基于纳米孔宏基因组测序的病原体和耐药基因快速精准诊断流程建立和应用

单分子纳米孔测序技术具有测序片段长，文库制备快速和数据实时采集分析的优势，已用于靶向方法从临床样本中识别感染性病原体。本研究利用临床样本探究了宏基因组测序，重点是纳米孔宏基因组测序在感染性疾病病原学诊断中的临床可行性，搭建一条完整的病原体和耐药基因诊断流程，评估该流程用作临床即时检验的潜力。

我们使用Illumina宏基因组测序检测胸腔脓液样本的结果提示，临床样本的复杂性，如临床标本来源复杂多样、大量宿主信息存在导致病原体信息太少、病原体信息混杂在正常共生菌群中难以区分，使宏基因组学测序面临挑战。通过引入宿主DNA耗竭的方法，增加测序数据中微生物序列的富集检出，可以改进宏基因组学方法，快速准确地表征病原体。本研究应用并评价了一种基于纳米孔适应采样（Nanopore adaptive sampling，NAS）技术的宿主DNA耗竭方法，无需额外的样本处理步骤，通过测序过程中程序控制，在不损失病原体DNA的前提下特异性去除宿主DNA。此方法在感染模拟样本中得到很好验证，适合对低丰度微生物的样本进行快速、实时检测。

基于NAS宿主DNA耗竭的方法，本研究建立了快速精确诊断感染病原体及耐药基因的宏基因组学流程，为临床病原体快速诊断提供新的思路和有力工具。我们的工作流程将高质量宏基因组提取方法、NAS测序、自动分析软件-RUARGpore 相结合，缩短同时检测病原体和耐药基因的周转时间。通过对肺泡灌洗液样本的验证，在4.5 h内完成低丰度微生物样本中病原体识别和耐药基因检测。与微生物培养结果相比显示100%的正/负百分比协议，结合自动化生物信息学分析软件-RUARGpore，该流程展现出作为临床感染诊断工具的巨大潜力。

综上所述，本研究基于宏基因组纳米孔测序技术，探索、建立并优化了一套新的感染性疾病病原体和耐药基因诊断流程，对其诊断性能进行了综合评估，验证了其应用于临床的巨大潜力。

Long-read sequencing technologies, such as the Oxford nanopore technologies (ONT) sequencing platforms, allow for the fast, real-time sequencing of complex clinic samples. This study explored the clinical feasibility of nanopore sequencing in the pathogenic diagnosis of infectious diseases, and present a workflow for pathogen diagnosis and antibiotic resistance genes (ARGs) detection, and evaluate the potential of this workflow for clinical point-of-care testing.

The results of using Illumina metagenomic sequencing to detect pleural pus samples suggest that, clinical specimens present a difficult challenge for metagenomics sequencing due to variable pathogen load, the presence of commensal tract flora, and the high ratio of host: pathogen nucleic acids present. By introducing host DNA depletion and increasing the enrichment of microbiome sequences, the metagenomics method would be improved to quickly and accurately identify pathogens. This study applied and evaluated a host DNA depletion method based on nanopore adaptive sampling (NAS) technology. Without additional sample processing steps, the host DNA was specifically removed without loss of pathogen DNA through program control in the sequencing process. This method has been well verified in mock community, and is suitable for rapid and real-time sequencing of samples with low abundance of microbiota.

Based on the method of NAS host DNA depletion, this study has established a metagenome workflow for rapid and accurate diagnosis of infectious pathogens and ARGs, providing a powerful tool for rapid diagnosis of clinical pathogens. Our workflow combines high-quality metagenome extraction methods, NAS real-time sequencing, and automatic analysis software-RUARGpore, which shorten the turnaround time for simultaneous detection of pathogens and ARGs. Through the validation of bronchoalveolar lavage fluid (BALF) samples, which completes bacterial pathogen and ARGs detection within 4.5 h. The described metagenome workflow had a perfect positive/negative percent agreement (100%) for traditional culture. Combined with the automated bioinformatics analysis software - RUARGPore, this workflow shows great potential as a rapid and accurate clinical diagnosis tool.

To summarize, based on the metagenome nanopore sequencing technology, we explored, established and optimized a new diagnostic workflow for pathogens and ARGs, comprehensively evaluated its diagnostic performance, and verified its huge potential for clinical application.

[1] CHARALAMPOUS T, KAY G L, RICHARDSON H, et al. Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection [J]. Nat Biotechnol, 2019, 37(7): 783-792.
[2] JAIN S, SELF W H, WUNDERINK R G, et al. Community-Acquired Pneumonia Requiring Hospitalization among US Adults [J]. New England Journal of Medicine, 2015, 373(5): 415-427.
[3] CHARALAMPOUS T, KAY G L, RICHARDSON H, et al. Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection [J]. Nature Biotechnology, 2019, 37(7): 783-+.
[4] ROSE T M. CODEHOP-mediated PCR - a powerful technique for the identification and characterization of viral genomes [J]. Virol J, 2005, 2: 20.
[5] TORSVIK V, OVREAS L. Microbial diversity and function in soil: from genes to ecosystems [J]. Curr Opin Microbiol, 2002, 5(3): 240-245.
[6] BRYNILDSRUD O B, ELDHOLM V, BOHLIN J, et al. Acquisition of virulence genes by a carrier strain gave rise to the ongoing epidemics of meningococcal disease in West Africa [J]. Proc Natl Acad Sci U S A, 2018, 115(21): 5510-5515.
[7] CHEN J, ZHAO Y, SHANG Y, et al. The clinical significance of simultaneous detection of pathogens from bronchoalveolar lavage fluid and blood samples by metagenomic next-generation sequencing in patients with severe pneumonia [J]. J Med Microbiol, 2021, 70(1).
[8] GU W, MILLER S, CHIU C Y. Clinical Metagenomic Next-Generation Sequencing for Pathogen Detection [J]. Annu Rev Pathol, 2019, 14: 319-338.
[9] LEGGETT R M, CLARK M D. A world of opportunities with nanopore sequencing [J]. Journal of Experimental Botany, 2017, 68(20): 5419-5429.
[10] MARX V. Nanopores: a sequencer in your backpack [J]. Nat Methods, 2015, 12(11): 1015-1018.
[11] LEWANDOWSKI K, XU Y, PULLAN S T, et al. Metagenomic Nanopore Sequencing of Influenza Virus Direct from Clinical Respiratory Samples [J]. J Clin Microbiol, 2019, 58(1).
[12] SANDERSON N D, STREET T L, FOSTER D, et al. Real-time analysis of nanopore￾based metagenomic sequencing from infected orthopaedic devices [J]. BMC Genomics, 2018, 19(1): 714.
[13] SCHMIDT K, MWAIGWISYA S, CROSSMAN L C, et al. Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing [J]. J Antimicrob Chemother, 2017, 72(1): 104-114.
[14] PENDLETON K M, ERB-DOWNWARD J R, BAO Y, et al. Rapid Pathogen Identification in Bacterial Pneumonia Using Real-Time Metagenomics [J]. Am J Respir Crit Care Med, 2017, 196(12): 1610-1612.
[15] MAROTZ C A, SANDERS J G, ZUNIGA C, et al. Improving saliva shotgun metagenomics by chemical host DNA depletion [J]. Microbiome, 2018, 6(1): 42.
[16] HASAN M R, RAWAT A, TANG P, et al. Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing [J]. J Clin Microbiol, 2016, 54(4): 919-927.
[17] ZELENIN S, HANSSON J, ARDABILI S, et al. Microfluidic-based isolation of bacteria from whole blood for sepsis diagnostics [J]. Biotechnol Lett, 2015, 37(4): 825-830.
[18] COUTO N, SCHUELE L, RAANGS E C, et al. Critical steps in clinical shotgun metagenomics for the concomitant detection and typing of microbial pathogens [J]. Sci Rep, 2018, 8(1): 13767.
[19] PITT M E, NGUYEN S H, DUARTE T P S, et al. Evaluating the genome and resistome of extensively drug-resistant Klebsiella pneumoniae using native DNA and RNA Nanopore sequencing [J]. Gigascience, 2020, 9(2).
[20] CHENG J, HU H, KANG Y, et al. Identification of pathogens in culture-negative infective endocarditis cases by metagenomic analysis [J]. Ann Clin Microbiol Antimicrob, 2018, 17(1): 43.
[21] 罗越, 胡洋洋, 张兴, 等. 《中国宏基因组学第二代测序技术检测感染病原体的临床应用专家共识》解读 [J]. 河北医科大学学报, 2021, 42(07): 745-749.
[22] 李曼诗, 黄巍峰, 陆一涵. 感染性疾病的微生物宏基因组测序结果判读的比较[J]. 复旦学报(医学版), 2021, 48(05): 624-629.
[23] 李颖, 麻锦敏. 宏基因组学测序技术在中重症感染中的临床应用专家共识（第一版） [J]. 感染、炎症、修复, 2020, 21(02): 75-81.
[24] 何军. 宏基因组测序技术检测感染性病原体江苏专家共识(2020 版) [J]. 临床检验杂志, 2020, 38(09): 641-645.
[25] MARTIN S, HEAVENS D, LAN Y, et al. Nanopore adaptive sampling: a tool for enrichment of low abundance species in metagenomic samples [J]. Genome Biol, 2022, 23(1): 11.
[26] GAN M, WU B, YAN G, et al. Combined nanopore adaptive sequencing and enzyme￾based host depletion efficiently enriched microbial sequences and identified missing respiratory pathogens [J]. BMC Genomics, 2021, 22(1): 732.
[27] ZHONG N S, ZHENG B J, LI Y M, et al. Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People's Republic of China, in February, 2003 [J]. Lancet, 2003, 362(9393): 1353-1358.
[28] KSIAZEK T G, ERDMAN D, GOLDSMITH C S, et al. A novel coronavirus associated with severe acute respiratory syndrome [J]. New England Journal of Medicine, 2003,142参考文献348(20): 1953-1966.
[29] ASSIRI A M, BIGGS H M, ABEDI G R, et al. Increase in middle east respiratory syndrome-coronavirus cases in Saudi Arabia linked to hospital outbreak with continued circulation of recombinant virus, July 1-August 31, 2015 [J]. Open Forum Infectious Diseases, 2016, 3(3).
[30] AL-DORZI H M, ALDAWOOD A S, KHAN R, et al. The critical care response to a hospital outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infection: an observational study [J]. Annals of Intensive Care, 2016, 6(1).
[31] DIALLO M S K, TOURE A, SOW M S, et al. Understanding Long-term Evolution and Predictors of Sequelae of Ebola Virus Disease Survivors in Guinea: A 48-Month Prospective, Longitudinal Cohort Study (PostEboGui) [J]. Clin Infect Dis, 2021, 73(12): 2166-2174.
[32] DE PIJPER C A, SCHNYDER J L, STIJNIS C, et al. A review of severe thrombocytopenia in Zika patients – Pathophysiology, treatment and outcome [J].Travel Medicine and Infectious Disease, 2022, 45.
[33] SAFFORD T G, WHITMORE E H, HAMILTON L C. Scientists, presidents, and pandemics—comparing the science–politics nexus during the Zika virus and COVID-19 outbreaks [J]. Social Science Quarterly, 2021, 102(6): 2482-2498.
[34] FAROOQ H Z, DAVIES E, BROWN B, et al. Real-world SARS CoV-2 testing in Northern England during the first wave of the COVID-19 pandemic [J]. Journal of Infection, 2021, 83(1): 84-91.
[35] CHAMPIGNEULLE B, HANCCO I, RENAN R, et al. High-altitude environment and COVID-19: SARS-CoV-2 seropositivity in the highest city in the world [J]. High Alt Med Biol, 2021.
[36] MAGLANGIT F, YU Y, DENG H. Bacterial pathogens: Threat or treat (a review on bioactive natural products from bacterial pathogens) [J]. Natural Product Reports, 2021, 38(4): 782-821.
[37] DISEASES G B D, INJURIES C. Global burden of 369 diseases and injuries in 204 countries and territories, 1990-2019: a systematic analysis for the Global Burden of Disease Study 2019 [J]. Lancet, 2020, 396(10258): 1204-1222.
[38] RAJAPAKSHA P, ELBOURNE A, GANGADOO S, et al. A review of methods for the detection of pathogenic microorganisms [J]. Analyst, 2019, 144(2): 396-411.
[39] LI P, NIU W, FANG Y, et al. Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Rapid and Specific Identification of Carbapenem￾Resistant Acinetobacter baumannii Strains Harboring blaOXA-23, and the Epidemiological Survey of Clinical Isolates [J]. Microbial Drug Resistance, 2020, 26(12): 1458-1465.
[40] BODULEV O L, SAKHAROV I Y. Isothermal Nucleic Acid Amplification Techniques and Their Use in Bioanalysis [J]. Biochemistry (Moscow), 2020, 85(2): 147-166.
[41] TIAN B, FOCK J, MINERO G A S, et al. Ultrasensitive Real-Time Rolling Circle Amplification Detection Enhanced by Nicking-Induced Tandem-Acting Polymerases [J]. Analytical Chemistry, 2019, 91(15): 10102-10109.
[42] SIMNER P J, MILLER H B, BREITWIESER F P, et al. Development and optimization of metagenomic next-generation sequencing methods for cerebrospinal fluid diagnostics [J]. Journal of Clinical Microbiology, 2018, 56(9).
[43] LI Y, XIU L, LIU J, et al. A multiplex assay for characterization of antimicrobial resistance in Neisseria gonorrhoeae using multi-PCR coupled with mass spectrometry [J]. Journal of Antimicrobial Chemotherapy, 2020, 75(10): 2817-2825.
[44] ZHANG C, XIU L, XIAO Y, et al. Simultaneous detection of key bacterial pathogens related to pneumonia and meningitis using multiplex PCR coupled with mass spectrometry [J]. Frontiers in Cellular and Infection Microbiology, 2018, 8(APR).
[45] ZHANG C, XIAO Y, DU J, et al. Application of multiplex PCR coupled with matrix￾assisted laser desorption ionization-time of flight analysis for simultaneous detection of 21 common respiratory viruses [J]. Journal of Clinical Microbiology, 2015, 53(8): 2549-2554.
[46] HERNÁNDEZ HERNÁNDEZ O, GUTIÉRREZ-ESCOLANO A L, CANCIO￾LONCHES C, et al. Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry [J]. Food Microbiology, 2022, 102.
[47] XIU L, ZHANG C, LI Y, et al. High-resolution melting analysis for rapid detection of the internationally spreading ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone [J]. Journal of Antimicrobial Chemotherapy, 2020, 75(1): 106-109.
[48] NACCACHE S N, FEDERMAN S, VEERARAGHAVAN N, et al. A cloud-compatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples [J]. Genome Research, 2014, 24(7): 1180-1192.
[49] VAN DIJK E L. Ten years of next-generation sequencing technology [J]. Trends in genetics : TIG, 2014, 30(9): 418-426.
[50] ZHAO L, ZHANG H, KOHNEN M V, et al. Analysis of transcriptome and epitranscriptome in plants using pacbio iso-seq and nanopore-based direct RNA sequencing [J]. Frontiers in Genetics, 2019, 10(MAR).
[51] EID J, FEHR A, GRAY J, et al. Real-time DNA sequencing from single polymerase molecules [J]. Science, 2009, 323(5910): 133-138.
[52] GIUSTI B, STICCHI E, DE CARIO R, et al. Genetic bases of bicuspid aortic valve: The contribution of traditional and high-throughput sequencing approaches on research and diagnosis [J]. Frontiers in Physiology, 2017, 8(AUG).
[53] WANG Y, YANG Q, WANG Z. The evolution of nanopore sequencing [J]. Frontiers in Genetics, 2014, 5(DEC).
[54] NIEDRINGHAUS T P, MILANOVA D, KERBY M B, et al. Landscape of next-generation sequencing technologies [J]. Analytical Chemistry, 2011, 83(12): 4327-4341.
[55] REUTER J A, SPACEK D V, SNYDER M P. High-Throughput Sequencing Technologies [J]. Molecular Cell, 2015, 58(4): 586-597.
[56] YAHARA K, SUZUKI M, HIRABAYASHI A, et al. Long-read metagenomics using PromethION uncovers oral bacteriophages and their interaction with host bacteria [J]. Nature Communications, 2021, 12(1).
[57] IMAI K, TAMURA K, TANIGAKI T, et al. Whole genome sequencing of influenza A and B viruses with the MinION sequencer in the clinical setting: A pilot study [J]. Frontiers in Microbiology, 2018, 9(NOV).
[58] MCINTYRE A B R, RIZZARDI L, YU A M, et al. Nanopore sequencing in microgravity [J]. npj Microgravity, 2016, 2.
[59] CASTRO-WALLACE S L, CHIU C Y, JOHN K K, et al. Nanopore DNA Sequencing and Genome Assembly on the International Space Station [J]. Scientific Reports, 2017, 7(1).
[60] JAIN M, KOREN S, MIGA K H, et al. Nanopore sequencing and assembly of a human genome with ultra-long reads [J]. Nature Biotechnology, 2018, 36(4): 338-345.
[61] PENNISI E. Search for pore-fection [J]. Science, 2012, 336(6081): 534-537.
[62] BADENES M L, FERNÁNDEZ I MARTÍ A, RÍOS G, et al. Application of genomic technologies to the breeding of trees [J]. Frontiers in Genetics, 2016, 7(NOV).
[63] SEREIKA M, KIRKEGAARD R H, KARST S M, et al. Oxford Nanopore R10.4 long￾read sequencing enables near-perfect bacterial genomes from pure cultures and metagenomes without short-read or reference polishing [J]. bioRxiv, 2021.
[64] ROYCHOWDHURY S, CHINNAIYAN A M. Translating cancer genomes and transcriptomes for precision oncology [J]. CA Cancer Journal for Clinicians, 2016, 66(1): 75-88.
[65] DE VLAMINCK I, KHUSH K K, STREHL C, et al. XTemporal response of the human virome to immunosuppression and antiviral therapy [J]. Cell, 2013, 155(5): 1178.
[66] ARUMUGAM M, RAES J, PELLETIER E, et al. Enterotypes of the human gut microbiome [J]. Nature, 2011, 473(7346): 174-180.
[67] TENG L, LEE S, GINN A, et al. Genomic comparison reveals natural occurrence of clinically relevant multidrugresistant extended-spectrum-β-lactamaseproducing Escherichia coli strains [J]. Applied and Environmental Microbiology, 2019, 85(13).
[68] DÍAZ-VIRAQUÉ F, PITA S, GREIF G, et al. Nanopore Sequencing Significantly Improves Genome Assembly of the Protozoan Parasite Trypanosoma cruzi [J]. Genome Biology and Evolution, 2019, 11(7): 1952-1957.
[69] MOSS E L, MAGHINI D G, BHATT A S. Complete, closed bacterial genomes from microbiomes using nanopore sequencing [J]. Nature Biotechnology, 2020, 38(6): 701-707.
[70] STARK R, GRZELAK M, HADFIELD J. RNA sequencing: the teenage years [J]. Nature Reviews Genetics, 2019, 20(11): 631-656.
[71] GARALDE D R, SNELL E A, JACHIMOWICZ D, et al. Highly parallel direct RN A sequencing on an array of nanopores [J]. Nature Methods, 2018, 15(3): 201-206.
[72] WORKMAN R E, TANG A D, TANG P S, et al. Nanopore native RNA sequencing of a human poly(A) transcriptome [J]. Nature Methods, 2019, 16(12): 1297-1305.
[73] ZHANG C, XIU L, LI Y, et al. Multiplex PCR and Nanopore Sequencing of Genes Associated with Antimicrobial Resistance in Neisseria gonorrhoeae Directly from Clinical Samples [J]. Clinical Chemistry, 2021, 67(4): 610-620.
[74] SEEDORF H, GRIFFIN N W, RIDAURA V K, et al. Bacteria from diverse habitats colonize and compete in the mouse gut [J]. Cell, 2014, 159(2): 253-266.
[75] DETHLEFSEN L, RELMAN D A. Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation [J]. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(SUPPL. 1): 4554-4561.
[76] DJEMIEL C, DEQUIEDT S, KARIMI B, et al. BIOCOM-PIPE: a new user-friendly metabarcoding pipeline for the characterization of microbial diversity from 16S, 18S and 23S rRNA gene amplicons [J]. BMC Bioinformatics, 2020, 21(1).
[77] QUICK J, GRUBAUGH N D, PULLAN S T, et al. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples [J]. Nature Protocols, 2017, 12(6): 1261-1266.
[78] MOORE S C, PENRICE-RANDAL R, ALRUWAILI M, et al. Amplicon-based detection and sequencing of SARS-CoV-2 in nasopharyngeal swabs from patients with COVID-19 and identification of deletions in the viral genome that encode proteins involved in interferon antagonism [J]. Viruses, 2020, 12(10).
[79] CHEW K L, OCTAVIA S, JUREEN R, et al. Targeted amplification and MinION nanopore sequencing of key azole and echinocandin resistance determinants of clinically relevant Candida spp. from blood culture bottles [J]. Letters in Applied Microbiology, 2021, 73(3): 286-293.
[80] YANG Y, CHE Y, LIU L, et al. Rapid absolute quantification of pathogens and ARGs by nanopore sequencing [J]. Science of the Total Environment, 2022, 809.
[81] ALILI R, BELDA E, LE P, et al. Exploring semi-quantitative metagenomic studies using oxford nanopore sequencing: A computational and experimental protocol [J]. Genes, 2021, 12(10).
[82] KARLSSON F H, TREMAROLI V, NOOKAEW I, et al. Gut metagenome in European women with normal, impaired and diabetic glucose control [J]. Nature, 2013, 498(7452): 99-103.
[83] CAUSSY C, HSU C, LO M T, et al. Link between gut-microbiome derived metabolite and shared gene-effects with hepatic steatosis and fibrosis in NAFLD [J]. Hepatology, 2018, 68(3): 918-932.
[84] MBALA-KINGEBENI P, VILLABONA-ARENAS C J, VIDAL N, et al. Rapid Confirmation of the Zaire Ebola Virus in the Outbreak of the Equateur Province in the Democratic Republic of Congo: Implications for Public Health Interventions [J]. Clinical Infectious Diseases, 2019, 68(2): 330-333.
[85] HOENEN T, GROSETH A, ROSENKE K, et al. Nanopore sequencing as a rapidly deployable Ebola outbreak tool [J]. Emerging Infectious Diseases, 2016, 22(2): 331-334.
[86] YANG M, COUSINEAU A, LIU X, et al. Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION – Promising Strategies for Multiplex Identification of Viable Pathogens in Food [J]. Frontiers in Microbiology, 2020, 11.
[87] ZHANG L, ZHANG C, ZENG Y, et al. Emergence and characterization of a ceftriaxone-resistant neisseria gonorrhoeae fc428 clone evolving moderate-level resistance to azithromycin in Shenzhen, China [J]. Infection and Drug Resistance, 2021, 14: 4271-4276.
[88] PETERSEN L M, MARTIN I W, MOSCHETTI W E, et al. Third-generation sequencing in the clinical laboratory: Exploring the advantages and challenges of nanopore sequencing [J]. Journal of Clinical Microbiology, 2020, 58(1).
[89] QUICK J, LOMAN N J, DURAFFOUR S, et al. Real-time, portable genome sequencing for Ebola surveillance [J]. Nature, 2016, 530(7589): 228-232.
[90] FARIA N R, SABINO E C, NUNES M R T, et al. Mobile real-time surveillance of Zika virus in Brazil [J]. Genome Medicine, 2016, 8(1).
[91] LU R, ZHAO X, LI J, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding [J]. The Lancet, 2020, 395(10224): 565-574.
[92] KIM D, LEE J Y, YANG J S, et al. The Architecture of SARS-CoV-2 Transcriptome [J]. Cell, 2020, 181(4): 914-921.e910.
[93] BULL R A, ADIKARI T N, FERGUSON J M, et al. Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis [J]. Nature Communications, 2020, 11(1).
[94] WU F, ZHAO S, YU B, et al. A new coronavirus associated with human respiratory disease in China [J]. Nature, 2020, 579(7798): 265-269.
[95] MAHMOOD T B, SAHA A, HOSSAN M I, et al. A next generation sequencing (NGS) analysis to reveal genomic and proteomic mutation landscapes of SARS-CoV-2 in South Asia [J]. Current Research in Microbial Sciences, 2021, 2.
[96] BADUA C L D C, BALDO K A T, MEDINA P M B. Genomic and proteomic mutation landscapes of SARS-CoV-2 [J]. Journal of Medical Virology, 2021, 93(3): 1702-1721.
[97] CHAN W M, IP J D, CHU A W H, et al. Identification of nsp1 gene as the target of SARS-CoV-2 real-time RT-PCR using nanopore whole-genome sequencing [J]. Journal of Medical Virology, 2020, 92(11): 2725-2734.
[98] WANG M, FU A, HU B, et al. Nanopore Targeted Sequencing for the Accurate and Comprehensive Detection of SARS-CoV-2 and Other Respiratory Viruses [J]. Small, 2020, 16(32).
[99] OLSEN R J, CHRISTENSEN P A, LONG S W, et al. Trajectory of Growth of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants in Houston, Texas, January through May 2021, Based on 12,476 Genome Sequences [J]. Am J Pathol, 2021, 191(10): 1754-1773.
[100]RUAN Z, ZOU S, WANG Z, et al. Toward accurate diagnosis and surveillance of bacterial infections using enhanced strain-level metagenomic next-generation sequencing of infected body fluids [J]. Brief Bioinform, 2022, 23(2).
[101]GU W, DENG X, LEE M, et al. Rapid pathogen detection by metagenomic next￾generation sequencing of infected body fluids [J]. Nat Med, 2021, 27(1): 115-124.
[102]LEGGETT R M, ALCON-GINER C, HEAVENS D, et al. Rapid MinION profiling of preterm microbiota and antimicrobial-resistant pathogens [J]. Nat Microbiol, 2020, 5(3): 430-442.
[103]CHNG K R, LI C, BERTRAND D, et al. Cartography of opportunistic pathogens and antibiotic resistance genes in a tertiary hospital environment [J]. Nat Med, 2020, 26(6): 941-951.
[104]MLYNARCZYK-BONIKOWSKA B, MAJEWSKA A, MALEJCZYK M, et al. Multiresistant Neisseria gonorrhoeae: a new threat in second decade of the XXI century [J]. Med Microbiol Immunol, 2020, 209(2): 95-108.
[105]ZHANG C, WANG F, ZHU C S, et al. Determining antimicrobial resistance profiles and identifying novel mutations of Neisseria gonorrhoeae genomes obtained by multiplexed MinION sequencing [J]. Science China-Life Sciences, 2020, 63(7): 1063-1070.
[106]KARST S M, ZIELS R M, KIRKEGAARD R H, et al. High-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing [J]. Nat Methods, 2021, 18(2): 165-169.
[107]DALBERTO P F, DE SOUZA E V, ABBADI B L, et al. Handling the Hurdles on the Way to Anti-tuberculosis Drug Development [J]. Front Chem, 2020, 8: 586294.
[108]OH S, TRIFONOV L, YADAV V D, et al. Tuberculosis Drug Discovery: A Decade of Hit Assessment for Defined Targets [J]. Front Cell Infect Microbiol, 2021, 11: 611304.
[109]CABIBBE A M, SPITALERI A, BATTAGLIA S, et al. Application of Targeted Next￾Generation Sequencing Assay on a Portable Sequencing Platform for Culture-Free Detection of Drug-Resistant Tuberculosis from Clinical Samples [J]. J Clin Microbiol, 2020, 58(10).
[110]TAXT A M, AVERSHINA E, FRYE S A, et al. Rapid identification of pathogens, antibiotic resistance genes and plasmids in blood cultures by nanopore sequencing [J]. Sci Rep, 2020, 10(1): 7622.
[111]ZHOU M, WU Y, KUDINHA T, et al. Comprehensive Pathogen Identification, Antibiotic Resistance, and Virulence Genes Prediction Directly From Simulated Blood Samples and Positive Blood Cultures by Nanopore Metagenomic Sequencing [J]. Front Genet, 2021, 12: 620009.
[112]FENG Y, ZOU S, CHEN H, et al. BacWGSTdb 2.0: a one-stop repository for bacterial whole-genome sequence typing and source tracking [J]. Nucleic Acids Res, 2021, 49(D1): D644-D650.
[113]CHEN H, JIANG T, WU J, et al. Genomic and phylogenetic analysis of a multidrug￾resistant mcr-1-carrying Klebsiella pneumoniae recovered from a urinary tract infection in China [J]. J Glob Antimicrob Resist, 2021, 27: 222-224.
[114]YUE M, LIU D, HU X, et al. Genomic characterisation of a multidrug-resistant Escherichia coli strain carrying the mcr-1 gene recovered from a paediatric patient in China [J]. J Glob Antimicrob Resist, 2021, 24: 370-372.
[115]RUNTUWENE L R, TUDA J S B, MONGAN A E, et al. Nanopore sequencing of drug￾resistance-associated genes in malaria parasites, Plasmodium falciparum [J]. Scientific Reports, 2018, 8.
[116]LOURENCO T G B, HELLER D, SILVA-BOGHOSSIAN C M, et al. Microbial signature profiles of periodontally healthy and diseased patients [J]. Journal of Clinical Periodontology, 2014, 41(11): 1027-1036.
[117]NAYFACH S, SHI Z J, SESHADRI R, et al. New insights from uncultivated genomes of the global human gut microbiome [J]. Nature, 2019, 568(7753): 505-+.
[118]HEIKEMA A P, HORST-KREFT D, BOERS S A, et al. Comparison of Illumina versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota [J]. Genes, 2020, 11(9).
[119]YANG L B, HAIDAR G, ZIA H, et al. Metagenomic identification of severe pneumonia pathogens in mechanically-ventilated patients: a feasibility and clinical validity study [J]. Respiratory Research, 2019, 20(1).
[120]BENITEZ-PAEZ A, SANZ Y. Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION (TM) portable nanopore sequencer [J]. Gigascience, 2017, 6(7).
[121]CUSCO A, CATOZZI C, VINES J, et al. Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon [J]. F1000Res, 2018, 7: 1755.
[122]BUNYAVANICH S, SCHADT E E. Systems biology of asthma and allergic diseases: a multiscale approach [J]. J Allergy Clin Immunol, 2015, 135(1): 31-42.
[123]HUANG Y J, CHARLSON E S, COLLMAN R G, et al. The role of the lung microbiome in health and disease. A National Heart, Lung, and Blood Institute workshop report [J]. Am J Respir Crit Care Med, 2013, 187(12): 1382-1387.
[124]DEAMER D, AKESON M, BRANTON D. Three decades of nanopore sequencing [J]. Nat Biotechnol, 2016, 34(5): 518-524.
[125]JAIN M, OLSEN H E, PATEN B, et al. The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community [J]. Genome Biol, 2016, 17(1): 239.
[126]AMARASINGHE S L, SU S, DONG X, et al. Opportunities and challenges in long￾read sequencing data analysis [J]. Genome Biol, 2020, 21(1): 30.
[127]RUAN J, LI H. Fast and accurate long-read assembly with wtdbg2 [J]. Nat Methods, 2020, 17(2): 155-158.
[128]FENG Z, CLEMENTE J C, WONG B, et al. Detecting and phasing minor single￾nucleotide variants from long-read sequencing data [J]. Nat Commun, 2021, 12(1): 3032.
[129]LIEBERMAN K R, CHERF G M, DOODY M J, et al. Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase [J]. J Am Chem Soc, 2010, 132(50): 17961-17972.
[130]JAWORSKI E, ROUTH A. Parallel ClickSeq and Nanopore sequencing elucidates the rapid evolution of defective-interfering RNAs in Flock House virus [J]. PLoS Pathog, 2017, 13(5): e1006365.
[131]YUEN Z W, SRIVASTAVA A, DANIEL R, et al. Systematic benchmarking of tools for CpG methylation detection from nanopore sequencing [J]. Nat Commun, 2021, 12(1): 3438.
[132]XIONG X, KELKAR Y D, GEDEN C J, et al. Long-Read Assembly and Annotation of the Parasitoid Wasp Muscidifurax raptorellus, a Biological Control Agent for Filth Flies [J]. Front Genet, 2021, 12: 748135.
[133]PAVLISCAK L A, NIRMALA J, SINGH V K, et al. Tracing Viral Transmission and Evolution of Bovine Leukemia Virus through Long Read Oxford Nanopore Sequencing of the Proviral Genome [J]. Pathogens, 2021, 10(9).
[134]MILLER D E, SULOVARI A, WANG T, et al. Targeted long-read sequencing identifies missing disease-causing variation [J]. Am J Hum Genet, 2021, 108(8): 1436-1449.
[135]PROPS R, KERCKHOF F M, RUBBENS P, et al. Absolute quantification of microbial taxon abundances [J]. ISME J, 2017, 11(2): 584-587.
[136]HU Y, FANG L, CHEN X, et al. LIQA: long-read isoform quantification and analysis [J]. Genome Biol, 2021, 22(1): 182.
[137]SCHRINNER S D, MARI R S, EBLER J, et al. Haplotype threading: accurate polyploid phasing from long reads [J]. Genome Biol, 2020, 21(1): 252.
[138]NYGAARD A B, TUNSJO H S, MEISAL R, et al. A preliminary study on the potential of Nanopore MinION and Illumina MiSeq 16S rRNA gene sequencing to characterize building-dust microbiomes [J]. Scientific Reports, 2020, 10(1).
[139]GONCALVES A T, COLLIPAL-MATAMAL R, VALENZUELA-MUNOZ V, et al. Nanopore sequencing of microbial communities reveals the potential role of sea lice as a reservoir for fish pathogens [J]. Scientific Reports, 2020, 10(1).
[140]THOMAS T, GILBERT J, MEYER F. Metagenomics - a guide from sampling to data analysis [J]. Microb Inform Exp, 2012, 2(1): 3.
[141]WILSON M R, NACCACHE S N, SAMAYOA E, et al. Actionable Diagnosis of Neuroleptospirosis by Next-Generation Sequencing [J]. New England Journal of Medicine, 2014, 370(25): 2408-2417.
[142]ASAI N, SUEMATSU H, HAGIHARA M, et al. The etiology and bacteriology of healthcare-associated empyema are quite different from those of community-acquired empyema [J]. Journal of Infection and Chemotherapy, 2017, 23(10): 661-667.
[143]LASKEN R S, MCLEAN J S. Recent advances in genomic DNA sequencing of microbial species from single cells [J]. Nature Reviews Genetics, 2014, 15(9): 577-584.
[144]FRANCHETTI L, SCHUMANN D M, TAMM M, et al. Multiplex bacterial polymerase chain reaction in a cohort of patients with pleural effusion [J]. BMC Infectious Diseases, 2020, 20(1).
[145]BLASCHKE A J, HEYREND C, BYINGTON C L, et al. Molecular analysis improves pathogen identification and epidemiologic study of pediatric parapneumonic empyema [J]. Pediatric Infectious Disease Journal, 2011, 30(4): 289-294.
[146]DYRHOVDEN R, NYGAARD R M, PATEL R, et al. The bacterial aetiology of pleural empyema. A descriptive and comparative metagenomic study [J]. Clinical Microbiology and Infection, 2019, 25(8): 981-986.
[147]BROOKS J P, EDWARDS D J, HARWICH M D, JR., et al. The truth about metagenomics: Quantifying and counteracting bias in 16S rRNA studies Ecological and evolutionary microbiology [J]. BMC Microbiology, 2015, 15(1).
[148]COTE I, ANDERSEN M E, ANKLEY G T, et al. The next generation of risk assessment multi-year study–highlights of findings, applications to risk assessment, and future directions [J]. Environmental Health Perspectives, 2016, 124(11): 1671-1682.
[149]LIGHT R W. Parapneumonic effusions and empyema [J]. Proceedings of the American Thoracic Society, 2006, 3(1): 75-80.
[150]BOLGER A M, LOHSE M, USADEL B. Trimmomatic: A flexible trimmer for Illumina sequence data [J]. Bioinformatics, 2014, 30(15): 2114-2120.
[151]LANGMEAD B, SALZBERG S L. Fast gapped-read alignment with Bowtie 2 [J]. Nature Methods, 2012, 9(4): 357-359.
[152]TRUONG D T, FRANZOSA E A, TICKLE T L, et al. MetaPhlAn2 for enhanced metagenomic taxonomic profiling [J]. Nat Methods, 2015, 12(10): 902-903.
[153]LI D, LUO R, LIU C M, et al. MEGAHIT v1.0: A fast and scalable metagenome assembler driven by advanced methodologies and community practices [J]. Methods, 2016, 102: 3-11.
[154]SEEMANN T. Prokka: Rapid prokaryotic genome annotation [J]. Bioinformatics, 2014, 30(14): 2068-2069.
[155]FU L, NIU B, ZHU Z, et al. CD-HIT: Accelerated for clustering the next-generation sequencing data [J]. Bioinformatics, 2012, 28(23): 3150-3152.
[156]PATRO R, DUGGAL G, LOVE M I, et al. Salmon provides fast and bias-aware quantification of transcript expression [J]. Nature Methods, 2017, 14(4): 417-419.
[157]ALCOCK B P, RAPHENYA A R, LAU T T Y, et al. CARD 2020: Antibiotic resistome surveillance with the comprehensive antibiotic resistance database [J]. Nucleic Acids Research, 2020, 48(D1): D517-D525.
[158]SEGATA N, IZARD J, WALDRON L, et al. Metagenomic biomarker discovery and explanation [J]. Genome Biology, 2011, 12(6).
[159]FORSBERG K J, PATEL S, GIBSON M K, et al. Bacterial phylogeny structures soil resistomes across habitats [J]. Nature, 2014, 509(7502): 612-616.
[160]HASSAN M, CARGILL T, HARRISS E, et al. The microbiology of pleural infection in adults: A systematic review [J]. European Respiratory Journal, 2019, 54(3).
[161]POPOWICZ N D, LANSLEY S M, CHEAH H M, et al. Human pleural fluid is a potent growth medium for Streptococcus pneumoniae [J]. PLoS One, 2017, 12(11).
[162]THOMAS R, RAHMAN N M, MASKELL N A, et al. Pleural effusions and pneumothorax: Beyond simple plumbing [J]. Respirology, 2020, 25(9): 963-971.
[163]ZHANG L, LI J, LIANG J, et al. The effect of Cyclic-di-GMP on biofilm formation by Pseudomonas aeruginosa in a novel empyema model [J]. Ann Trans Med, 2020, 8(18): 1146.
[164]BOASE S, VALENTINE R, SINGHAL D, et al. A sheep model to investigate the role of fungal biofilms in sinusitis: Fungal and bacterial synergy [J]. International Forum of Allergy and Rhinology, 2011, 1(5): 340-347.
[165]RAMAGE G, MOWAT E, JONES B, et al. Our Current Understanding of Fungal Biofilms Fungal biofilms Gordon Ramage et al [J]. Critical Reviews in Microbiology, 2009, 35(4): 340-355.
[166]DE RUDDER C, CALATAYUD ARROYO M, LEBEER S, et al. Modelling upper respiratory tract diseases: getting grips on host-microbe interactions in chronic rhinosinusitis using in vitro technologies [J]. Microbiome, 2018, 6(1): 75.
[167]KOBASHI Y, MOURI K, YAGI S, et al. Clinical analysis of cases of empyema due to Streptococcus milleri group [J]. Japanese Journal of Infectious Diseases, 2008, 61(6): 484-486.
[168]MENZIES S M, RAHMAN N M, WRIGHTSON J M, et al. Blood culture bottle culture of pleural fluid in pleural infection [J]. Thorax, 2011, 66(8): 658-662.
[169]BRIMS F, POPOWICZ N, ROSENSTENGEL A, et al. Bacteriology and clinical outcomes of patients with culture-positive pleural infection in Western Australia: A 6-year analysis [J]. Respirology, 2019, 24(2): 171-178.
[170]KOMA Y, INOUE S, ODA N, et al. Clinical characteristics and outcomes of patients with community-acquired, health-care-associated and hospital-acquired empyema [J]. Clinical Respiratory Journal, 2017, 11(6): 781-788.
[171]LOWDER M, UNGE A, MARAHA N, et al. Effect of starvation and the viable-but￾nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506 [J]. Applied and Environmental Microbiology, 2000, 66(8): 3160-3165.
[172]POMMEPUY M, BUTIN M, DERRIEN A, et al. Retention of enteropathogenicity by viable but nonculturable Escherichia coli exposed to seawater and sunlight [J]. Applied and Environmental Microbiology, 1996, 62(12): 4621-4626.
[173]COBO F, CALATRAVA E, RODRÍGUEZ-GRANGER J, et al. A rare case of pleural effusion due to Prevotella dentalis [J]. Anaerobe, 2018, 54: 144-145.
[174]SEVILLE L A, PATTERSON A J, SCOTT K P, et al. Distribution of tetracycline and erythromycin resistance genes among human oral and fecal metagenomic DNA [J]. Microbial Drug Resistance, 2009, 15(3): 159-166.
[175]QUICK J, QUINLAN A R, LOMAN N J. A reference bacterial genome dataset generated on the MinION portable single-molecule nanopore sequencer [J]. Gigascience, 2014, 3: 22.
[176]LOMAN N J, QUINLAN A R. Poretools: a toolkit for analyzing nanopore sequence data [J]. Bioinformatics, 2014, 30(23): 3399-3401.
[177]WHITTLE E, YONKUS J A, JERALDO P, et al. Optimizing Nanopore Sequencing for Rapid Detection of Microbial Species and Antimicrobial Resistance in Patients at Risk of Surgical Site Infections [J]. mSphere, 2022, 7(1): e0096421.
[178]FEEHERY G R, YIGIT E, OYOLA S O, et al. A method for selectively enriching microbial DNA from contaminating vertebrate host DNA [J]. PLoS One, 2013, 8(10): e76096.
[179]JIANG W, ZHAO X, GABRIELI T, et al. Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters [J]. Nat Commun, 2015, 6: 8101.
[180]GILPATRICK T, LEE I, GRAHAM J E, et al. Targeted nanopore sequencing with Cas9-guided adapter ligation [J]. Nat Biotechnol, 2020, 38(4): 433-438.
[181]PAYNE A, HOLMES N, CLARKE T, et al. Readfish enables targeted nanopore sequencing of gigabase-sized genomes [J]. Nat Biotechnol, 2021, 39(4): 442-450.
[182]LOOSE M, MALLA S, STOUT M. Real-time selective sequencing using nanopore technology [J]. Nat Methods, 2016, 13(9): 751-754.
[183]PATEL A, DOGAN H, PAYNE A, et al. Rapid-CNS2: rapid comprehensive adaptive nanopore-sequencing of CNS tumors, a proof-of-concept study [J]. Acta Neuropathologica, 2022, 143(5): 609-612.
[184]MARQUET M, ZOLLKAU J, PASTUSCHEK J, et al. Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore's adaptive sequencing [J]. Sci Rep, 2022, 12(1): 4000.
[185]PEREIRA-MARQUES J, HOUT A, FERREIRA R M, et al. Impact of Host DNA and Sequencing Depth on the Taxonomic Resolution of Whole Metagenome Sequencing for Microbiome Analysis [J]. Front Microbiol, 2019, 10: 1277.
[186]GWEON H, SCHONLAU M, STEINER S H. The k conditional nearest neighbor algorithm for classification and class probability estimation [J]. PeerJ Comput Sci, 2019, 5: e194.
[187]YAP A. "Call Me Your Clinical Reader-Witness" [J]. Perm J, 2020, 25: 1-3.
[188]HERAVI F S, ZAKRZEWSKI M, VICKERY K, et al. Metatranscriptomic Analysis Reveals Active Bacterial Communities in Diabetic Foot Infections [J]. Front Microbiol, 2020, 11: 1688.
[189]LI H. Minimap2: pairwise alignment for nucleotide sequences [J]. Bioinformatics, 2018, 34(18): 3094-3100.
[190]ETHERINGTON G J, RAMIREZ-GONZALEZ R H, MACLEAN D. bio-samtools 2: a package for analysis and visualization of sequence and alignment data with SAMtools in Ruby [J]. Bioinformatics, 2015, 31(15): 2565-2567.
[191]KIM D, SONG L, BREITWIESER F P, et al. Centrifuge: rapid and sensitive classification of metagenomic sequences [J]. Genome Res, 2016, 26(12): 1721-1729.
[192]SIMNER P J, MILLER S, CARROLL K C. Understanding the Promises and Hurdles of Metagenomic Next-Generation Sequencing as a Diagnostic Tool for Infectious Diseases [J]. Clin Infect Dis, 2018, 66(5): 778-788.
[193]DENG X, ACHARI A, FEDERMAN S, et al. Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance [J]. Nat Microbiol, 2020, 5(3): 443-454.
[194]BURNHAM P, KIM M S, AGBOR-ENOH S, et al. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma [J]. Sci Rep, 2016, 6: 27859.
[195]YATERA K, NOGUCHI S, MUKAE H. The microbiome in the lower respiratory tract [J]. Respir Investig, 2018, 56(6): 432-439.
[196]TROPINI C, EARLE K A, HUANG K C, et al. The Gut Microbiome: Connecting Spatial Organization to Function [J]. Cell Host Microbe, 2017, 21(4): 433-442.
[197]PEREZ-MUNOZ M E, ARRIETA M C, RAMER-TAIT A E, et al. A critical assessment of the "sterile womb" and "in utero colonization" hypotheses: implications for research on the pioneer infant microbiome [J]. Microbiome, 2017, 5.
[198]JOHNSON J S, SPAKOWICZ D J, HONG B Y, et al. Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis [J]. Nat Commun, 2019, 10(1): 5029.
[199]THOENDEL M, JERALDO P R, GREENWOOD-QUAINTANCE K E, et al. Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing [J]. J Microbiol Methods, 2016, 127: 141-145.
[200]KOVAKA S, FAN Y, NI B, et al. Targeted nanopore sequencing by real-time mapping of raw electrical signal with UNCALLED [J]. Nat Biotechnol, 2021, 39(4): 431-441.
[201]PAYNE A, HOLMES N, RAKYAN V, et al. BulkVis: a graphical viewer for Oxford nanopore bulk FAST5 files [J]. Bioinformatics, 2019, 35(13): 2193-2198.
[202]TAN T, LITTLE P, STOKES T, et al. Antibiotic prescribing for self limiting respiratory tract infections in primary care: summary of NICE guidance [J]. BMJ, 2008, 337: a437.
[203]TRIGODET F, LOLANS K, FOGARTY E, et al. High molecular weight DNA extraction strategies for long-read sequencing of complex metagenomes [J]. Mol Ecol Resour, 2022, 22(5): 1786-1802.
[204]SHAIBER A, WILLIS A D, DELMONT T O, et al. Functional and genetic markers of niche partitioning among enigmatic members of the human oral microbiome [J]. Genome Biology, 2020, 21(1).
[205]YUAN S, COHEN D B, RAVEL J, et al. Evaluation of methods for the extraction and purification of DNA from the human microbiome [J]. PLoS One, 2012, 7(3): e33865.
[206]BOLGER A M, LOHSE M, USADEL B. Trimmomatic: a flexible trimmer for Illumina sequence data [J]. Bioinformatics, 2014, 30(15): 2114-2120.
[207]LANGDON W B. Performance of genetic programming optimised Bowtie2 on genome comparison and analytic testing (GCAT) benchmarks [J]. BioData Min, 2015, 8(1): 1.
[208]YANG Y, LI B, JU F, et al. Exploring variation of antibiotic resistance genes in activated sludge over a four-year period through a metagenomic approach [J]. Environ Sci Technol, 2013, 47(18): 10197-10205.
[209]QUICK J, ASHTON P, CALUS S, et al. Rapid draft sequencing and real-time nanopore sequencing in a hospital outbreak of Salmonella [J]. Genome Biol, 2015, 16: 114.
[210]MA L, XIA Y, LI B, et al. Metagenomic Assembly Reveals Hosts of Antibiotic Resistance Genes and the Shared Resistome in Pig, Chicken, and Human Feces [J]. Environ Sci Technol, 2016, 50(1): 420-427.
[211]PEARMAN W S, FREED N E, SILANDER O K. Testing the advantages and disadvantages of short- and long- read eukaryotic metagenomics using simulated reads [J]. BMC Bioinformatics, 2020, 21(1): 220.
[212]MC I J. Emergency Pathology Service [J]. Lancet, 1946, 1(6401): 669.
[213]GORZYNSKI J E, GOENKA S D, SHAFIN K, et al. Ultrarapid Nanopore Genome Sequencing in a Critical Care Setting [J]. N Engl J Med, 2022, 386(7): 700-702.
[214]YONKUS J A, WHITTLE E, ALVA-RUIZ R, et al. "Answers in hours": A prospective clinical study using nanopore sequencing for bile duct cultures [J]. Surgery, 2022, 171(3): 693-702.
[215]NICHOLLS S M, QUICK J C, TANG S, et al. Ultra-deep, long-read nanopore sequencing of mock microbial community standards [J]. Gigascience, 2019, 8(5).
[216]HERAVI F S, ZAKRZEWSKI M, VICKERY K, et al. Host DNA depletion efficiency of microbiome DNA enrichment methods in infected tissue samples [J]. J Microbiol Methods, 2020, 170: 105856.
[217]AKACIN I, ERSOY S, DOLUCA O, et al. Comparing the significance of the utilization of next generation and third generation sequencing technologies in microbial metagenomics [J]. Microbiol Res, 2022, 264: 127154.
[218]YOUSAFZAI M T, KARIM S, QURESHI S, et al. Effectiveness of typhoid conjugate vaccine against culture-confirmed Salmonella enterica serotype Typhi in an extensively drug-resistant outbreak setting of Hyderabad, Pakistan: a cohort study [J]. Lancet Glob Health, 2021, 9(8): e1154-e1162.
[219]CRUMP J A, OO W T. Salmonella Typhi Vi polysaccharide conjugate vaccine protects infants and children against typhoid fever [J]. Lancet, 2021, 398(10301): 643-644.
[220]NAIR S, CHATTAWAY M, LANGRIDGE G C, et al. ESBL-producing strains isolated from imported cases of enteric fever in England and Wales reveal multiple chromosomal integrations of blaCTX-M-15 in XDR Salmonella Typhi [J]. J Antimicrob Chemother, 2021, 76(6): 1459-1466.
[221]BANKEVICH A, NURK S, ANTIPOV D, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing [J]. J Comput Biol, 2012, 19(5): 455-477.
[222]WICK R R, JUDD L M, GORRIE C L, et al. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads [J]. PLoS Comput Biol, 2017, 13(6): e1005595.
[223]WALKER B J, ABEEL T, SHEA T, et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement [J]. PLoS One, 2014, 9(11): e112963.
[224]LARSEN M V, COSENTINO S, RASMUSSEN S, et al. Multilocus sequence typing of total-genome-sequenced bacteria [J]. J Clin Microbiol, 2012, 50(4): 1355-1361.
[225]CARATTOLI A, ZANKARI E, GARCIA-FERNANDEZ A, et al. In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing [J]. Antimicrobial Agents and Chemotherapy, 2014, 58(7): 3895-3903.
[226]ALCOCK B P, RAPHENYA A R, LAU T T Y, et al. CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database [J]. Nucleic Acids Res, 2020, 48(D1): D517-D525.
[227]ALIKHAN N F, PETTY N K, BEN ZAKOUR N L, et al. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons [J]. BMC Genomics, 2011, 12.
[228]CAMACHO C, COULOURIS G, AVAGYAN V, et al. BLAST+: architecture and applications [J]. BMC Bioinformatics, 2009, 10: 421.
[229]KAAS R S, LEEKITCHAROENPHON P, AARESTRUP F M, et al. Solving the problem of comparing whole bacterial genomes across different sequencing platforms [J]. PLoS One, 2014, 9(8): e104984.
[230]LETUNIC I, BORK P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation [J]. Nucleic Acids Res, 2021, 49(W1): W293-W296.
[231]CENTERS FOR DISEASE C, PREVENTION. Notes from the field: Multistate outbreak of human Salmonella typhimurium infections linked to contact with pet hedgehogs - United States, 2011-2013 [J]. MMWR Morb Mortal Wkly Rep, 2013, 62(4): 73.
[232]NAIR S, ASHTON P, DOUMITH M, et al. WGS for surveillance of antimicrobial resistance: a pilot study to detect the prevalence and mechanism of resistance to azithromycin in a UK population of non-typhoidal Salmonella [J]. J Antimicrob Chemother, 2016, 71(12): 3400-3408.
[233]FACCONE D, LUCERO C, ALBORNOZ E, et al. Emergence of azithromycin resistance mediated by the mph(A) gene in Salmonella Typhimurium clinical isolates in Latin America [J]. J Glob Antimicrob Resist, 2018, 13: 237-239.
[234]WANG J, LI Y, XU X, et al. Antimicrobial Resistance of Salmonella enterica Serovar Typhimurium in Shanghai, China [J]. Front Microbiol, 2017, 8: 510.
[235]DONG N, LI Y, ZHAO J, et al. The phenotypic and molecular characteristics of antimicrobial resistance of Salmonella enterica subsp. enterica serovar Typhimurium in Henan Province, China [J]. BMC Infect Dis, 2020, 20(1): 511.
[236]HONG Y P, WANG Y W, HUANG I H, et al. Genetic Relationships among Multidrug￾Resistant Salmonella enterica Serovar Typhimurium Strains from Humans and Animals [J]. Antimicrobial Agents and Chemotherapy, 2018, 62(5).
[237]DARTON T C, TUYEN H T, THE H C, et al. Azithromycin Resistance in Shigella spp. in Southeast Asia [J]. Antimicrobial Agents and Chemotherapy, 2018, 62(4).
[238]NGUYEN M C P, WOERTHER P L, BOUVET M, et al. Escherichia coli as Reservoir for Macrolide Resistance Genes [J]. Emerging Infectious Diseases, 2009, 15(10): 1648-1650.
[239]YU G C, WANG L G, HAN Y Y, et al. clusterProfiler: an R Package for Comparing Biological Themes Among Gene Clusters [J]. Omics-a Journal of Integrative Biology, 2012, 16(5): 284-287.
[240]WANG Y, TONG M K, CHOW K H, et al. Occurrence of Highly Conjugative IncX3 Epidemic Plasmid Carrying bla NDM in Enterobacteriaceae Isolates in Geographically Widespread Areas [J]. Front Microbiol, 2018, 9: 2272.
[241]WANG Y, TIAN G B, ZHANG R, et al. Prevalence, risk factors, outcomes, and molecular epidemiology of mcr-1-positive Enterobacteriaceae in patients and healthy adults from China: an epidemiological and clinical study [J]. Lancet Infect Dis, 2017, 17(4): 390-399.
[242]BUSTAMANTE P, IREDELL J R. Carriage of type II toxin-antitoxin systems by the growing group of IncX plasmids [J]. Plasmid, 2017, 91: 19-27.