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Full-Length 16S rRNA Gene Analysis Using Long-Read Nanopore Sequencing for Rapid Identification of Bacteria from Clinical Specimens

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Nanopore Sequencing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2632))

Abstract

Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene is a practical and reliable measure for taxonomic profiling of bacterial communities. This chapter describes the detailed workflow for full-length 16S rRNA gene amplicon analysis using nanopore sequencing and bioinformatics pipelines to analyze nanopore sequencing data for taxonomic assignment. This approach offers a higher taxonomic resolution for bacterial identification from clinical specimens with a markedly reduced timeframe and improved versatility.

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Data Availability

The exact pipeline commands, sequence files, and other supplementary materials are available at figshare (https://doi.org/10.6084/m9.figshare.20367651).

References

  1. Chiu CY, Miller SA (2019) Clinical metagenomics. Nat Rev Genet 20(6):341–355. https://doi.org/10.1038/s41576-019-0113-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Loman NJ, Misra RV, Dallman TJ, Constantinidou C, Gharbia SE, Wain J, Pallen MJ (2012) Performance comparison of benchtop high-throughput sequencing platforms. Nat Biotechnol 30(5):434–439. https://doi.org/10.1038/nbt.2198

    Article  CAS  PubMed  Google Scholar 

  3. Didelot X, Bowden R, Wilson DJ, Peto TEA, Crook DW (2012) Transforming clinical microbiology with bacterial genome sequencing. Nat Rev Genet 13(9):601–612. https://doi.org/10.1038/nrg3226

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Langille MG, Zaneveld J, Caporaso JG, McDonald D, Knights D, Reyes JA, Clemente JC, Burkepile DE, Vega Thurber RL, Knight R, Beiko RG, Huttenhower C (2013) Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nat Biotechnol 31(9):814–821. https://doi.org/10.1038/nbt.2676

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Johnson JS, Spakowicz DJ, Hong BY, Petersen LM, Demkowicz P, Chen L, Leopold SR, Hanson BM, Agresta HO, Gerstein M, Sodergren E, Weinstock GM (2019) Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis. Nat Commun 10(1):5029. https://doi.org/10.1038/s41467-019-13036-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Clarridge JE 3rd (2004) Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin Microbiol Rev 17(4):840–862. https://doi.org/10.1128/CMR.17.4.840-862.2004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Srinivasan R, Karaoz U, Volegova M, MacKichan J, Kato-Maeda M, Miller S, Nadarajan R, Brodie EL, Lynch SV (2015) Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens. PLoS One 10(2):e0117617. https://doi.org/10.1371/journal.pone.0117617

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ravi RK, Walton K, Khosroheidari M (2018) MiSeq: a next generation sequencing platform for genomic analysis. Methods Mol Biol 1706:223–232. https://doi.org/10.1007/978-1-4939-7471-9_12

    Article  CAS  PubMed  Google Scholar 

  9. Kuczynski J, Lauber CL, Walters WA, Parfrey LW, Clemente JC, Gevers D, Knight R (2011) Experimental and analytical tools for studying the human microbiome. Nat Rev Genet 13(1):47–58. https://doi.org/10.1038/nrg3129

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Deamer D, Akeson M, Branton D (2016) Three decades of nanopore sequencing. Nat Biotechnol 34(5):518–524. https://doi.org/10.1038/nbt.3423

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kono N, Arakawa K (2019) Nanopore sequencing: review of potential applications in functional genomics. Develop Growth Differ 61(5):316–326. https://doi.org/10.1111/dgd.12608

    Article  Google Scholar 

  12. Mitsuhashi S, Kryukov K, Nakagawa S, Takeuchi JS, Shiraishi Y, Asano K, Imanishi T (2017) A portable system for rapid bacterial composition analysis using a nanopore-based sequencer and laptop computer. Sci Rep 7(1):5657. https://doi.org/10.1038/s41598-017-05772-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Nakagawa S, Inoue S, Kryukov K, Yamagishi J, Ohno A, Hayashida K, Nakazwe R, Kalumbi M, Mwenya D, Asami N, Sugimoto C, Mutengo MM, Imanishi T (2019) Rapid sequencing-based diagnosis of infectious bacterial species from meningitis patients in Zambia. Clin Transl Immunology 8(11):e01087. https://doi.org/10.1002/cti2.1087

    Article  PubMed  PubMed Central  Google Scholar 

  14. Tanaka H, Matsuo Y, Nakagawa S, Nishi K, Okamoto A, Kai S, Iwai T, Tabata Y, Tajima T, Komatsu Y, Satoh M, Kryukov K, Imanishi T, Hirota K (2019) Real-time diagnostic analysis of MinION-based metagenomic sequencing in clinical microbiology evaluation: a case report. JA Clin Rep 5(1):24. https://doi.org/10.1186/s40981-019-0244-z

    Article  PubMed  PubMed Central  Google Scholar 

  15. Komiya S, Matsuo Y, Nakagawa S, Morimoto Y, Kryukov K, Okada H, Hirota K (2022) MinION, a portable long-read sequencer, enables rapid vaginal microbiota analysis in a clinical setting. BMC Med Genet 15(1):68. https://doi.org/10.1186/s12920-022-01218-8

    Article  CAS  Google Scholar 

  16. Ishino M, Omi M, Araki-Sasaki K, Oba S, Yamada H, Matsuo Y, Hirota K, Takahashi K (2022) Successful identification of Granulicatella adiacens in postoperative acute infectious endophthalmitis using a bacterial 16S ribosomal RNA gene-sequencing platform with MinIONâ„¢: a case report. Am J Ophthalmol Case Rep 26:101524. https://doi.org/10.1016/j.ajoc.2022.101524

    Article  PubMed  PubMed Central  Google Scholar 

  17. Omi M, Matsuo Y, Araki-Sasaki K, Oba S, Yamada H, Hirota K, Takahashi K (2022) 16S rRNA nanopore sequencing for the diagnosis of ocular infection: a feasibility study. BMJ Open Ophthalmol 7:e000910. https://doi.org/10.1136/bmjophth-2021-000910

    Article  PubMed  PubMed Central  Google Scholar 

  18. Kai S, Matsuo Y, Nakagawa S, Kryukov K, Matsukawa S, Tanaka H, Iwai T, Imanishi T, Hirota K (2019) Rapid bacterial identification by direct PCR amplification of 16S rRNA genes using the MinION nanopore sequencer. FEBS Open Bio 9(3):548–557. https://doi.org/10.1002/2211-5463.12590

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Matsuo Y, Komiya S, Yasumizu Y, Yasuoka Y, Mizushima K, Takagi T, Kryukov K, Fukuda A, Morimoto Y, Naito Y, Okada H, Bono H, Nakagawa S, Hirota K (2021) Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION nanopore sequencing confers species-level resolution. BMC Microbiol 21(1):35. https://doi.org/10.1186/s12866-021-02094-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Karst SM, Ziels RM, Kirkegaard RH, Sorensen EA, McDonald D, Zhu Q, Knight R, Albertsen M (2021) High-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing. Nat Methods 18(2):165–169. https://doi.org/10.1038/s41592-020-01041-y

    Article  CAS  PubMed  Google Scholar 

  21. Santos A, van Aerle R, Barrientos L, Martinez-Urtaza J (2020) Computational methods for 16S metabarcoding studies using Nanopore sequencing data. Comput Struct Biotechnol J 18:296–305. https://doi.org/10.1016/j.csbj.2020.01.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. EPI2ME workflows (registration required). https://epi2me.nanoporetech.com/workflows

  23. Zhang Z, Schwartz S, Wagner L, Miller W (2000) A greedy algorithm for aligning DNA sequences. J Comput Biol 7(1–2):203–214. https://doi.org/10.1089/10665270050081478

    Article  CAS  PubMed  Google Scholar 

  24. 16S ribosomal RNA (Bacteria and Archaea type strains) [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information (NCBI); Available from: https://www.ncbi.nlm.nih.gov/refseq/targetedloci/

  25. SeqKit. https://bioinf.shenwei.me/seqkit/

  26. Shen W, Le S, Li Y, Hu F (2016) SeqKit: a cross-platform and ultrafast toolkit for FASTA/Q file manipulation. PLoS One 11(10):e0163962. https://doi.org/10.1371/journal.pone.0163962

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. EPI2ME Desktop Agent (registration required). https://epi2me.nanoporetech.com/software

  28. Conda. https://docs.conda.io/en/latest/

  29. VSEARCH. https://github.com/torognes/vsearch

  30. Rognes T, Flouri T, Nichols B, Quince C, Mahe F (2016) VSEARCH: a versatile open source tool for metagenomics. PeerJ 4:e2584. https://doi.org/10.7717/peerj.2584

    Article  PubMed  PubMed Central  Google Scholar 

  31. Medaka. https://github.com/nanoporetech/medaka

  32. Tabata Y, Matsuo Y, Fujii Y, Ohta A, Hirota K (2022) Rapid detection of single nucleotide polymorphisms using the MinION nanopore sequencer: a feasibility study for perioperative precision medicine. JA Clin Rep 8(1):17. https://doi.org/10.1186/s40981-022-00506-7

    Article  PubMed  PubMed Central  Google Scholar 

  33. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glockner FO (2013) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41(Database issue):D590–D596. https://doi.org/10.1093/nar/gks1219

    Article  CAS  PubMed  Google Scholar 

  34. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75(23):7537–7541. https://doi.org/10.1128/AEM.01541-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Silva reference files. https://mothur.org/wiki/silva_reference_files/

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Authors and Affiliations

  1. Department of Human Stress Response Science, Institute of Biomedical Science, Kansai Medical University, Osaka, Japan

    Yoshiyuki Matsuo

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  1. Yoshiyuki Matsuo
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Correspondence to Yoshiyuki Matsuo .

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Editors and Affiliations

  1. Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan

    Kazuharu Arakawa

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Matsuo, Y. (2023). Full-Length 16S rRNA Gene Analysis Using Long-Read Nanopore Sequencing for Rapid Identification of Bacteria from Clinical Specimens. In: Arakawa, K. (eds) Nanopore Sequencing. Methods in Molecular Biology, vol 2632. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2996-3_14

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  • DOI: https://doi.org/10.1007/978-1-0716-2996-3_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2995-6

  • Online ISBN: 978-1-0716-2996-3

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