Research Article

A Low-cost High-throughput Targeted Sequencing for the Accurate Detection of Respiratory Tract Pathogen

Changyan Ju, Chengbosen Zhou, Zhezhi Deng, Jingwei Gao, Weizhao Jiang, Hanbing Zeng, Haiwei Huang, Yongxiang Duan and David X Deng*

Published: 12 March, 2024 | Volume 8 - Issue 1 | Pages: 001-007

Introduction: The current gold standard for SARS-CoV-2 diagnosis by real-time RT-PCR has limitations of gene numbers that can be detected. In this study, we developed a low-cost and high-throughput next-generation sequencing technology that can overcome the limitations of RT-PCR. 
Methodology: A targeted sequencing panel (TSP) consisting of approximately 500 amplicons was designed that can simultaneously detect a broad range of gene loci of SARS-CoV-2 and genes for the most common viruses of respiratory infectious viruses in a single run of up to 96 samples. 448 samples and 31 control samples were examined independently with both TSP and RT-PCR, results were compared for accuracy and other indicators. 
Results:  TSP identified 50 SARS-CoV-2 positive samples with a 99.33% match to RT-PCR results. It is not surprising that TSP also identified multiple viral infections from 96 samples, whereas RT-PCR could not. TSP demonstrated its ability to conclude diagnosis for those undecided from RT-PCR tests. 
Conclusion: Our data demonstrated that TSP is a fast and accurate test for detecting multiple pathogen infections of the respiratory tract.

Read Full Article HTML DOI: 10.29328/journal.ijcv.1001056 Cite this Article Read Full Article PDF


RT-PCR; Targeted sequencing; SARS-CoV-2; Respiratory tract pathogen


  1. Malik YA. Properties of Coronavirus and SARS-CoV-2. Malays J Pathol. 2020 Apr;42(1):3-11. PMID: 32342926.
  2. Machkovech HM, Hahn AM, Garonzik Wang J, Grubaugh ND, Halfmann PJ, Johnson MC, Lemieux JE, O'Connor DH, Piantadosi A, Wei W, Friedrich TC. Persistent SARS-CoV-2 infection: significance and implications. Lancet Infect Dis. 2024 Feb 7:S1473-3099(23)00815-0. doi: 10.1016/S1473-3099(23)00815-0. Epub ahead of print. PMID: 38340735.
  3. Tam D, Lorenzo-Leal AC, Hernández LR, Bach H. Targeting SARS-CoV-2 Non-Structural Proteins. Int J Mol Sci. 2023 Aug 20;24(16):13002. doi: 10.3390/ijms241613002. PMID: 37629182; PMCID: PMC10455537.
  4. Hassanin AA, Haidar Abbas Raza S, Ahmed Ujjan J, Aysh ALrashidi A, Sitohy BM, Al-Surhanee AA, Saad AM, Mohamed Al-Hazani T, Osman Atallah O, Al Syaad KM, Ezzat Ahmed A, Swelum AA, El-Saadony MT, Sitohy MZ. Emergence, evolution, and vaccine production approaches of SARS-CoV-2 virus: Benefits of getting vaccinated and common questions. Saudi J Biol Sci. 2022 Apr;29(4):1981-1997. doi: 10.1016/j.sjbs.2021.12.020. Epub 2021 Dec 13. PMID: 34924802; PMCID: PMC8667566.
  5. Noori R, Sardar M. An outlook on potential protein targets of COVID-19 as a druggable site. Mol Biol Rep. 2022 Nov;49(11):10729-10748. doi: 10.1007/s11033-022-07724-3. Epub 2022 Jul 6. PMID: 35790657; PMCID: PMC9256362.
  6. Young M, Crook H, Scott J, Edison P. Covid-19: virology, variants, and vaccines. BMJ Med. 2022 Apr 1;1(1):e000040. doi: 10.1136/bmjmed-2021-000040. PMID: 36936563; PMCID: PMC9951271.
  7. Xu J, Chen J, Wen F, Liu K, Chen Y. Detection methods and dynamic characteristics of specific antibodies in patients with COVID-19: A review of the early literature. Heliyon. 2024 Jan 24;10(3):e24580. doi: 10.1016/j.heliyon.2024.e24580. PMID: 38317938; PMCID: PMC10839880.
  8. Caixeta DC, Paranhos LR, Blumenberg C, Garcia-Júnior MA, Guevara-Vega M, Taveira EB, Nunes MAC, Cunha TM, Jardim ACG, Flores-Mir C, Sabino-Silva R. Salivary SARS-CoV-2 RNA for diagnosis of COVID-19 patients: a systematic revisew and meta-analysis of diagnostic accuracy. Jpn Dent Sci Rev. 2023 Jun 21;59:219–38. doi: 10.1016/j.jdsr.2023.06.004. Epub ahead of print. PMID: 37360001; PMCID: PMC10284464.
  9. Kanji JN, Zelyas N, MacDonald C, Pabbaraju K, Khan MN, Prasad A, Hu J, Diggle M, Berenger BM, Tipples G. False negative rate of COVID-19 PCR testing: a discordant testing analysis. Virol J. 2021 Jan 9;18(1):13. doi: 10.1186/s12985-021-01489-0. PMID: 33422083; PMCID: PMC7794619.
  10. Su YCF, Anderson DE, Young BE, Linster M, Zhu F, Jayakumar J, Zhuang Y, Kalimuddin S, Low JGH, Tan CW, Chia WN, Mak TM, Octavia S, Chavatte JM, Lee RTC, Pada S, Tan SY, Sun L, Yan GZ, Maurer-Stroh S, Mendenhall IH, Leo YS, Lye DC, Wang LF, Smith GJD. Discovery and Genomic Characterization of a 382-Nucleotide Deletion in ORF7b and ORF8 during the Early Evolution of SARS-CoV-2. mBio. 2020 Jul 21;11(4):e01610-20. doi: 10.1128/mBio.01610-20. PMID: 32694143; PMCID: PMC7374062.
  11. Phan T. Genetic diversity and evolution of SARS-CoV-2. Infect Genet Evol. 2020 Jul;81:104260. doi: 10.1016/j.meegid.2020.104260. Epub 2020 Feb 21. PMID: 32092483; PMCID: PMC7106203.
  12. Khailany RA, Safdar M, Ozaslan M. Genomic characterization of a novel SARS-CoV-2. Gene Rep. 2020 Jun;19:100682. doi: 10.1016/j.genrep.2020.100682. Epub 2020 Apr 16. PMID: 32300673; PMCID: PMC7161481.
  13. Pachetti M, Marini B, Benedetti F, Giudici F, Mauro E, Storici P, Masciovecchio C, Angeletti S, Ciccozzi M, Gallo RC, Zella D, Ippodrino R. Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant. J Transl Med. 2020 Apr 22;18(1):179. doi: 10.1186/s12967-020-02344-6. PMID: 32321524; PMCID: PMC7174922.
  14. Stowe J, Tessier E, Zhao H, Guy R, Muller-Pebody B, Zambon M, Andrews N, Ramsay M, Lopez Bernal J. Interactions between SARS-CoV-2 and influenza, and the impact of coinfection on disease severity: a test-negative design. Int J Epidemiol. 2021 Aug 30;50(4):1124-1133. doi: 10.1093/ije/dyab081. PMID: 33942104; PMCID: PMC8135706.
  15. Garcia-Vidal C, Sanjuan G, Moreno-García E, Puerta-Alcalde P, Garcia-Pouton N, Chumbita M, Fernandez-Pittol M, Pitart C, Inciarte A, Bodro M, Morata L, Ambrosioni J, Grafia I, Meira F, Macaya I, Cardozo C, Casals C, Tellez A, Castro P, Marco F, García F, Mensa J, Martínez JA, Soriano A; COVID-19 Researchers Group. Incidence of co-infections and superinfections in hospitalized patients with COVID-19: a retrospective cohort study. Clin Microbiol Infect. 2021 Jan;27(1):83-88. doi: 10.1016/j.cmi.2020.07.041. Epub 2020 Jul 31. PMID: 32745596; PMCID: PMC7836762.
  16. Singhal T. A Review of Coronavirus Disease-2019 (COVID-19). Indian J Pediatr. 2020 Apr;87(4):281-286. doi: 10.1007/s12098-020-03263-6. Epub 2020 Mar 13. PMID: 32166607; PMCID: PMC7090728.
  17. Herrera-Plasencia PM, Enoki-Miñano E, Ruiz-Barrueto MYA. Riesgos, contaminación y prevención frente al COVID-19 en el quehacer odontológico: una revisión [Risks, contamination and prevention against COVID-19 in dental work: a review]. Rev Salud Publica (Bogota). 2020 Sep 1;22(5):560-565. Spanish. doi: 10.15446/rsap.V22n5.86065. PMID: 36753227.
  18. Kaye AD, Okeagu CN, Pham AD, Silva RA, Hurley JJ, Arron BL, Sarfraz N, Lee HN, Ghali GE, Gamble JW, Liu H, Urman RD, Cornett EM. Economic impact of COVID-19 pandemic on healthcare facilities and systems: International perspectives. Best Pract Res Clin Anaesthesiol. 2021 Oct;35(3):293-306. doi: 10.1016/j.bpa.2020.11.009. Epub 2020 Nov 17. PMID: 34511220; PMCID: PMC7670225.
  19. Ma L, Zhu H, Jiang Y, Kong X, Gao P, Liu Y, Zhao M, Deng G, Cao Y. Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B Virus. Int J Anal Chem. 2024 Feb 29;2024:4950391. doi: 10.1155/2024/4950391. PMID: 38456096; PMCID: PMC10919977.
  20. Samsunder N, Devnarain N, Sivro A, Kharsany ABM. The Performance of Diagnostic Tests for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in the South African Population: A Scoping Review. Trop Med Infect Dis. 2023 Dec 1;8(12):514. doi: 10.3390/tropicalmed8120514. PMID: 38133446; PMCID: PMC10748306.
  21. Vasei M, Jafari E, Falah Azad V, Safavi M, Sotoudeh M. Molecular Diagnosis of COVID-19; Biosafety and Pre-analytical Recommendations. Iran J Pathol. 2023 Summer;18(3):244-256. doi: 10.30699/IJP.2023.1988405.3061. Epub 2023 Jul 16. PMID: 37942195; PMCID: PMC10628373.
  22. Carpenter RE, Tamrakar VK, Almas S, Sharma A, Sharma R. SARS-CoV-2 Next Generation Sequencing (NGS) data from clinical isolates from the East Texas Region of the United States. Data Brief. 2023 Aug;49:109312. doi: 10.1016/j.dib.2023.109312. Epub 2023 Jun 14. PMID: 37346925; PMCID: PMC10264325.
  23. Chan AP, Siddique A, Desplat Y, Choi Y, Ranganathan S, Choudhary KS, Khalid MF, Diaz J, Bezney J, DeAscanis D, George Z, Wong S, Selleck W, Bowers J, Zismann V, Reining L, Highlander S, Brown K, Armstrong JR, Hakak Y, Schork NJ. A CRISPR-enhanced metagenomic NGS test to improve pandemic preparedness. Cell Rep Methods. 2023 Apr 18;3(5):100463. doi: 10.1016/j.crmeth.2023.100463. PMID: 37323571; PMCID: PMC10110940.
  24. Tartanian AC, Mulroney N, Poselenzny K, Akroush M, Unger T, Helseth DL, Jr., Sabatini LM, Bouma M, Larkin PMK. NGS implementation for monitoring SARS-CoV-2 variants in Chicagoland: An institutional perspective, successes and challenges. Front Public Health. 2023;11:1177695. doi:10.3389/fpubh.2023.1177695 [Pubmed: 37151582]
  25. Wang X, Stelzer-Braid S, Scotch M, Rawlinson WD. Detection of respiratory viruses directly from clinical samples using next-generation sequencing: A literature review of recent advances and potential for routine clinical use. Rev Med Virol. 2022 Sep;32(5):e2375. doi: 10.1002/rmv.2375. Epub 2022 Jul 1. PMID: 35775736; PMCID: PMC9539958.
  26. Nafea AM, Wang Y, Wang D, Salama AM, Aziz MA, Xu S, Tong Y. Application of next-generation sequencing to identify different pathogens. Front Microbiol. 2023;14:1329330.doi:10.3389/fmicb.2023.1329330 [Pubmed: 38348304]
  27. Li F, Wang Y, Zhang Y, Shi P, Cao L, Su L, Zhu Q, Wang L, Lu R, Tan W, Shen J. Etiology of Severe Pneumonia in Children in Alveolar Lavage Fluid Using a High-Throughput Gene Targeted Amplicon Sequencing Assay. Front Pediatr. 2021; 9:659164.doi:10.3389/fped.2021.659164 [Pubmed: 34249808]
  28. Gaston DC, Miller HB, Fissel JA, Jacobs E, Gough E, Wu J, Klein EY, Carroll KC, Simner PJ. Evaluation of Metagenomic and Targeted Next-Generation Sequencing Workflows for Detection of Respiratory Pathogens from Bronchoalveolar Lavage Fluid Specimens. J Clin Microbiol. 2022 Jul 20;60(7):e0052622. doi: 10.1128/jcm.00526-22. Epub 2022 Jun 13. PMID: 35695488; PMCID: PMC9297812.
  29. Chiara M, D'Erchia AM, Gissi C, Manzari C, Parisi A, Resta N, Zambelli F, Picardi E, Pavesi G, Horner DS, Pesole G. Next generation sequencing of SARS-CoV-2 genomes: challenges, applications and opportunities. Brief Bioinform. 2021 Mar 22;22(2):616-630. doi: 10.1093/bib/bbaa297. PMID: 33279989; PMCID: PMC7799330.
  30. Thorburn F, Bennett S, Modha S, Murdoch D, Gunson R, Murcia PR. The use of next generation sequencing in the diagnosis and typing of respiratory infections. J Clin Virol. 2015 Aug;69:96-100. doi: 10.1016/j.jcv.2015.06.082. Epub 2015 Jun 18. Erratum in: J Clin Virol. 2015 Sep;70:128. PMID: 26209388; PMCID: PMC4533236.


Similar Articles

Recently Viewed

Read More

Most Viewed

Read More

Help ?