Establishment and evaluation of a multiplex PCR amplification-based sequencing method for respiratory virus type A
Abstract
Aim: This study aimed to establish a simple whole-genome sequencing method for respiratory virus type A (RSVA) used in genomic epidemiological studies. Methods: Multiple primers were designed to amplify overlapping amplicons specific to RSVA. The amplicons were sequenced using Illumina and Nanopore sequencing platforms and the sequencing performances of the two platforms were also compared. Results: The study shows that the developed method could recover almost full genomes of RSVA when Ct values were less than 33 for clinical samples. Conclusion: In this study, a whole-genome sequencing method was developed for RSVA based on multiplex PCR.
Papers of special note have been highlighted as: • of interest
References
- 1. . Molecular epidemiology of respiratory syncytial virus among children and adults in India 2016 to 2018. Virus Genes 57(6), 489–501 (2021).
- 2. Toward unified molecular surveillance of RSV: a proposal for genotype definition. Influenza Other Respir. Viruses. 14(3), 274–285 (2020).
- 3. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in children younger than 5 years in 2019: a systematic analysis. Lancet 399(10340), 2047–2064 (2022). • Describes the prevalence of respiratory syncytial virus.
- 4. . Respiratory syncytial virus infection in elderly and high-risk adults. N. Engl. J. Med. 352(17), 1749–1759 (2005).
- 5. Comparison of infections with respiratory syncytial virus between children and adults: a multicenter surveillance from 2015 to 2019 in Beijing, China. Eur. J. Clin. Microbiol. Infect. Dis. 41(12), 1387–1397 (2022).
- 6. . Systematic review of the respiratory syncytial virus (RSV) prevalence, genotype distribution, and seasonality in children from the Middle East and North Africa (MENA) region. Microorganisms. 8(5), 713 (2020).
- 7. Changes in endemic patterns of respiratory syncytial virus infection in pediatric patients under the pressure of nonpharmaceutical interventions for COVID-19 in Beijing, China. J. Med. Virol. 95(1), e28411 (2023).
- 8. . Transmission of paediatric respiratory syncytial virus and influenza in the wake of the COVID-19 pandemic. Eurosurveillance. 26(29), 2100186 (2021).
- 9. Rapid outbreak sequencing of Ebola virus in Sierra Leone identifies transmission chains linked to sporadic cases. Virus Evol. 2(1), vew016 (2016). • Provide inspiration for multiplex PCR sequencing methods.
- 10. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nat. Protoc. 12(6), 1261–1276 (2017). • Provide inspiration for multiplex PCR sequencing methods.
- 11. Coast-to-coast spread of SARS-CoV-2 during the early epidemic in the United States. Cell 181(5), 990–996.e5 (2020). • Provide inspiration for multiplex PCR sequencing methods.
- 12. . MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 20(4), 1160–1166 (2017).
- 13. . Analysis of RNA sequencing data using CLC Genomics Workbench. Methods Mol. Biol. 2102, 61–113 (2020).
- 14. Systematic benchmarking of nanopore Q20+ kit in SARS-CoV-2 whole genome sequencing. Front. Microbiol. 13, 973367 (2022).
- 15. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 47(W1), W636–W641 (2019).
- 16. . MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol. Biol. Evol. 38(7), 3022–3027 (2021).
- 17. . Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 49(W1), W293–W296 (2021).
- 18. Rapid genomic characterization of SARS-CoV-2 by direct amplicon-based sequencing through comparison of MinION and Illumina iSeq100TM System. Front. Microbiol. 11, 571328 (2020). • Describe the differences between Illumina and Nanopore.
- 19. Assessment of a multiplex PCR and Nanopore-based method for dengue virus sequencing in Indonesia. Virol. J. 17(1), 24 (2020). • Describe the differences between Illumina and Nanopore.
- 20. . To interpret the SARS-CoV-2 test, consider the cycle threshold value. Clin. Infect. Dis. 71(16), 2252–2254 (2020).
- 21. Assessment of two-pool multiplex long-amplicon nanopore sequencing of SARS-CoV-2. J. Med. Virol. 94(1), 327–334 (2022).
- 22. Detailed evolutionary analyses of the F gene in the respiratory syncytial virus subgroup A. Viruses. 13(12), 2525 (2021).
- 23. . Functional features of the respiratory syncytial virus G protein. Viruses. 13(7), 1214 (2021).
- 24. Genetic diversity and its impact on disease severity in respiratory syncytial virus subtype-A and -B bronchiolitis before and after pandemic restrictions in Rome. J. Infect. S0163-4453(23)00381-X (2023).