in the country due to the low number of samples taken into the study from 1st and 2nd COVID-19 waves.

CONCLUSION

Conclusively, the document summarized the advantagesof Nanopore Technology, it’s establishment by enabling whole genome sequencing facility in a low resource setting county to contribute to SARS-CoV-2 surveillance locally and further empowered by global surveillance. The present study declared the emergence, prevalence, and distribution of variants locally, which were related to different COVID-19 waves in Sri Lanka. Ongoing sequencing efforts are critical to monitor subtypes and assist in identifying emerging variants of concern which will contribute to the global effort toward elucidating the molecular epidemic profile of the virus

ACKNOWLEDGMENT

We thank Dr. Palitha Abeykoon, former WHO Special Envoy, Dr. Anil Jasinghe, former DGHS for assistance and guidance towards establishment of gene sequencing at Reference laboratory in Sri Lanka and the staff of the Department of Virology, Medical Research Institute, Colombo 8.

REFERENCES

1. Dyson L, Hill EM, Moore S, Curran-Sebastian J, Tildesley MJ, Lythgoe KA, et al. Possible future waves of SARS-CoV-2 infection generated by variants of concern with a range of characteristics. Nat Commun. 2021; 12(1): 5730. doi: 10.1038/s41467-021-25915-7. PMID: 34593807; PMCID: PMC8484271.

2. World Health Organization. Guidance for surveillance of SARS-CoV-2 variants: Interim guidance. WHO Headquarters, 2021.

3. Yavarian J, Nejati A, Salimi V, Shafiei Jandaghi NZ, Sadeghi K, Abedi A, et al. Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemic. PLoS One. 2022; 17(5): e0267847. doi: 10.1371/journal.pone.0267847. PMID: 35499994; PMCID: PMC9060343.

4. Cheng XW, Li J, Zhang L, Hu WJ, Zong L, Xu X, et al. Identification of SARS-CoV-2 Variants and Their Clinical Significance in Hefei, China. Front Med (Lausanne). 2022; 8: 784632. doi: 10.3389/ fmed.2021.784632. PMID: 35083244; PMCID: PMC8784789.

5. Yakovleva A, Kovalenko G, Redlinger M, Liulchuk MG, Bortz E, Zadorozhna VI, et al. Tracking SARS-COV-2 variants using Nanopore sequencing in Ukraine in 2021. Sci Rep. 2022; 12(1): 15749. doi: 10.1038/s41598-022-19414-y. Erratum in: Sci Rep. 2023; 13(1): 2555. PMID: 36131001; PMCID: PMC9491264.

6. https://en.wikipedia.org”Whole genome sequencings. Wikipedia.

7. Tshiabuila D, Giandhari J, Pillay S, Ramphal U, Ramphal Y, Maharaj A, et al. Comparison of SARS-CoV-2 sequencing using the ONT GridION and the Illumina MiSeq. BMC Genomics. 2022; 23(1): 319. doi: 10.1186/s12864-022-08541-5. PMID: 35459088; PMCID: PMC9026045.

8. https://www.sciencedirect.com”Nanopore Sequencing.

9. Request form and the Instructions to send the sample for SARS-CoV-2 gene sequencing testing. General Circular. 2021, Ministry of Health, Sri Lanka.

10. Juanola-Falgarona M, Peñarrubia L, Jiménez-Guzmán S, Porco R, Congost-Teixidor C, Varo-Velázquez M, et al. Ct values as a diagnostic tool for monitoring SARS-CoV-2 viral load using the QIAstat-Dx® Respiratory SARS-CoV-2 Panel. Int J Infect Dis. 2022; 122: 930-935. doi: 10.1016/j.ijid.2022.07.022. Epub 2022 Jul 12. PMID: 35840097; PMCID: PMC9273520.

11. https://www.qiagen.com/virus.”QIAamp Viral RNA mini kit 250, QIAGEN group.

12. 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(1): bpaa014. doi: 10.1093/biomethods/bpaa014. PMID: 33029559; PMCID: PMC7454405.

13. https://www.cdc.gov/coronavirus/2019-ncov/variants/%20 cdcrole-%20surveillance.”CDC’s Role in tracking emerging COVID-19 Variants.

14. Harvey WT, Carabelli AM, Jackson B, Gupta RK, Thomson EC, Harrison EM, et al. COVID-19 Genomics UK (COG-UK) Consortium; Peacock SJ, Robertson DL. SARS-CoV-2 variants, spike mutations and immune escape. Nat Rev Microbiol. 2021; 19(7): 409-424. doi: 10.1038/ s41579-021-00573-0. Epub 2021 Jun 1. PMID: 34075212; PMCID: PMC8167834.

15. Ou J, Zhou Z, Dai R, Zhang J, Zhao S, Wu X, et al. V367F Mutation in SARS-CoV-2 Spike RBD Emerging during the Early Transmission Phase Enhances Viral Infectivity through Increased Human ACE2 Receptor Binding Affinity. J Virol. 2021; 95(16): e0061721. doi: 10.1128/JVI.00617-21. Epub 2021 Jul 26. PMID: 34105996; PMCID: PMC8373230.

16. Goswami C, Sheldon M, Bixby C, Keddache M, Bogdanowicz A, Wang Y, et al. Identification of SARS-CoV-2 variants using viral sequencing for the Centers for Disease Control and Prevention genomic surveillance program. BMC Infect Dis. 2022; 22(1): 404. doi: 10.1186/s12879- 022-07374-7. PMID: 35468749; PMCID: PMC9035976.

17. Shen L, Dien Bard J, Biegel JA, Judkins AR, Gai X. Comprehensive Genome Analysis of 6,000 USA SARS-CoV-2 Isolates Reveals Haplotype Signatures and Localized Transmission Patterns by State and by Country. Front Microbiol. 2020; 11: 573430. doi: 10.3389/ fmicb.2020.573430. PMID: 33013809; PMCID: PMC7509426.

18. Page AJ, Mather AE, Le-Viet T, Meader EJ, Alikhan NF, Kay GL, et al. Large-scale sequencing of SARS-CoV-2 genomes from one region allows detailed epidemiology and enables local outbreak management. Microb Genom. 2021; 7(6): 000589. doi: 10.1099/ mgen.0.000589. PMID: 34184982; PMCID: PMC8461472.

19. Dächert C, Muenchhoff M, Graf A, Autenrieth H, Bender S, Mairhofer H, et al. Rapid and sensitive identification of omicron by variantspecific PCR and nanopore sequencing: paradigm for diagnostics of emerging SARS-CoV-2 variants. Med Microbiol Immunol. 2022; 211(1): 71-77. doi: 10.1007/s00430-022-00728-7. PMID: 35061086; PMCID: PMC8780046.

20. Bull RA, Adikari TN, Ferguson JM, Hammond JM, Stevanovski I, Beukers AG, et al. Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis. Nat Commun. 2020; 11(1): 6272. doi: 10.1038/s41467-020-20075-6. PMID: 33298935; PMCID: PMC7726558.

21. Lanfear R, Schalamun M, Kainer D, Wang W, Schwessinger B. MinIONQC: fast and simple quality control for MinION sequencing data. Bioinformatics. 2019; 35(3): 523-525. doi: 10.1093/ bioinformatics/bty654. PMID: 30052755; PMCID: PMC6361240.

22. Cheng VCC, Ip JD, Chu AWH, Tam AR, Chan WM, Abdullah SMU, et al. Rapid Spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Omicron Subvariant BA.2 in a Single-Source Community Outbreak. Clin Infect Dis. 2022; 75(1): e44-e49. doi: 10.1093/cid/ciac203. PMID: 35271728; PMCID: PMC8992238.

23. https://www.fda.gov/”SARS -CoV-2 Viral Mutations: Impact on COVID-19 Tests. FDA. 2023.

24. https://www.who.int/”TAG-VE statement on Omicron sub lineages BQ.1 and XBB. WHO Statement, 2022

25. https://www.usnews.com”CDC: Omicron Subvariants BA.4.6, BF.7 increasing while BA.5 declines. By Cecelia Smith-Schoenwalder. 2022

Abeynayake JI , Chathuranga GP, Fernando MAY, Sahoo MK (2023) Surveillance of Emerging SARS-CoV-2 Variants by Nanopore Technologybased Genome Sequencing. Clin Res Infect Dis 7(1): 1058.

Received : 13 May 2023
Accepted : 27 May 2023
Published : 29 May 2023
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Journal of Human Nutrition and Food Science
ISSN : 2333-6706
Launched : 2013
JSM Regenerative Medicine and Bioengineering
ISSN : 2379-0490
Launched : 2013
JSM Spine
ISSN : 2578-3181
Launched : 2016
Archives of Palliative Care
ISSN : 2573-1165
Launched : 2016
JSM Nutritional Disorders
ISSN : 2578-3203
Launched : 2017
Annals of Neurodegenerative Disorders
ISSN : 2476-2032
Launched : 2016
Journal of Fever
ISSN : 2641-7782
Launched : 2017
JSM Bone Marrow Research
ISSN : 2578-3351
Launched : 2016
JSM Mathematics and Statistics
ISSN : 2578-3173
Launched : 2014
Journal of Autoimmunity and Research
ISSN : 2573-1173
Launched : 2014
JSM Arthritis
ISSN : 2475-9155
Launched : 2016
JSM Head and Neck Cancer-Cases and Reviews
ISSN : 2573-1610
Launched : 2016
JSM General Surgery Cases and Images
ISSN : 2573-1564
Launched : 2016
JSM Anatomy and Physiology
ISSN : 2573-1262
Launched : 2016
JSM Dental Surgery
ISSN : 2573-1548
Launched : 2016
Annals of Emergency Surgery
ISSN : 2573-1017
Launched : 2016
Annals of Mens Health and Wellness
ISSN : 2641-7707
Launched : 2017
Journal of Preventive Medicine and Health Care
ISSN : 2576-0084
Launched : 2018
Journal of Chronic Diseases and Management
ISSN : 2573-1300
Launched : 2016
Annals of Vaccines and Immunization
ISSN : 2378-9379
Launched : 2014
JSM Heart Surgery Cases and Images
ISSN : 2578-3157
Launched : 2016
Annals of Reproductive Medicine and Treatment
ISSN : 2573-1092
Launched : 2016
JSM Brain Science
ISSN : 2573-1289
Launched : 2016
JSM Biomarkers
ISSN : 2578-3815
Launched : 2014
JSM Biology
ISSN : 2475-9392
Launched : 2016
Archives of Stem Cell and Research
ISSN : 2578-3580
Launched : 2014
Annals of Clinical and Medical Microbiology
ISSN : 2578-3629
Launched : 2014
JSM Pediatric Surgery
ISSN : 2578-3149
Launched : 2017
Journal of Memory Disorder and Rehabilitation
ISSN : 2578-319X
Launched : 2016
JSM Tropical Medicine and Research
ISSN : 2578-3165
Launched : 2016
JSM Head and Face Medicine
ISSN : 2578-3793
Launched : 2016
JSM Cardiothoracic Surgery
ISSN : 2573-1297
Launched : 2016
JSM Bone and Joint Diseases
ISSN : 2578-3351
Launched : 2017
JSM Bioavailability and Bioequivalence
ISSN : 2641-7812
Launched : 2017
JSM Atherosclerosis
ISSN : 2573-1270
Launched : 2016
Journal of Genitourinary Disorders
ISSN : 2641-7790
Launched : 2017
Journal of Fractures and Sprains
ISSN : 2578-3831
Launched : 2016
Journal of Autism and Epilepsy
ISSN : 2641-7774
Launched : 2016
Annals of Marine Biology and Research
ISSN : 2573-105X
Launched : 2014
JSM Health Education & Primary Health Care
ISSN : 2578-3777
Launched : 2016
JSM Communication Disorders
ISSN : 2578-3807
Launched : 2016
Annals of Musculoskeletal Disorders
ISSN : 2578-3599
Launched : 2016
Annals of Virology and Research
ISSN : 2573-1122
Launched : 2014
JSM Renal Medicine
ISSN : 2573-1637
Launched : 2016
Journal of Muscle Health
ISSN : 2578-3823
Launched : 2016
JSM Genetics and Genomics
ISSN : 2334-1823
Launched : 2013
JSM Anxiety and Depression
ISSN : 2475-9139
Launched : 2016
Clinical Journal of Heart Diseases
ISSN : 2641-7766
Launched : 2016
Annals of Medicinal Chemistry and Research
ISSN : 2378-9336
Launched : 2014
JSM Pain and Management
ISSN : 2578-3378
Launched : 2016
JSM Women's Health
ISSN : 2578-3696
Launched : 2016
Clinical Research in HIV or AIDS
ISSN : 2374-0094
Launched : 2013
Journal of Endocrinology, Diabetes and Obesity
ISSN : 2333-6692
Launched : 2013
Journal of Substance Abuse and Alcoholism
ISSN : 2373-9363
Launched : 2013
JSM Neurosurgery and Spine
ISSN : 2373-9479
Launched : 2013
Journal of Liver and Clinical Research
ISSN : 2379-0830
Launched : 2014
Journal of Drug Design and Research
ISSN : 2379-089X
Launched : 2014
JSM Clinical Oncology and Research
ISSN : 2373-938X
Launched : 2013
JSM Bioinformatics, Genomics and Proteomics
ISSN : 2576-1102
Launched : 2014
JSM Chemistry
ISSN : 2334-1831
Launched : 2013
Journal of Trauma and Care
ISSN : 2573-1246
Launched : 2014
JSM Surgical Oncology and Research
ISSN : 2578-3688
Launched : 2016
Annals of Food Processing and Preservation
ISSN : 2573-1033
Launched : 2016
Journal of Radiology and Radiation Therapy
ISSN : 2333-7095
Launched : 2013
JSM Physical Medicine and Rehabilitation
ISSN : 2578-3572
Launched : 2016
Annals of Clinical Pathology
ISSN : 2373-9282
Launched : 2013
Annals of Cardiovascular Diseases
ISSN : 2641-7731
Launched : 2016
Journal of Behavior
ISSN : 2576-0076
Launched : 2016
Annals of Clinical and Experimental Metabolism
ISSN : 2572-2492
Launched : 2016
JSM Microbiology
ISSN : 2333-6455
Launched : 2013
Journal of Urology and Research
ISSN : 2379-951X
Launched : 2014
Journal of Family Medicine and Community Health
ISSN : 2379-0547
Launched : 2013
Annals of Pregnancy and Care
ISSN : 2578-336X
Launched : 2017
JSM Cell and Developmental Biology
ISSN : 2379-061X
Launched : 2013
Annals of Aquaculture and Research
ISSN : 2379-0881
Launched : 2014
Clinical Research in Pulmonology
ISSN : 2333-6625
Launched : 2013
Journal of Immunology and Clinical Research
ISSN : 2333-6714
Launched : 2013
Annals of Forensic Research and Analysis
ISSN : 2378-9476
Launched : 2014
JSM Biochemistry and Molecular Biology
ISSN : 2333-7109
Launched : 2013
Annals of Breast Cancer Research
ISSN : 2641-7685
Launched : 2016
Annals of Gerontology and Geriatric Research
ISSN : 2378-9409
Launched : 2014
Journal of Sleep Medicine and Disorders
ISSN : 2379-0822
Launched : 2014
JSM Burns and Trauma
ISSN : 2475-9406
Launched : 2016
Chemical Engineering and Process Techniques
ISSN : 2333-6633
Launched : 2013
Annals of Clinical Cytology and Pathology
ISSN : 2475-9430
Launched : 2014
JSM Allergy and Asthma
ISSN : 2573-1254
Launched : 2016
Journal of Neurological Disorders and Stroke
ISSN : 2334-2307
Launched : 2013
Annals of Sports Medicine and Research
ISSN : 2379-0571
Launched : 2014
JSM Sexual Medicine
ISSN : 2578-3718
Launched : 2016
Annals of Vascular Medicine and Research
ISSN : 2378-9344
Launched : 2014
JSM Biotechnology and Biomedical Engineering
ISSN : 2333-7117
Launched : 2013
Journal of Hematology and Transfusion
ISSN : 2333-6684
Launched : 2013
JSM Environmental Science and Ecology
ISSN : 2333-7141
Launched : 2013
Journal of Cardiology and Clinical Research
ISSN : 2333-6676
Launched : 2013
JSM Nanotechnology and Nanomedicine
ISSN : 2334-1815
Launched : 2013
Journal of Ear, Nose and Throat Disorders
ISSN : 2475-9473
Launched : 2016
JSM Ophthalmology
ISSN : 2333-6447
Launched : 2013
Journal of Pharmacology and Clinical Toxicology
ISSN : 2333-7079
Launched : 2013
Annals of Psychiatry and Mental Health
ISSN : 2374-0124
Launched : 2013
Medical Journal of Obstetrics and Gynecology
ISSN : 2333-6439
Launched : 2013
Annals of Pediatrics and Child Health
ISSN : 2373-9312
Launched : 2013
JSM Clinical Pharmaceutics
ISSN : 2379-9498
Launched : 2014
JSM Foot and Ankle
ISSN : 2475-9112
Launched : 2016
JSM Alzheimer's Disease and Related Dementia
ISSN : 2378-9565
Launched : 2014
Journal of Addiction Medicine and Therapy
ISSN : 2333-665X
Launched : 2013
Journal of Veterinary Medicine and Research
ISSN : 2378-931X
Launched : 2013
Annals of Public Health and Research
ISSN : 2378-9328
Launched : 2014
Annals of Orthopedics and Rheumatology
ISSN : 2373-9290
Launched : 2013
Journal of Clinical Nephrology and Research
ISSN : 2379-0652
Launched : 2014
Annals of Community Medicine and Practice
ISSN : 2475-9465
Launched : 2014
Annals of Biometrics and Biostatistics
ISSN : 2374-0116
Launched : 2013
JSM Clinical Case Reports
ISSN : 2373-9819
Launched : 2013
Journal of Cancer Biology and Research
ISSN : 2373-9436
Launched : 2013
Journal of Surgery and Transplantation Science
ISSN : 2379-0911
Launched : 2013
Journal of Dermatology and Clinical Research
ISSN : 2373-9371
Launched : 2013
JSM Gastroenterology and Hepatology
ISSN : 2373-9487
Launched : 2013
Annals of Nursing and Practice
ISSN : 2379-9501
Launched : 2014
JSM Dentistry
ISSN : 2333-7133
Launched : 2013
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