Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island

Abstract

Short-read, high-throughput sequencing technology cannot identify the chromosomal position of repetitive insertion sequences that typically flank horizontally acquired genes such as bacterial virulence genes and antibiotic resistance genes. The MinION nanopore sequencer can produce long sequencing reads on a device similar in size to a USB memory stick. Here we apply a MinION sequencer to resolve the structure and chromosomal insertion site of a composite antibiotic resistance island in Salmonella Typhi Haplotype 58. Nanopore sequencing data from a single 18-h run was used to create a scaffold for an assembly generated from short-read Illumina data. Our results demonstrate the potential of the MinION device in clinical laboratories to fully characterize the epidemic spread of bacterial pathogens.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1
Figure 2: Genetic organization of the S. Typhi chromosomal resistance island.
Figure 3: Comparison of the S. Typhi chromosomal resistance island with two closely related plasmids.

Accession codes

Primary accessions

European Nucleotide Archive

NCBI Reference Sequence

References

  1. Underwood, A.P. et al. Public health value of next-generation DNA sequencing of enterohemorrhagic Escherichia coli isolates from an outbreak. J. Clin. Microbiol. 51, 232–237 (2013).

    Article  Google Scholar 

  2. Wain, J. & Mavrogiorgou, E. Next-generation sequencing in clinical microbiology. Expert Rev. Mol. Diagn. 13, 225–227 (2013).

    Article  CAS  Google Scholar 

  3. Thomson, N. et al. The role of prophage-like elements in the diversity of Salmonella enterica serovars. J. Mol. Biol. 339, 279–300 (2004).

    Article  CAS  Google Scholar 

  4. Livermore, D.M. & Wain, J. Revolutionising bacteriology to improve treatment outcomes and antibiotic stewardship. Infect Chemother. 45, 1–10 (2013).

    Article  CAS  Google Scholar 

  5. Clarke, J. et al. Continuous base identification for single-molecule nanopore DNA sequencing. Nat. Nanotechnol. 4, 265–270 (2009).

    Article  CAS  Google Scholar 

  6. Buckle, G.C., Walker, C.L. & Black, R.E. Typhoid fever and paratyphoid fever: Systematic review to estimate global morbidity and mortality for 2010. J. Glob. Health 2, 010401 (2012).

    Article  Google Scholar 

  7. Wain, J., Hendriksen, R., Mikoleit, M., Keddy, K. & Ochiai, R. Typhoid fever. Lancet 10.1016/S0140-6736(13)62708-7 (21 October 2014).

  8. Roumagnac, P. et al. Evolutionary history of Salmonella typhi. Science 314, 1301–1304 (2006).

    Article  CAS  Google Scholar 

  9. Kariuki, S. et al. Typhoid in Kenya is associated with a dominant multidrug-resistant Salmonella enterica serovar Typhi haplotype that is also widespread in Southeast Asia. J. Clin. Microbiol. 48, 2171–2176 (2010).

    Article  CAS  Google Scholar 

  10. Holt, K.E. et al. Temporal fluctuation of multidrug resistant salmonella typhi haplotypes in the mekong river delta region of Vietnam. PLoS Negl. Trop. Dis. 5, e929 (2011).

    Article  Google Scholar 

  11. Holt, K.E. et al. High-resolution genotyping of the endemic Salmonella Typhi population during a Vi (typhoid) vaccination trial in Kolkata. PLoS Negl. Trop. Dis. 6, e1490 (2012).

    Article  Google Scholar 

  12. Holt, K.E. et al. High-throughput sequencing provides insights into genome variation and evolution in Salmonella Typhi. Nat. Genet. 40, 987–993 (2008).

    Article  CAS  Google Scholar 

  13. Holt, K.E. et al. Emergence of a globally dominant IncHI1 plasmid type associated with multiple drug resistant typhoid. PLoS Negl. Trop. Dis. 5, e1245 (2011).

    Article  Google Scholar 

  14. Le, T.A. et al. Clonal expansion and microevolution of quinolone-resistant Salmonella enterica serotype typhi in Vietnam from 1996 to 2004. J. Clin. Microbiol. 45, 3485–3492 (2007).

    Article  CAS  Google Scholar 

  15. Phan, M.D. et al. Variation in Salmonella enterica serovar typhi IncHI1 plasmids during the global spread of resistant typhoid fever. Antimicrob. Agents Chemother. 53, 716–727 (2009).

    Article  CAS  Google Scholar 

  16. Frith, M.C., Hamada, M. & Horton, P. Parameters for accurate genome alignment. BMC Bioinformatics 11, 80 (2010).

    Article  Google Scholar 

  17. Adey, A. et al. Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition. Genome Biol. 11, R119 (2010).

    Article  CAS  Google Scholar 

  18. Bankevich, A. et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19, 455–477 (2012).

    Article  CAS  Google Scholar 

  19. Szczepanowski, R. et al. The 120 592 bp IncF plasmid pRSB107 isolated from a sewage-treatment plant encodes nine different antibiotic-resistance determinants, two iron-acquisition systems and other putative virulence-associated functions. Microbiology 151, 1095–1111 (2005).

    Article  CAS  Google Scholar 

  20. Watson, M. et al. poRe: an R package for the visualization and analysis of nanopore sequencing data. Bioinformatics doi:10.1093/bioinformatics/btu590 (29 August 2014).

  21. Loman, N.J. & Quinlan, A.R. Poretools: a toolkit for analyzing nanopore sequence data. Bioinformatics 30, 3399–3401 (2014).

    Article  CAS  Google Scholar 

  22. Quick, J., Quinlan, A.R. & Loman, N.J. A reference bacterial genome dataset generated on the MinION portable single-molecule nanopore sequencer. GigaScience 3, 22 (2014).

    Article  Google Scholar 

  23. Mikheyev, A.S. & Tin, M.M.Y. A first look at the Oxford Nanopore MinION sequencer. Mol. Ecol. Res. 14, 1097–1102 (2014).

    Article  CAS  Google Scholar 

  24. Kim, K.E. et al. Long-read, whole genome shotgun sequence data for five model organisms. Preprint at http://biorxiv.org/content/early/2014/10/23/008037 (2014).

  25. Chin, C.S. et al. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat. Methods 10, 563–569 (2013).

    Article  CAS  Google Scholar 

  26. Parkhill, J. et al. Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18. Nature 413, 848–852 (2001).

    Article  CAS  Google Scholar 

  27. Holt, K.E. et al. Pseudogene accumulation in the evolutionary histories of Salmonella enterica serovars Paratyphi A and Typhi. BMC Genomics 10, 36 (2009).

    Article  Google Scholar 

  28. Grimont, A. & Weill, F. Antigenic Formulae of the Salmonella Serovars 9th edn. (World Health Organization, Geneva, 2007).

  29. Callow, B. A new phage-typing scheme for Salmonella typhi-murium . J. Hyg. (Lond.) 57, 346–359 (1959).

    Article  CAS  Google Scholar 

  30. Cock, P.J. et al. Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics 25, 1422–1423 (2009).

    Article  CAS  Google Scholar 

  31. Ewing, B. & Green, P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 8, 175–185 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

M. Day (Salmonella Reference Unit, Public Health England) for antibiotic susceptibility tests. J.O'G. and J.W. were funded by the University of East Anglia. We would like to thank Oxford Nanopore Technologies Ltd. for including us in the MinION Access Programme. We would also like to thank L. Nederbragt for a thorough review and contributions toward the presentation of Figure 1.

Author information

Authors and Affiliations

Authors

Contributions

P.M.A., S.N., T.D., J.W. and J.O'G. conceived the study, performed the analysis and wrote the first draft of the manuscript. J.O'G. and S.M. performed the MinION sequencing. P.M.A. and T.D. performed the bioinformatics analysis. S.N. performed the PCR analysis and coordinated the Illumina sequencing. P.M.A., T.D., S.R., W.R., J.W. and J.O'G. analyzed the resistance island structure and insertion site and devised the figures. All authors contributed to editing and data analysis of the final manuscript.

Corresponding authors

Correspondence to Tim Dallman or Justin O'Grady.

Ethics declarations

Competing interests

J.O'G. is a participant of Oxford Nanopore's MinION Access Programme (MAP) and received the MinION device and flowcells used for this study free of charge.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1–3 (PDF 345 kb)

Supplementary Software (ZIP 9 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ashton, P., Nair, S., Dallman, T. et al. MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island. Nat Biotechnol 33, 296–300 (2015). https://doi.org/10.1038/nbt.3103

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.3103

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing