Third-Generation Sequencing in the Clinical Laboratory: Exploring the Advantages and Challenges of Nanopore Sequencing

Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so far include high cost, the length of time to results, and the need for technical and bioinformatics expertise. However, the recent technological innovation of nanopore sequencing from Oxford Nanopore Technologies (ONT) has the potential to address these challenges. ABSTRACT Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so far include high cost, the length of time to results, and the need for technical and bioinformatics expertise. However, the recent technological innovation of nanopore sequencing from Oxford Nanopore Technologies (ONT) has the potential to address these challenges. ONT sequencing is an attractive platform for clinical laboratories to adopt due to its low cost, rapid turnaround time, and user-friendly bioinformatics pipelines. However, this method still faces the problem of base-calling accuracy compared to other platforms. This review highlights the general challenges of pathogen detection in clinical specimens by metagenomic sequencing, the advantages and disadvantages of the ONT platform, and how research to date supports the potential future use of nanopore sequencing in infectious disease diagnostics.

[1]  K. Ikebuchi,et al.  Rapid identification of pathogens from positive blood culture bottles with the MinION nanopore sequencer , 2018, Journal of medical microbiology.

[2]  Doug Stryke,et al.  Clinical Metagenomic Sequencing for Diagnosis of Meningitis and Encephalitis. , 2019, The New England journal of medicine.

[3]  Zamin Iqbal,et al.  Resolving plasmid structures in Enterobacteriaceae using the MinION nanopore sequencer: assessment of MinION and MinION/Illumina hybrid data assembly approaches , 2017, Microbial genomics.

[4]  Douglas J. Botkin,et al.  Nanopore DNA Sequencing and Genome Assembly on the International Space Station , 2016, bioRxiv.

[5]  S. Abbott,et al.  16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls , 2007, Journal of Clinical Microbiology.

[6]  P. Worning,et al.  Emergence of a vancomycin-variable Enterococcus faecium ST1421 strain containing a deletion in vanX , 2018, The Journal of antimicrobial chemotherapy.

[7]  J. Thaden,et al.  Epidemiology and Mechanisms of Resistance of Extensively Drug Resistant Gram-Negative Bacteria , 2019, Antibiotics.

[8]  Sunando Roy,et al.  Whole-genome Sequencing Provides Data for Stratifying Infection Prevention and Control Management of Nosocomial Influenza A , 2019, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[9]  G. Vercellotti,et al.  Commensal and Pathogenic Members of the Dental Calculus Microbiome of Badia Pozzeveri Individuals from the 11th to 19th Centuries , 2019, Genes.

[10]  Julian Parkhill,et al.  Early insights into the potential of the Oxford Nanopore MinION for the detection of antimicrobial resistance genes , 2015, The Journal of antimicrobial chemotherapy.

[11]  K. Chu,et al.  Campylobacter fetus meningitis confirmed by a 16S rRNA gene analysis using the MinION nanopore sequencer, South Korea, 2016 , 2017, Emerging Microbes & Infections.

[12]  Shujiro Okuda,et al.  Next generation sequencing‐based gene panel tests for the management of solid tumors , 2018, Cancer science.

[13]  Alexander Payne,et al.  BulkVis: a graphical viewer for Oxford nanopore bulk FAST5 files , 2018, Bioinform..

[14]  John R Tyson,et al.  MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome , 2018, Genome research.

[15]  Jeremy Swann,et al.  Real-time analysis of nanopore-based metagenomic sequencing from infected orthopaedic devices , 2018, BMC Genomics.

[16]  K. Vickery,et al.  Biofilm contamination of high‐touched surfaces in intensive care units: epidemiology and potential impacts , 2019, Letters in applied microbiology.

[17]  R. Kirkegaard,et al.  Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers and Nanopore sequencing , 2019, bioRxiv.

[18]  John L. Spouge,et al.  Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi , 2012, Proceedings of the National Academy of Sciences.

[19]  N. Verma,et al.  Genome Analysis of Shigella flexneri Serotype 3b Strain SFL1520 Reveals Significant Horizontal Gene Acquisitions Including a Multidrug Resistance Cassette , 2019, Genome biology and evolution.

[20]  Ruichao Li,et al.  Resolution of dynamic MDR structures among the plasmidome of Salmonella using MinION single-molecule, long-read sequencing , 2018, The Journal of antimicrobial chemotherapy.

[21]  Wei Gu,et al.  Clinical Metagenomic Next-Generation Sequencing for Pathogen Detection. , 2019, Annual Review of Pathology.

[22]  Mathias C. Walter,et al.  MinION as part of a biomedical rapidly deployable laboratory. , 2017, Journal of biotechnology.

[23]  S. Brisse,et al.  Complete Genome Sequences of Bordetella pertussis Clinical Isolate FR5810 and Reference Strain Tohama from Combined Oxford Nanopore and Illumina Sequencing , 2018, Microbiology Resource Announcements.

[24]  Julian Parkhill,et al.  Comparison of bacterial genome assembly software for MinION data and their applicability to medical microbiology , 2016, bioRxiv.

[25]  Alvin T. Liem,et al.  Bacterial and viral identification and differentiation by amplicon sequencing on the MinION nanopore sequencer , 2015, GigaScience.

[26]  J P Flandrois,et al.  16S rRNA sequencing in routine bacterial identification: a 30-month experiment. , 2006, Journal of microbiological methods.

[27]  Eddy J. Bautista,et al.  Community characteristics of the gut microbiomes of competitive cyclists , 2017, Microbiome.

[28]  Jacquelyn S. Meisel,et al.  Strain- and Species-Level Variation in the Microbiome of Diabetic Wounds Is Associated with Clinical Outcomes and Therapeutic Efficacy. , 2019, Cell host & microbe.

[29]  Timothy D. Read,et al.  Genome-Based Prediction of Bacterial Antibiotic Resistance , 2018, Journal of Clinical Microbiology.

[30]  Z. Zong,et al.  The co-transfer of plasmid-borne colistin-resistant genes mcr-1 and mcr-3.5, the carbapenemase gene blaNDM-5 and the 16S methylase gene rmtB from Escherichia coli , 2019, Scientific Reports.

[31]  T. Taniguchi,et al.  Whole Genome Sequencing of Influenza A and B Viruses With the MinION Sequencer in the Clinical Setting: A Pilot Study , 2018, Front. Microbiol..

[32]  Nobuaki Kono,et al.  Nanopore sequencing: Review of potential applications in functional genomics , 2019, Development, growth & differentiation.

[33]  G. Olsen,et al.  Critical Evaluation of Two Primers Commonly Used for Amplification of Bacterial 16S rRNA Genes , 2008, Applied and Environmental Microbiology.

[34]  Ryan R. Wick,et al.  Performance of neural network basecalling tools for Oxford Nanopore sequencing , 2019, Genome Biology.

[35]  P. Schreckenberger,et al.  The Role of 16S rRNA Gene Sequencing in Identification of Microorganisms Misidentified by Conventional Methods , 2005, Journal of Clinical Microbiology.

[36]  J. Chun,et al.  Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. , 2014, International Journal of Systematic and Evolutionary Microbiology.

[37]  G. Braus,et al.  One Juliet and four Romeos: VeA and its methyltransferases , 2015, Front. Microbiol..

[38]  Robert Schlaberg,et al.  Validation of Metagenomic Next-Generation Sequencing Tests for Universal Pathogen Detection. , 2017, Archives of pathology & laboratory medicine.

[39]  J. Gilbert,et al.  Introducing the Microbiome into Precision Medicine. , 2017, Trends in pharmacological sciences.

[40]  J. Ronholm,et al.  Metagenomics: The Next Culture-Independent Game Changer , 2017, Front. Microbiol..

[41]  D. Relman,et al.  Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation , 2010, Proceedings of the National Academy of Sciences.

[42]  Doug Stryke,et al.  Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis , 2015, Genome Medicine.

[43]  T. Dallman,et al.  MinION nanopore sequencing identifies the position and structure of bacterial antibiotic resistance determinants in a multidrug-resistant strain of enteroaggregative Escherichia coli , 2018, Microbial genomics.

[44]  Kai Zhou,et al.  Application of next generation sequencing in clinical microbiology and infection prevention. , 2017, Journal of biotechnology.

[45]  S. Koren,et al.  Nanopore sequencing and assembly of a human genome with ultra-long reads , 2017, bioRxiv.

[46]  Heike Sichtig,et al.  FDA-ARGOS is a database with public quality-controlled reference genomes for diagnostic use and regulatory science , 2019, Nature Communications.

[47]  John P. Dekker,et al.  Rapid Nanopore Sequencing of Plasmids and Resistance Gene Detection in Clinical Isolates , 2017, Journal of Clinical Microbiology.

[48]  W. Bolosky,et al.  Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid , 2018, bioRxiv.

[49]  S. Salzberg,et al.  First Draft Genome Sequence of the Pathogenic Fungus Lomentospora prolificans (Formerly Scedosporium prolificans) , 2017, G3: Genes, Genomes, Genetics.

[50]  Lyle G. Whyte,et al.  In Situ Field Sequencing and Life Detection in Remote (79°26′N) Canadian High Arctic Permafrost Ice Wedge Microbial Communities , 2017, Front. Microbiol..

[51]  G. Weinstock,et al.  Making the Leap from Research Laboratory to Clinic: Challenges and Opportunities for Next-Generation Sequencing in Infectious Disease Diagnostics , 2015, mBio.

[52]  Francesca Giordano,et al.  Oxford Nanopore MinION Sequencing and Genome Assembly , 2016, Genom. Proteom. Bioinform..

[53]  Richard M. Leggett,et al.  Rapid Diagnosis of Lower Respiratory Infection using Nanopore-based Clinical Metagenomics , 2018, bioRxiv.

[54]  Yun Sung Cho,et al.  Chromosome-scale assembly comparison of the Korean Reference Genome KOREF from PromethION and PacBio with Hi-C mapping information , 2019, bioRxiv.

[55]  Niklas Krumm,et al.  One Codex: A Sensitive and Accurate Data Platform for Genomic Microbial Identification , 2015, bioRxiv.

[56]  D. Osmon.  Microbiology and Antimicrobial Challenges of Prosthetic Joint Infection. , 2017, The Journal of the American Academy of Orthopaedic Surgeons.

[57]  Niaz Banaei,et al.  Next-Generation Sequencing for Infectious Disease Diagnosis and Management: A Report of the Association for Molecular Pathology. , 2015, The Journal of molecular diagnostics : JMD.

[58]  Huang-Ming Hu,et al.  Identification of pathogens in culture-negative infective endocarditis cases by metagenomic analysis , 2018, Annals of Clinical Microbiology and Antimicrobials.

[59]  G. Reifenberger,et al.  Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics , 2018, Virchows Archiv.

[60]  Pranita D. Tamma,et al.  Applying Rapid Whole-Genome Sequencing To Predict Phenotypic Antimicrobial Susceptibility Testing Results among Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates , 2018, Antimicrobial Agents and Chemotherapy.

[61]  S. Salzberg,et al.  Development and Optimization of Metagenomic Next-Generation Sequencing Methods for Cerebrospinal Fluid Diagnostics , 2018, Journal of Clinical Microbiology.

[62]  Hanlee P. Ji,et al.  Next-generation DNA sequencing , 2008, Nature Biotechnology.

[63]  M. P. Francino,et al.  Antibiotics and the Human Gut Microbiome: Dysbioses and Accumulation of Resistances , 2016, Front. Microbiol..

[64]  I. Karunasagar,et al.  Whole-genome-based analysis reveals multiclone Serratia marcescens outbreaks in a non-Neonatal Intensive Care Unit setting in a tertiary care hospital in India. , 2019, Journal of medical microbiology.

[65]  Savitri Sharma,et al.  New insights into culture negative endophthalmitis by unbiased next generation sequencing , 2019, Scientific Reports.

[66]  David A. Matthews,et al.  Real-time, portable genome sequencing for Ebola surveillance , 2016, Nature.

[67]  S. O’Brien,et al.  SmileFinder: a resampling-based approach to evaluate signatures of selection from genome-wide sets of matching allele frequency data in two or more diploid populations , 2015, GigaScience.

[68]  Ryan R. Wick,et al.  Completing bacterial genome assemblies with multiplex MinION sequencing , 2017, bioRxiv.

[69]  Tiago F Jesus,et al.  Critical steps in clinical shotgun metagenomics for the concomitant detection and typing of microbial pathogens , 2018, Scientific Reports.

[70]  T Laver,et al.  Assessing the performance of the Oxford Nanopore Technologies MinION , 2015, Biomolecular detection and quantification.

[71]  D. Relman.  The Human Microbiome and the Future Practice of Medicine. , 2015, JAMA.