Metagenomic Next-Generation Sequencing for Viral Pathogen Detection: A Comprehensive Review
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Abstract
Viruses, the most abundant biological entities on Earth, have remained largely uncharted, despite their significant ecological impact. Next-generation sequencing (NGS) technologies have catalyzed a revolution in viral discovery by enabling rapid retrieval of vast RNA and DNA sequences from diverse sources. Metagenomic next-generation sequencing (mNGS) surpasses traditional methods by facilitating the detection of both known and novel viruses without the need for pre-existing knowledge of genomes. It encompasses various paradigms, including shotgun sequencing and targeted amplification, which allow for comprehensive viral detection and classification. The evolution of bioinformatics tools has bolstered mNGS applications, aiding in clinical diagnostics, environmental monitoring, and epidemiological studies. mNGS has proven particularly valuable in identifying pathogens linked to complex syndromes and emerging threats, such as SARS-CoV-2. By revealing viral genomics across clinical samples and environmental matrices, mNGS enhances our understanding of viral evolution and diversity, critical to public health surveillance and biosecurity. The advent of advanced sequencing techniques further improves detection accuracy and coverage, fostering the development of bioinformatic pipelines that refine analytical processes. Metagenomic technologies promise robust applications in clinical diagnostics and environmental virology, enabling proactive responses to emerging viral threats while addressing public health challenges through comprehensive and efficient pathogen monitoring and detection.
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References
I. Koonin EV, Starokadomskyy P. Are viruses alive? The replicator paradigm sheds decisive light on an old but misguided question. Stud Hist Philos Biol Biomed Sci. 2016;59:125-134. doi:10.1016/j.shpsc.2016.02.016
II. Bassi C, Guerriero P, Pierantoni M, Callegari E, Sabbioni S. Novel Virus Identification through Metagenomics: A Systematic Review. Life (Basel). 2022;12(12):2048. doi:10.3390/life12122048
III. Conradie T, Caparros-Martin JA, Egan S, et al. Exploring the Complexity of the Human Respiratory Virome through an In Silico Analysis of Shotgun Metagenomic Data Retrieved from Public Repositories. Viruses. 2024;16(6):953. doi:10.3390/v16060953
IV. Tremblay J, Schreiber L, Greer CW. High-resolution shotgun metagenomics: the more data, the better?. Brief Bioinform. 2022;23(6):bbac443. doi:10.1093/bib/bbac443
V. Koonin EV, Kuhn JH, Dolja VV, Krupovic M. Megataxonomy and global ecology of the virosphere. ISME J. 2024;18(1):wrad042. doi:10.1093/ismejo/wrad042
VI. Angel NZ, Sullivan MJ, Alsheikh-Hussain A, et al. Metagenomics: a new frontier for routine pathology testing of gastrointestinal pathogens. Gut Pathog. 2025;17(1):4. Published 2025 Jan 18. doi:10.1186/s13099-024-00673-1
VII. Reghu R, Karunakaran RL, Balaji R, Babu A, Arumugaperumal A. Unlocking Nature’s Code: The Power of Pan-Genomics in Biological Entities. MEDIN [Internet]. 2024 Dec. 24 [cited 2026 Jan. 1];. Available from: https://ojs.bonviewpress.com/index.php/MEDIN/article/view/3423
VIII. Ibañez-Lligoña M, Colomer-Castell S, González-Sánchez A, et al. Bioinformatic Tools for NGS-Based Metagenomics to Improve the Clinical Diagnosis of Emerging, Re-Emerging and New Viruses. Viruses. 2023;15(2):587. doi:10.3390/v15020587
IX. Russell T, Formiconi E, Casey M, McElroy M, Mallon PWG, Gautier VW. Viral Metagenomic Next-Generation Sequencing for One Health Discovery and Surveillance of (Re)Emerging Viruses: A Deep Review. Int J Mol Sci. 2025;26(19):9831. doi:10.3390/ijms26199831
X. Shi T, Chen H, Liu Y, Wu Y, Lin F. Clinical applications of metagenomic next-generation sequencing in the identification of pathogens in periprosthetic joint infections: a retrospective study. J Orthop Surg Res. 2024;19(1):301. doi:10.1186/s13018-024-04745-5
XI. Kim C, Pongpanich M, Porntaveetus T. Unraveling metagenomics through long-read sequencing: a comprehensive review. J Transl Med. 2024;22(1):111. doi:10.1186/s12967-024-04917-1
XII. Kumar KR, Cowley MJ, Davis RL. Next-Generation Sequencing and Emerging Technologies. Semin Thromb Hemost. 2024;50(7):1026-1038. doi:10.1055/s-0044-1786397
XIII. Khan AS, Mallet L, Blümel J, et al. Report of the third conference on next-generation sequencing for adventitious virus detection in biologics for humans and animals. Biologicals. 2023;83:101696. doi:10.1016/j.biologicals.2023.101696
XIV. Nafea AM, Wang Y, Wang D, et al. Application of next-generation sequencing to identify different pathogens. Front Microbiol. 2024;14:1329330doi:10.3389/fmicb.2023.1329330
XV. Jiang W, Ji W, Zhang Y, et al. An Update on Detection Technologies for SARS-CoV-2 Variants of Concern. Viruses. 2022;14(11):2324. doi:10.3390/v14112324
XVI. Kufner V, Plate A, Schmutz S, et al. Two Years of Viral Metagenomics in a Tertiary Diagnostics Unit: Evaluation of the First 105 Cases. Genes (Basel). 2019;10(9):661. doi:10.3390/genes10090661
XVII. Miller S, Chiu C. The Role of Metagenomics and Next-Generation Sequencing in Infectious Disease Diagnosis. Clin Chem. 2021;68(1):115-124. doi:10.1093/clinchem/hvab173
XVIII. Lindner BG, Gerhardt K, Feistel DJ, Rodriguez-R LM, Hatt JK, Konstantinidis KT. A user's guide to the bioinformatic analysis of shotgun metagenomic sequence data for bacterial pathogen detection. Int J Food Microbiol. 2024;410:110488. doi:10.1016/j.ijfoodmicro.2023.110488
XIX. Guajardo-Leiva, S.; Chnaiderman, J.; Gaggero, A.; Díez, B. Metagenomic Insights into the Sewage RNA Virosphere of a Large City. Viruses 2020, 12, 1050. https://doi.org/10.3390/v12091050
XX. Downie DL, Rao P, David-Ferdon C, et al. Literature Review of Pathogen Agnostic Molecular Testing of Clinical Specimens From Difficult-to-Diagnose Patients: Implications for Public Health. Health Secur. 2024;22(2):93-107. doi:10.1089/hs.2023.0100
XXI. Liu H, Zhang Y, Yang J, Liu Y, Chen J. Application of mNGS in the Etiological Analysis of Lower Respiratory Tract Infections and the Prediction of Drug Resistance. Microbiol Spectr. 2022 Feb 23;10(1):e0250221. doi: 10.1128/spectrum.02502-21.
XXII. Torres Montaguth OE, Buddle S, Morfopoulou S, Breuer J. Clinical metagenomics for diagnosis and surveillance of viral pathogens. Nat Rev Microbiol. 2026 Jan;24(1):61-75. doi: 10.1038/s41579-025-01223-5.
XXIII. Wieczynski DJ, Yoshimura KM, Denison ER, Geisen S, DeBruyn JM, Shaw AJ, Weston DJ, Pelletier DA, Wilhelm SW, Gibert JP. Viral infections likely mediate microbial controls on ecosystem responses to global warming. FEMS Microbiol Ecol. 2023 Feb 28;99(3):fiad016. doi: 10.1093/femsec/fiad016. PMID: 36828391.
XXIV. Gao Y, Lu Y, Dungait JAJ, Liu J, Lin S, Jia J, Yu G. The "Regulator" Function of Viruses on Ecosystem Carbon Cycling in the Anthropocene. Front Public Health. 10.3389/fpubh.2022.858615.
XXV. Hill V, Githinji G, Vogels CBF, Bento AI, Chaguza C, Carrington CVF, Grubaugh ND. Toward a global virus genomic surveillance network. Cell Host Microbe. 2023 Jun 14;31(6):861-873. doi: 10.1016/j.chom.2023.03.003. Epub 2023 Mar 6.
XXVI. Almansour AM, Alhadlaq MA, Alzahrani KO, Mukhtar LE, Alharbi AL, Alajel SM. The silent threat: Antimicrobial-resistant pathogens in food-producing animals and their impact on public health. Microorganisms. 2023 Aug 22;11(9):2127. mdpi.com
XXVII. Parker K, Wood H, Russell JA, Yarmosh D, Shteyman A, Bagnoli J, Knight B, Aspinwall JR, Jacobs J, Werking K, Winegar R. Development and Optimization of an Unbiased, Metagenomics-Based Pathogen Detection Workflow for Infectious Disease and Biosurveillance Applications. Trop Med Infect Dis. 2023 Feb 15;8(2):121. doi: 10.3390/tropicalmed8020121.
XXVIII. Lim YW, Haynes M, Furlan M, Robertson CE, Harris JK, Rohwer F. Purifying the impure: sequencing metagenomes and metatranscriptomes from complex animal-associated samples. J Vis Exp. 2014 Dec 22;(94):52117. doi: 10.3791/52117.
XXIX. Williams ML, Olomukoro AA, Emmons RV, Godage NH, Gionfriddo E. Matrix effects demystified: Strategies for resolving challenges in analytical separations of complex samples. J Sep Sci. 2023 Dec;46(23):e2300571. doi: 10.1002/jssc.202300571.
XXX. Cabanes-Creus M, Liao SHY, Gale Navarro R, Knight M, Nazareth D, Lau NS, Ly M, Zhu E, Roca-Pinilla R, Bugallo Delgado R, Vicente AF, Baltazar G, Westhaus A, Merjane J, Crawford M, McCaughan GW, Unzu C, González-Aseguinolaza G, Alexander IE, Pulitano C, Lisowski L. Harnessing whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation. Nat Commun. 2024 Mar 14;15(1):1876. doi: 10.1038/s41467-024-46194-y.
XXXI. Regnault B, Bigot T, Ma L, Pérot P, Temmam S, Eloit M. Deep Impact of Random Amplification and Library Construction Methods on Viral Metagenomics Results. Viruses. 2021 Feb 7;13(2):253. doi: 10.3390/v13020253.
XXXII. Zhang D, Lou X, Yan H, Pan J, Mao H, Tang H, Shu Y, Zhao Y, Liu L, Li J, Chen J, Zhang Y, Ma X. Metagenomic analysis of viral nucleic acid extraction methods in respiratory clinical samples. BMC Genomics. 2018 Oct 25;19(1):773. doi: 10.1186/s12864-018-5152-5.
XXXIII. Chen G, Qiu Y, Zhuang Q, Wang S, Wang T, Chen J, Wang K. Next-generation sequencing library preparation method for identification of RNA viruses on the Ion Torrent Sequencing Platform. Virus Genes. 2018 Aug;54(4):536-542. doi: 10.1007/s11262-018-1568-x.
XXXIV. Jia X, Hu L, Wu M, Ling Y, Wang W, Lu H, Yuan Z, Yi Z, Zhang X. A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification. Sci Rep. 2021 Feb 23;11(1):4405. doi: 10.1038/s41598-021-83812-x.
XXXV. Liu Y, Ma Y. Clinical applications of metagenomics next-generation sequencing in infectious diseases. J Zhejiang Univ Sci B. 2024 May 17;25(6):471-484. doi: 10.1631/jzus.B2300029.
XXXVI. Xiao Y, Zhang L, Yang B, Li M, Ren L, Wang J. Application of next generation sequencing technology on contamination monitoring in microbiology laboratory. Biosaf Health. 2019 Jun;1(1):25-31. doi: 10.1016/j.bsheal.2019.02.003.
XXXVII. Ribarska T, Bjørnstad PM, Sundaram AYM, Gilfillan GD. Optimization of enzymatic fragmentation is crucial to maximize genome coverage: a comparison of library preparation methods for Illumina sequencing. BMC Genomics. 2022 Feb 1;23(1):92. doi: 10.1186/s12864-022-08316-y.
XXXVIII. Szabó G, Eloe-Fadrosh EA, Pett-Ridge J, Woyke T. A genomic view of Earth's Nat Rev Genet. 2026 Jan;27(1):13-31. doi: 10.1038/s41576-025-00888-1.
XXXIX. Alrasheed, Rasheed Saud, Mohammed Mahrous Al-Mahrous, Raed Mohammed Alshahrani and Nadiah Rabee Alsaadi. “Safety and Decontamination Procedures for Infectious Sample Handling.” Journal Of Healthcare Sciences. 2024; 4(12): 1010-1015.
XL. Afonso CL, Afonso AM. Next-Generation Sequencing for the Detection of Microbial Agents in Avian Clinical Samples. Vet Sci. 2023 Dec 4;10(12):690. doi: 10.3390/vetsci10120690.
XLI. Lambert C, Braxton C, Charlebois RL, Deyati A, Duncan P, La Neve F, Malicki HD, Ribrioux S, Rozelle DK, Michaels B, Sun W, Yang Z, Khan AS. Considerations for Optimization of High-Throughput Sequencing Bioinformatics Pipelines for Virus Detection. Viruses. 2018 Sep 27;10(10):528. doi: 10.3390/v10100528.
XLII. Tatineni S, Hein GL. Plant Viruses of Agricultural Importance: Current and Future Perspectives of Virus Disease Management Strategies. Phytopathology. 2023 Feb;113(2):117-141. doi: 10.1094/PHYTO-05-22-0167-RVW.
XLIII. Sewe SO, Silva G, Sicat P, Seal SE, Visendi P. Trimming and Validation of Illumina Short Reads Using Trimmomatic, Trinity Assembly, and Assessment of RNA-Seq Data. Methods Mol Biol. 2022;2443:211-232. doi: 10.1007/978-1-0716-2067-0_11.