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Volume 6, Issue 3

nanoMLST: accurate multilocus sequence typing using Oxford Nanopore Technologies MinION with a dual-barcode approach to multiplex large numbers of samples Open Access

Abstract

Multilocus sequence typing (MLST) is one of the most commonly used methods for studying microbial lineage worldwide. However, the traditional MLST process using Sanger sequencing is time-consuming and expensive. We have designed a workflow that simultaneously sequenced seven full-length housekeeping genes of 96 meticillin-resistant isolates with dual-barcode multiplexing using just a single flow cell of an Oxford Nanopore Technologies MinION system, and then we performed bioinformatic analysis for strain typing. Fifty-one of the isolates comprising 34 sequence types had been characterized using Sanger sequencing. We demonstrate that the allele assignments obtained by our nanopore workflow (nanoMLST, available at https://github.com/jade-nhri/nanoMLST) were identical to those obtained by Sanger sequencing (359/359, with 100 % agreement rate). In addition, we estimate that our multiplex system is able to perform MLST for up to 1000 samples simultaneously; thus, providing a rapid and cost-effective solution for molecular typing.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): dual-barcode multiplexing , meticillin-resistant Staphylococcus aureus (MRSA) , MinION , molecular typing , multilocus sequence typing (MLST) and nanopore
Funding
This study was supported by the:
  • Ministry of Science and Technology, Taiwan (Award MOST 106-2923-B-400-001-MY3)
    • Principle Award Recipient: Yu-Chieh Liao
  • National Health Research Institutes (Award PH-107-PP-05)
    • Principle Award Recipient: Yu-Chieh Liao
  • National Health Research Institutes (Award IV-107-PP-07)
    • Principle Award Recipient: Feng-Jui Chen
© 2020 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution NonCommercial License.
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2020-02-17
2024-10-08
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References

  1. Lowy FD. Staphylococcus aureus infections. N Engl J Med 1998; 339:520–532 [View Article]
     [Google Scholar]
  2. Tong SYC, Davis JS, Eichenberger E, Holland TL, Fowler VG. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 2015; 28:603–661 [View Article]
     [Google Scholar]
  3. Tenover FC, Arbeit R, Archer G, Biddle J, Byrne S et al. Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureus . J Clin Microbiol 1994; 32:407–415 [View Article]
     [Google Scholar]
  4. Shopsin B, Gomez M, Montgomery SO, Smith DH, Waddington M et al. Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains. J Clin Microbiol 1999; 37:3556–3563 [View Article]
     [Google Scholar]
  5. Enright MC, Day NPJ, Davies CE, Peacock SJ, Spratt BG. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus . J Clin Microbiol 2000; 38:1008–1015 [View Article]
     [Google Scholar]
  6. Murchan S, Kaufmann ME, Deplano A, de Ryck R, Struelens M et al. Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains. J Clin Microbiol 2003; 41:1574–1585 [View Article]
     [Google Scholar]
  7. Lee GH, Pang S, Coombs GW. Misidentification of Staphylococcus aureus by the Cepheid Xpert MRSA/SA BC assay due to deletions in the spa gene. J Clin Microbiol 2018; 56:e00530-18 [View Article]
     [Google Scholar]
  8. Baum C, Haslinger-Loffler B, Westh H, Boye K, Peters G et al. Non-spa-typeable clinical Staphylococcus aureus strains are naturally occurring protein A mutants. J Clin Microbiol 2009; 47:3624–3629 [View Article]
     [Google Scholar]
  9. Hudson LO, Murphy CR, Spratt BG, Enright MC, Terpstra L et al. Differences in methicillin-resistant Staphylococcus aureus strains isolated from pediatric and adult patients from hospitals in a large county in California. J Clin Microbiol 2012; 50:573–579 [View Article]
     [Google Scholar]
  10. Maiden MCJ, Bygraves JA, Feil E, Morelli G, Russell JE et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 1998; 95:3140–3145 [View Article]
     [Google Scholar]
  11. Muñoz M, Camargo M, Ramírez JD. Estimating the intra-taxa diversity, population genetic structure, and evolutionary pathways of Cryptococcus neoformans and Cryptococcus gattii . Front Genet 2018; 9:148 [View Article]
     [Google Scholar]
  12. Jolley KA, Bray JE, Maiden MCJ. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res 2018; 3:124 [View Article]
     [Google Scholar]
  13. Boers SA, van der Reijden WA, Jansen R. High-throughput multilocus sequence typing: bringing molecular typing to the next level. PLoS One 2012; 7:e39630 [View Article]
     [Google Scholar]
  14. Singh P, Foley SL, Nayak R, Kwon YM. Multilocus sequence typing of Salmonella strains by high-throughput sequencing of selectively amplified target genes. J Microbiol Methods 2012; 88:127–133 [View Article]
     [Google Scholar]
  15. Takahashi H, Iwakawa A, Ohshima C, Kyoui D, Kumano S et al. A rapid typing method for Listeria monocytogenes based on high-throughput multilocus sequence typing (Hi-MLST). Int J Food Microbiol 2017; 243:84–89 [View Article]
     [Google Scholar]
  16. Zhang N, Wheeler D, Truglio M, Lazzarini C, Upritchard J et al. Multi-locus next-generation sequence typing of DNA extracted from pooled colonies detects multiple unrelated Candida albicans strains in a significant proportion of patient samples. Front Microbiol 2018; 9:1179 [View Article]
     [Google Scholar]
  17. Chen Y, Frazzitta AE, Litvintseva AP, Fang C, Mitchell TG et al. Next generation multilocus sequence typing (NGMLST) and the analytical software program MLSTEZ enable efficient, cost-effective, high-throughput, multilocus sequencing typing. Fungal Genet Biol 2015; 75:64–71 [View Article]
     [Google Scholar]
  18. Heather JM, Chain B. The sequence of sequencers: the history of sequencing DNA. Genomics 2016; 107:1–8 [View Article]
     [Google Scholar]
  19. Pérez-Losada M, Arenas M, Castro-Nallar E. Microbial sequence typing in the genomic era. Infect Genet Evol 2018; 63:346–359 [View Article]
     [Google Scholar]
  20. Cao MD, Ganesamoorthy D, Elliott AG, Zhang H, Cooper MA et al. Streaming algorithms for identification of pathogens and antibiotic resistance potential from real-time MinION(TM) sequencing. Gigascience 2016; 5:32 [View Article]
     [Google Scholar]
  21. Tarumoto N, Sakai J, Sujino K, Yamaguchi T, Ohta M et al. Use of the Oxford Nanopore MinION sequencer for MLST genotyping of vancomycin-resistant enterococci. J Hosp Infect 2017; 96:296–298 [View Article]
     [Google Scholar]
  22. Ardui S, Ameur A, Vermeesch JR, Hestand MS. Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics. Nucleic Acids Res 2018; 46:2159–2168 [View Article]
     [Google Scholar]
  23. Ho M, McDonald LC, Lauderdale TL, Yeh LL, Chen PC et al. Surveillance of antibiotic resistance in Taiwan, 1998. J Microbiol Immunol Infect 1999; 32:239–249
     [Google Scholar]
  24. Chen FJ, Huang IW, Wang CH, Chen PC, Wang HY et al. mecA-positive Staphylococcus aureus with low-level oxacillin MIC in Taiwan. J Clin Microbiol 2012; 50:1679–1683 [View Article]
     [Google Scholar]
  25. Corpet F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 1988; 16:10881–10890 [View Article]
     [Google Scholar]
  26. Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 2018; 34:3094–3100 [View Article]
     [Google Scholar]
  27. Vaser R, Sović I, Nagarajan N, Šikić M. Fast and accurate de novo genome assembly from long uncorrected reads. Genome Res 2017; 27:737–746 [View Article]
     [Google Scholar]
  28. Loman NJ, Quick J, Simpson JT. A complete bacterial genome assembled de novo using only nanopore sequencing data. Nat Methods 2015; 12:733–735 [View Article]
     [Google Scholar]
  29. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215:403–410 [View Article]
     [Google Scholar]
  30. Liao Y-C, Cheng H-W, Wu H-C, Kuo S-C, Lauderdale T-LY et al. Completing circular bacterial genomes with assembly complexity by using a sampling strategy from a single MinION run with barcoding. Front Microbiol 2019; 10:2068 [View Article]
     [Google Scholar]
  31. Urwin R, Maiden MCJ. Multi-locus sequence typing: a tool for global epidemiology. Trends Microbiol 2003; 11:479–487 [View Article]
     [Google Scholar]
  32. Srivathsan A, Baloğlu B, Wang W, Tan WX, Bertrand D et al. A MinION™-based pipeline for fast and cost-effective DNA barcoding. Mol Ecol Resour 2018; 18:1035–1049 [View Article]
     [Google Scholar]
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nanoMLST: accurate multilocus sequence typing using Oxford Nanopore Technologies MinION with a dual-barcode approach to multiplex large numbers of samples
M Gen 6, e000336 (2020); https://doi.org/10.1099/mgen.0.000336
/content/journal/mgen/10.1099/mgen.0.000336
/content/journal/mgen/10.1099/mgen.0.000336
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