Emerging role of metagenomic next-generation sequencing in infectious disease diagnostics: Clinical integration and future directions

Abstract

Infectious disease diagnostics has been transformed by metagenomic next-generation sequencing (mNGS), an unbiased approach that detects bacteria, viruses, fungi, and parasites in a single assay. By sequencing all nucleic acids in a sample, mNGS overcomes the narrow detection scope and slow turnaround of conventional tests, substantially improving pathogen detection. In conditions such as meningitis/encephalitis, sepsis, and pneumonia, mNGS frequently identifies etiologies missed by routine diagnostic tests, thereby facilitating earlier pathogen-directed therapy and, in selected settings, improving clinical management and outcomes. This approach is particularly valuable for immunocompromised, pediatric, and intensive care unit (ICU) patients with atypical infections. Currently, clinical mNGS workflows primarily rely on short-read sequencing platforms (e.g., Illumina), whereas long-read platforms (e.g., Nanopore, PacBio) offer advantages for rapid or high-resolution applications. Optimized bioinformatics and stringent quality control are essential for reliable results. Beyond clinical diagnostics, mNGS provides valuable genetic data on antimicrobial resistance (AMR) and pathogen phylogeny, supporting public health and outbreak surveillance (e.g., wastewater monitoring and variant tracking). Current challenges include distinguishing colonization from infection, interpreting sequencing data quantitatively, and reducing cost and turnaround time. Looking ahead, emerging strategies such as targeted panels, rapid automated workflows, and host‑response integration are expected to further shorten time‑to‑result and improve diagnostic specificity. Parallel progress in ethical and regulatory frameworks remains essential to ensure responsible implementation. To support clinical adoption, a standardized framework for clinical interpretation of mNGS results, together with associated training, has been developed and implemented. Overall, mNGS is likely to become an increasingly important component of infectious disease diagnostics, with ongoing innovations expected to broaden its clinical and epidemiological impact.

Keywords

bioinformatics pipelines / infectious disease diagnostics / metagenomic next-generation sequencing (mNGS) / public health surveillance / standardization of mNGS results

References

[1] Benoit P, Brazer N, de Lorenzi-Tognon M, Kelly E, Servellita V, Oseguera M, et al. Seven-year performance of a clinical metagenomic next-generation sequencing test for diagnosis of central nervous system infections. Nat Med. 2024; 30: 3522–3533.

[2] Wilson MR, Sample HA, Zorn KC, Arevalo S, Yu G, Neuhaus J. et al. Clinical metagenomic sequencing for diagnosis of meningitis and encephalitis. N Engl J Med. 2019; 380: 2327–2340.

[3] Rodino KG, Simner PJ. Status check: next-generation sequencing for infectious-disease diagnostics. J Clin Investig. 2024; 134:e178003.

[4] GBD 2019 Antimicrobial Resistance Collaborators. Global mortality associated with 33 bacterial pathogens in 2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet. 2022; 400: 2221–2248.

[5] Chiu CY, Miller SA. Clinical metagenomics. Nat Rev Genet. 2019; 20: 341–355.

[6] Ng DL, Granados AC, Santos YA, Servellita V, Goldgof GM, Meydan C, et al. A diagnostic host response biosignature for COVID-19 from RNA profiling of nasal swabs and blood. Sci Adv. 2021; 7:eabe5984.

[7] Wilson MR, Naccache SN, Samayoa E, Biagtan M, Bashir H, Yu G, et al. Actionable diagnosis of neuroleptospirosis by next-generation sequencing. N Engl J Med. 2014; 370: 2408–2417.

[8] Tang H, Chen Y, Tang X, Wei M, Hu J, Zhang X, et al. Yield of clinical metagenomics: insights from real-world practice for tissue infections. Ebiomedicine. 2025; 111:105536.

[9] Zinter MS, Dvorak CC, Mayday MY, Iwanaga K, Ly NP, Mcgarry ME, et al. Pulmonary metagenomic sequencing suggests missed infections in immunocompromised children. Clin Infect Dis. 2019; 68: 1847–1855.

[10] Lee HJ, Georgiadou A, Walther M, Nwakanma D, Stewart LB, Levin M, et al. Integrated pathogen load and dual transcriptome analysis of systemic host-pathogen interactions in severe malaria. Sci Transl Med. 2018; 10:eaar3619.

[11] Blauwkamp TA, Thair S, Rosen MJ, Blair L, Lindner MS, Vilfan ID, et al. Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease. Nat Microbiol. 2019; 4: 663–674.

[12] Lai LM, Chen Q, Liu Y, Zhao R, Cao ML, Yuan L. The value of metagenomic next-generation sequencing in the diagnosis of fever of unknown origin. Sci Rep. 2025; 15: 1963.

[13] Liu J, Zhang Q, Dong YQ, Yin J, Qiu YQ. Diagnostic accuracy of metagenomic next-generation sequencing in diagnosing infectious diseases: a meta-analysis. Sci Rep. 2022; 12:21032.

[14] Hao J, Li W, Wang Y, Zhao J, Chen Y. Clinical utility of metagenomic next-generation sequencing in pathogen detection for lower respiratory tract infections and impact on clinical outcomes in southernmost China. Front Cell Infect Microbiol. 2023; 13:1271952.

[15] Kodama F, Yamaguchi H, Park E, Tatemoto K, Sashika M, Nakao R, et al. A novel nairovirus associated with acute febrile illness in Hokkaido, Japan. Nat Commun. 2021; 12: 5539.

[16] Zhang XA, Li H, Jiang FC, Zhu F, Zhang YF, Chen JJ, et al. A zoonotic henipavirus in febrile patients in China. N Engl J Med. 2022; 387: 470–472.

[17] Liang X, Wang Q, Liu J, Ma J, Zhang Y, Wang M, et al. Coinfection of SARS-CoV-2 and influenza a (H3N2) detected in bronchoalveolar lavage fluid of a patient with long COVID using metagenomic next-generation sequencing: a case report. Front Cell Infect Microbiol. 2023; 13:1224794.

[18] Xie Y, Dai B, Zhou X, Liu H, Wu W, Yu F, et al. Diagnostic value of metagenomic next-generation sequencing for multi-pathogenic pneumonia in HIV-infected patients. Infect Drug Resist. 2023; 16: 607–618.

[19] Wu X, Sun T, He H, Xing L, Cheng Z, Geng S, et al. Effect of metagenomic next-generation sequencing on clinical outcomes of patients with severe community-acquired pneumonia in the ICU. Chest. 2025; 167: 362–373.

[20] Nafea AM, Wang Y, Wang D, Salama AM, Aziz MA, Xu S, et al. Application of next-generation sequencing to identify different pathogens. Front Microbiol. 2023; 14:1329330.

[21] Govender KN, Street TL, Sanderson ND, Eyre DW. Metagenomic sequencing as a pathogen-agnostic clinical diagnostic tool for infectious diseases: a systematic review and meta-analysis of diagnostic test accuracy studies. J Clin Microbiol. 2021; 59:e291620.

[22] Kan CM, Tsang HF, Pei XM, Ng S.S.M., Yim A.K.Y., Yu A.C.S., et al. Enhancing clinical utility: utilization of international standards and guidelines for metagenomic sequencing in infectious disease diagnosis. Int J Mol Sci. 2024; 25: 3333.

[23] Jiang Z, Gai W, Zhang X, Zheng Y, Jin X, Han Z, et al. Clinical performance of metagenomic next-generation sequencing for diagnosis of pulmonary Aspergillus infection and colonization. Front Cell Infect Microbiol. 2024; 14:1345706.

[24] Hodcroft EB, Zuber M, Nadeau S, Vaughan TG, Crawford K.H.D., Althaus CL, et al. Spread of a SARS-CoV-2 variant through Europe in the summer of 2020. Nature. 2021; 595: 707–712.

[25] Viana R, Moyo S, Amoako DG, Tegally H, Scheepers C, Althaus CL, et al. Rapid epidemic expansion of the SARS-CoV-2 omicron variant in southern Africa. Nature. 2022; 603: 679–686.

[26] Tan JK, Servellita V, Stryke D, Kelly E, Streithorst J, Sumimoto N, et al. Laboratory validation of a clinical metagenomic next-generation sequencing assay for respiratory virus detection and discovery. Nat Commun. 2024; 15: 9016.

[27] Yi X, Lu H, Liu X, He J, Li B, Wang Z, et al. Unravelling the enigma of the human microbiome: evolution and selection of sequencing technologies. Microb Biotechnol. 2024; 17:e14364.

[28] Peng S, Liang R, Mao S, Tang W, Tang M, Ai Y, et al. 558. Cost-effectiveness of introducing metagenomic next-generation sequencing (mNGS) for the diagnosis of suspected respiratory infections patients in China. Open Forum Infect Dis. 2023; 10: d500–d627.

[29] Sung YH, Ju YK, Lee HJ, Park SM, Suh JW, Kim JY, et al. Clinical performance of real-time nanopore metagenomic sequencing for rapid identification of bacterial pathogens in cerebrospinal fluid: a pilot study. Sci Rep. 2025; 15: 3493.

[30] Han D, Yu F, Zhang D, Hu J, Zhang X, Xiang D, et al. Molecular rapid diagnostic testing for bloodstream infections: nanopore targeted sequencing with pathogen-specific primers. J Infect. 2024; 88:106166.

[31] Zhu X, Yan S, Yuan F, Wan S. The applications of nanopore sequencing technology in pathogenic microorganism detection. Can J Infect Dis Med Microbiol. 2020; 2020:6675206.

[32] Wang Y, Zhao Y, Bollas A, Wang Y, Au KF. Nanopore sequencing technology, bioinformatics and applications. Nat Biotechnol. 2021; 39: 1348–1365.

[33] Udaondo Z, Sittikankaew K, Uengwetwanit T, Wongsurawat T, Sonthirod C, Jenjaroenpun P, et al. Comparative analysis of PacBio and Oxford Nanopore sequencing technologies for transcriptomic landscape identification of Penaeus monodon. Life. 2021; 11: 862.

[34] Pacheco MA, Cepeda AS, Miller EA, Beckerman S, Oswald M, London E, et al. A new long-read mitochondrial-genome protocol (PacBio HiFi) for haemosporidian parasites: a tool for population and biodiversity studies. Malar J. 2024; 23: 134.

[35] Han Y, He J, Li M, Peng Y, Jiang H, Zhao J, et al. Unlocking the potential of metagenomics with the PacBio high-fidelity sequencing technology. Microorganisms. 2024; 12: 2482.

[36] Hong SJ, Shin GH, Kim JT. Fabrication and application of turmeric extract-incorporated oleogels structured with xanthan gum and soy lecithin by emulsion template. Gels. 2024; 10: 84.

[37] Simner PJ, Miller S, Carroll KC. Understanding the promises and hurdles of metagenomic next-generation sequencing as a diagnostic tool for infectious diseases. Clin Infect Dis. 2018; 66: 778–788.

[38] Liang M, Fan Y, Zhang D, Yang L, Wang X, Wang S, et al. Metagenomic next-generation sequencing for accurate diagnosis and management of lower respiratory tract infections. Int J Infect Dis. 2022; 122: 921–929.

[39] Cao L, Yang H, Huang Z, Lu C, Chen F, Zhang J, et al. Direct prediction of antimicrobial resistance in Pseudomonas aeruginosa by metagenomic next-generation sequencing. Front Microbiol. 2024; 15:1413434.

[40] Tang W, Zhang Y, Luo C, Zhou L, Zhang Z, Tang X, et al. Clinical application of metagenomic next-generation sequencing for suspected infections in patients with primary immunodeficiency disease. Front Immunol. 2021; 12:696403.

[41] Chen S, Kang Y, Li D, Li Z. Diagnostic performance of metagenomic next-generation sequencing for the detection of pathogens in bronchoalveolar lavage fluid in patients with pulmonary infections: systematic review and meta-analysis. Int J Infect Dis. 2022; 122: 867–873.

[42] Islam Sajib M.S., Brunker K, Oravcova K, Everest P, Murphy ME, Forde T. Advances in host depletion and pathogen enrichment methods for rapid sequencing-based diagnosis of bloodstream infection. J Mol Diagn. 2024; 26: 741–753.

[43] Kim M, Parrish R.C., Tisza MJ, Shah VS, Tran T, Ross M, et al. Host DNA depletion on frozen human respiratory samples enables successful metagenomic sequencing for microbiome studies. Commun Biol. 2024; 7:1590.

[44] Yuan J, Ma L, Du J, Sun H, Li S, Zhou G, et al. Host DNA depletion assisted metagenomic sequencing of bronchoalveolar lavage fluids for diagnosis of pulmonary tuberculosis. Ann Clin Microbiol Antimicrob. 2025; 24: 13.

[45] Hall MB, Coin LJM. Pangenome databases improve host removal and mycobacteria classification from clinical metagenomic data. Gigascience. 2024; 13:giae010.

[46] Kalantar KL, Carvalho T, de Bourcy C.F.A., Dimitrov B, Dingle G, Egger R, et al. IDseq—an open source cloud-based pipeline and analysis service for metagenomic pathogen detection and monitoring. Gigascience. 2020; 9:giaa111.

[47] Alcolea-Medina A, Alder C, Snell LB, Charalampous T, Aydin A, Nebbia G, et al. Unified metagenomic method for rapid detection of microorganisms in clinical samples. Commun Med. 2024; 4: 135.

[48] Lu D, Kalantar KL, Glascock AL, Chu VT, Guerrero ES, Bernick N, et al. Simultaneous detection of pathogens and antimicrobial resistance genes with the open source, cloud-based, CZ ID platform. Genome Med. 2025; 17: 46.

[49] de Vries JJC, Brown JR, Fischer N, Sidorov IA, Morfopoulou S, Huang J, et al. Benchmark of thirteen bioinformatic pipelines for metagenomic virus diagnostics using datasets from clinical samples. J Clin Virol. 2021; 141:104908.

[50] Mzava O, Cheng AP, Chang A, Smalling S, Djomnang KL, Lenz J, et al. A metagenomic DNA sequencing assay that is robust against environmental DNA contamination. Nat Commun. 2021; 13: 4197.

[51] Gauthier NPG, Chan W, Locher K, Smailus D, Coope R, Charles M, et al. Validation of an automated, end-to-end metagenomic sequencing assay for agnostic detection of respiratory viruses. J Infect Dis. 2024; 230: e1245–e1253.

[52] Leung CM, Li D, Xin Y, Law WC, Zhang Y, Ting HF, et al. MegaPath: sensitive and rapid pathogen detection using metagenomic NGS data. BMC Genomics. 2020; 21: 500.

[53] Han D, Diao Z, Lai H, Han Y, Xie J, Zhang R, et al. Multilaboratory assessment of metagenomic next-generation sequencing for unbiased microbe detection. J Adv Res. 2022; 38: 213–222.

[54] Liu L, Hakhverdyan M, Wallgren P, Vanneste K, Fu Q, Lucas P, et al. An interlaboratory proficiency test using metagenomic sequencing as a diagnostic tool for the detection of RNA viruses in swine fecal material. Microbiol Spectr. 2024; 12:e420823.

[55] O'Sullivan DM, Doyle RM, Temisak S, Redshaw N, Whale AS, Logan G, et al. An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities. Sci Rep. 2021; 11:10590.

[56] Chen W, Liu G, Cui L, Tian F, Zhang J, Zhao J, et al. Evaluation of metagenomic and pathogen-targeted next-generation sequencing for diagnosis of meningitis and encephalitis in adults: a multicenter prospective observational cohort study in China. J Infect. 2024; 88:106143.

[57] Greninger AL, Naccache SN, Federman S, Yu G, Mbala P, Bres V, et al. Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis. Genome Med. 2015; 7: 99.

[58] Parks DH, Chuvochina M, Waite DW, Rinke C, Skarshewski A, Chaumeil PA, et al. A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nat Biotechnol. 2018; 36: 996–1004.

[59] Chaumeil PA, Mussig AJ, Hugenholtz P, Parks DH. GTDB-Tk: a toolkit to classify genomes with the genome taxonomy database. Bioinformatics. 2019; 36: 1925–1927.

[60] Amos GCA, Logan A, Anwar S, Fritzsche M, Mate R, Bleazard T, et al. Developing standards for the microbiome field. Microbiome. 2020; 8: 98.

[61] Chinese Thoracic Society. Consensus of clinical pathways of metagenomic next-generation sequencing test in diagnosis of lower respiratory tract infections in China. Zhonghua Jie He He Hu Xi Za Zhi. 2023; 46: 322–335. (in Chinese with an English abstract).

[62] 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.

[63] Miao Q, Ma Y, Wang Q, Pan J, Zhang Y, Jin W, et al. Microbiological diagnostic performance of metagenomic next-generation sequencing when applied to clinical practice. Clin Infect Dis. 2018; 67: S231–S240.

[64] Miao Q, Liang T, Pei N, Liu C, Pan J, Li N, et al. Evaluation of respiratory samples in etiology diagnosis and microbiome characterization by metagenomic sequencing. Respir Res. 2022; 23: 345.

[65] Ramachandran PS, Ramesh A, Creswell FV, Wapniarski A, Narendra R, Quinn CM, et al. Integrating central nervous system metagenomics and host response for diagnosis of tuberculosis meningitis and its mimics. Nat Commun. 2022; 13: 1675.

[66] Kalantar KL, Neyton L, Abdelghany M, Mick E, Jauregui A, Caldera S, et al. Integrated host-microbe plasma metagenomics for sepsis diagnosis in a prospective cohort of critically ill adults. Nat Microbiol. 2022; 7: 1805–1816.

[67] Xu F, Chen C, Lu S, Xue M, Ding H, Song Y, et al. Impact of metagenomics next-generation sequencing on etiological diagnosis and early outcomes in sepsis. J Transl Med. 2025; 23: 394.

[68] Zhao Y, Zhang W, Zhang X. Application of metagenomic next-generation sequencing in the diagnosis of infectious diseases. Front Cell Infect Microbiol. 2024; 14:1458316.

[69] Yuan L, Zhu XY, Lai LM, Chen Q, Liu Y, Zhao R. Clinical application and evaluation of metagenomic next-generation sequencing in pathogen detection for suspected central nervous system infections. Sci Rep. 2024; 14:16961.

[70] Lin K, Zhang HC, Zhang Y, Zhou Y, Fu ZF, Wang HY, et al. Clinical application and drug-use-guidance value of metagenomic next-generation sequencing in central nervous system infection. Am J Transl Res. 2023; 15: 47–62.

[71] Wei L, Luo J, Wu W, Yin J, Sun Z, Xu X, et al. Clinical diagnostic value of metagenomic next-generation sequencing in patients with acute infection in emergency department. Heliyon. 2024; 10:e35802.

[72] Zhu Y, Miao H, Zhang J, Jiang Z, Chu X, Xu Y, et al. Role of plasma and blood-cell co-metagenomic sequencing in precise diagnosis and severity evaluation of sepsis, a prospective cohort study in sepsis patients. J Infect. 2025; 90:106434.

[73] Jin W, Pan J, Miao Q, Ma Y, Zhang Y, Huang Y, et al. Diagnostic accuracy of metagenomic next-generation sequencing for active tuberculosis in clinical practice at a tertiary general hospital. Ann Transl Med. 2020; 8: 1065.

[74] Wang L, Chen Y, Wang Q, Pan J, Bao R, Jin W, et al. Comparison of molecular testing methods for diagnosing non-tuberculous mycobacterial infections. Eur J Clin Microbiol Infect Dis. 2025; 44: 109–116.

[75] Olearo F, Zein SE, Portillo ME, Zapf A, Rohde H, Berbari EF, et al. Diagnostic accuracy of 16S rDNA PCR, multiplex PCR and metagenomic next-generation sequencing in periprosthetic joint infections: a systematic review and meta-analysis. Clin Microbiol Infect. 2025; 31: 1115–1125.

[76] Hu H, Ding H, Lyu J, Chen Y, Huang C, Zhang C, et al. Detection of rare microorganisms in bone and joint infections by metagenomic next-generation sequencing. Bone Joint Res. 2024; 13: 401–410.

[77] Qian Z, Xia H, Zhou J, Wang R, Zhu D, Chen L, et al. Performance of metagenomic next-generation sequencing of cell-free DNA from vitreous and aqueous humor for diagnoses of intraocular infections. J Infect Dis. 2024; 229: 252–261.

[78] Wang Q, Miao Q, Pan J, Jin W, Ma Y, Zhang Y, et al. The clinical value of metagenomic next-generation sequencing in the microbiological diagnosis of skin and soft tissue infections. Int J Infect Dis. 2020; 100: 414–420.

[79] Daher M, Iordanov R, Al Mohajer M, Sohail MR, Staggers KA, Hamdi AM. Clinical utility of metagenomic next-generation sequencing in fever of undetermined origin. Ther Adv Infect Dis. 2024; 11:2028479785.

[80] Lai Y, Chen B, Chen S, Shen Y. Experience of implementing metagenomic next-generation sequencing in patients with suspected pulmonary infection in clinical practice. Sci Rep. 2025; 15:9579.

[81] Wang J, Xu H, Wang X, Lan J. Rapid diagnosis of non-tuberculous mycobacterial pulmonary diseases by metagenomic next-generation sequencing in non-referral hospitals. Front Cell Infect Microbiol. 2023; 12:1083497.

[82] Zhang Y, Cui P, Zhang HC, Wu HL, Ye MZ, Zhu YM, et al. Clinical application and evaluation of metagenomic next-generation sequencing in suspected adult central nervous system infection. J Transl Med. 2020; 18: 199.

[83] Jin X, Li J, Shao M, Lv X, Ji N, Zhu Y, et al. Improving suspected pulmonary infection diagnosis by bronchoalveolar lavage fluid metagenomic next-generation sequencing: a multicenter retrospective study. Microbiol Spectr. 2022; 10:e0247321.

[84] Gu W, Miller S, Chiu CY. Clinical metagenomic next-generation sequencing for pathogen detection. Ann Rev Pathol Mech Dis. 2019; 14: 319–338.

[85] Lei H, Liao J, Lin Y, Liu T, Lei W, Gao W. Application of metagenomic next-generation sequencing in treatment guidance for deep neck space abscess. BMC Microbiol. 2025; 25: 166.

[86] Liu Y, Qin S, Lan C, Huang Q, Zhang P, Cao W. Effectiveness of metagenomic next-generation sequencing in the diagnosis of infectious diseases: a systematic review and meta-analysis. Int J Infect Dis. 2024; 142:106996.

[87] Lv M, Zhu C, Zhu C, Yao J, Xie L, Zhang C, et al. Clinical values of metagenomic next-generation sequencing in patients with severe pneumonia: a systematic review and meta-analysis. Front Cell Infect Microbiol. 2023; 13:1106859.

[88] Eisenhofer R, Minich JJ, Marotz C, Cooper A, Knight R, Weyrich LS. Contamination in low microbial biomass microbiome studies: issues and recommendations. Trends Microbiol. 2019; 27: 105–117.

[89] Salter SJ, Cox MJ, Turek EM, Calus ST, Cookson WO, Moffatt MF, et al. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 2014; 12: 87.

[90] Chen M, Chen Y, He X, Hao R, Jiang X, Ji Y, et al. A case of Bartonella vinsonii endocarditis. Diagn Microbiol Infect Dis. 2024; 110:116535.

[91] Liu J, Xu Z, Bai Y, Feng J, Xu L, Li F. Streptomyces albireticuli lung infection managed as a pulmonary air cyst: a case report and literature review. Front Cell Infect Microbiol. 2024; 13:1296491.

[92] Pérot P, Fourgeaud J, Rouzaud C, Regnault B, Da Rocha N, Fontaine H, et al. Circovirus hepatitis infection in heart-lung transplant patient, france. Emerg Infect Dis. 2023; 29: 286–293.

[93] Mick E, Tsitsiklis A, Kamm J, Kalantar KL, Caldera S, Lyden A, et al. Integrated host/microbe metagenomics enables accurate lower respiratory tract infection diagnosis in critically ill children. J Clin Investig. 2023; 133:e165904.

[94] Klig JE. Office pediatrics: current perspectives on the outpatient evaluation and management of lower respiratory infections in children. Curr Opin Pediatrics. 2006; 18: 71–76.

[95] Lisha W, Jiao Q, Mengyuan C, Jiajia Q, Tianbin T, Yilan W, et al. Clinical evaluation of negative mNGS reports in sterile body fluids and tissues. Microbiol Spectr. 2025; 13:e0201324.

[96] Quick J, Loman NJ, Duraffour S, Simpson JT, Severi E, Cowley L, et al. Real-time, portable genome sequencing for Ebola surveillance. Nature. 2016; 530: 228–232.

[97] Wenger AM, Peluso P, Rowell WJ, Chang PC, Hall RJ, Concepcion GT, et al. Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome. Nat Biotechnol. 2019; 37: 1155–1162.

[98] Kanyerezi S, Guerfali FZ, Anzaku AA, Babaleye OA, Calvert-Joshua T, Nguinkal JA, et al. Wastewater metagenomics in Africa: opportunities and challenges. PLOS Glob Public Health. 2024; 4:e4044.

[99] Hendriksen RS, Munk P, Njage P, van Bunnik B, Mcnally L, Lukjancenko O, et al. Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage. Nat Commun. 2019; 10: 1124.

[100] Zhao M, Shi Y, Zhang C, Lu M, Shen M, Xie L, et al. Diagnostic value of metagenomic next-generation sequencing using bronchoalveolar lavage fluid samples for pathogen detection in children with severe or refractory pneumonia. Microbiol Spectr. 2025; 13:e108724.

[101] Chen Y, Wang J, Niu T. Clinical and diagnostic values of metagenomic next-generation sequencing for infection in hematology patients: a systematic review and meta-analysis. BMC Infect Dis. 2024; 24: 167.

[102] Wang C, Hu J, Gu Y, Wang X, Chen Y, Yuan W. Application of next-generation metagenomic sequencing in the diagnosis and treatment of acute spinal infections. Heliyon. 2023; 9:e13951.

[103] Wu J, Song W, Yan H, Luo C, Hu W, Xie L, et al. Metagenomic next-generation sequencing in detecting pathogens in pediatric oncology patients with suspected bloodstream infections. Pediatr Res. 2024; 95: 843–851.

[104] Yek C, Pacheco AR, Vanaerschot M, Bohl JA, Fahsbender E, Aranda-Díaz A, et al. Metagenomic pathogen sequencing in resource-scarce settings: lessons learned and the road ahead. Front Epidemiol. 2022; 2:926695.

[105] Inzaule SC, Tessema SK, Kebede Y, Ogwell Ouma AE, Nkengasong JN. Genomic-informed pathogen surveillance in Africa: opportunities and challenges. Lancet Infect Dis. 2021; 21: e281–e289.

RIGHTS & PERMISSIONS

2026 The Author(s). mLife published by John Wiley & Sons Australia, Ltd on behalf of Institute of Microbiology, Chinese Academy of Sciences.