1887

Abstract

Illumina sequencing platforms have enabled widespread bacterial whole genome sequencing. While Illumina data is appropriate for many analyses, its short read length limits its ability to resolve genomic structure. This has major implications for tracking the spread of mobile genetic elements, including those which carry antimicrobial resistance determinants. Fully resolving a bacterial genome requires long-read sequencing such as those generated by Oxford Nanopore Technologies (ONT) platforms. Here we describe our use of the ONT MinION to sequence 12 isolates of Klebsiella pneumoniae on a single flow cell. We assembled each genome using a combination of ONT reads and previously available Illumina reads, and little to no manual intervention was needed to achieve fully resolved assemblies using the Unicycler hybrid assembler. Assembling only ONT reads with Canu was less effective, resulting in fewer resolved genomes and higher error rates even following error correction with Nanopolish. We demonstrate that multiplexed ONT sequencing is a valuable tool for high-throughput bacterial genome finishing. Specifically, we advocate the use of Illumina sequencing as a first analysis step, followed by ONT reads as needed to resolve genomic structure.

Loading

Article metrics loading...

/content/journal/mgen/10.1099/mgen.0.000132
2017-09-14
2019-08-20
Loading full text...

Full text loading...

/deliver/fulltext/mgen/3/10/mgen000132.html?itemId=/content/journal/mgen/10.1099/mgen.0.000132&mimeType=html&fmt=ahah

References

  1. Holt KE, Wertheim H, Zadoks RN, Baker S, Whitehouse CA et al. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proc Natl Acad Sci USA 2015;112:E3574E3581 [CrossRef][PubMed]
    [Google Scholar]
  2. Inouye M, Dashnow H, Raven LA, Schultz MB, Pope BJ et al. SRST2: rapid genomic surveillance for public health and hospital microbiology labs. Genome Med 2014;6:90 [CrossRef][PubMed]
    [Google Scholar]
  3. Kwong JC, Mccallum N, Sintchenko V, Howden BP. Whole genome sequencing in clinical and public health microbiology. Pathology 2015;47:199–210 [CrossRef][PubMed]
    [Google Scholar]
  4. Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: quality assessment tool for genome assemblies. Bioinformatics 2013;29:1072–1075 [CrossRef][PubMed]
    [Google Scholar]
  5. He S, Hickman AB, Varani AM, Siguier P, Chandler M et al. Insertion sequence IS26 reorganizes plasmids in clinically isolated multidrug-resistant bacteria by replicative transposition. MBio 2015;6:e00762-15 [CrossRef][PubMed]
    [Google Scholar]
  6. Arredondo-Alonso S, van Schaik W, Willems RJ, Schurch AC. On the (im)possibility to reconstruct plasmids from whole genome short-read sequencing data. bioRxiv 2016;1–18
    [Google Scholar]
  7. Sheppard AE, Stoesser N, Wilson DJ, Sebra R, Kasarskis A et al. Nested Russian doll-like genetic mobility drives rapid dissemination of the carbapenem resistance gene blaKPC. Antimicrob Agents Chemother 2016;60:3767–3778 [CrossRef][PubMed]
    [Google Scholar]
  8. Wong VK, Baker S, Pickard DJ, Parkhill J, Page AJ et al. Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events. Nat Genet 2015;47:632–639 [CrossRef][PubMed]
    [Google Scholar]
  9. Ashton PM, Nair S, Dallman T, Rubino S, Rabsch W et al. MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island. Nat Biotechnol 2015;33:296–300 [CrossRef][PubMed]
    [Google Scholar]
  10. Conlan S, Thomas PJ, Deming C, Park M, Lau AF et al. Single-molecule sequencing to track plasmid diversity of hospital-associated carbapenemase-producing Enterobacteriaceae. Sci Transl Med 2014;6:254ra126 [CrossRef][PubMed]
    [Google Scholar]
  11. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res 2017;27:722–736 [CrossRef][PubMed]
    [Google Scholar]
  12. Loman NJ, Quick J, Simpson JT. A complete bacterial genome assembled de novo using only nanopore sequencing data. Nat Methods 2015;12:733–735 [CrossRef][PubMed]
    [Google Scholar]
  13. Risse J, Thomson M, Patrick S, Blakely G, Koutsovoulos G et al. A single chromosome assembly of Bacteroides fragilis strain BE1 from Illumina and MinION nanopore sequencing data. Gigascience 2015;4:60 [CrossRef][PubMed]
    [Google Scholar]
  14. Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 2017;13:e1005595 [CrossRef][PubMed]
    [Google Scholar]
  15. Gorrie CL, Mirceta M, Wick RR, Edwards DJ, Thomson NR et al. Gastrointestinal carriage is a major reservoir of K. pneumoniae infection in intensive care patients. Clin Infect Dis 2017;208–215 [CrossRef][PubMed]
    [Google Scholar]
  16. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 2014;9:e112963 [CrossRef][PubMed]
    [Google Scholar]
  17. Li H. Minimap and miniasm: fast mapping and de novo assembly for noisy long sequences. arXiv 2015;32:1–7 [CrossRef][PubMed]
    [Google Scholar]
  18. Wick RR, Schultz MB, Zobel J, Holt KE. Bandage: interactive visualization of de novo genome assemblies. Bioinformatics 2015;31:3350–3352 [CrossRef][PubMed]
    [Google Scholar]
  19. Simpson JT, Wong K, Jackman SD, Schein JE, Jones SJ et al. ABySS: a parallel assembler for short read sequence data. Genome Res 2009;19:1117–1123 [CrossRef][PubMed]
    [Google Scholar]
  20. Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 2008;18:821–829 [CrossRef][PubMed]
    [Google Scholar]
  21. Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M et al. Versatile and open software for comparing large genomes. Genome Biol 2004;5:R12 [CrossRef][PubMed]
    [Google Scholar]
  22. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J et al. BLAST+: architecture and applications. BMC Bioinformatics 2009;10:421 [CrossRef][PubMed]
    [Google Scholar]
  23. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods 2012;9:357–359 [CrossRef][PubMed]
    [Google Scholar]
  24. Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv 2013;3:1303.3997
    [Google Scholar]
  25. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J et al. The sequence alignment/map format and SAMtools. Bioinformatics 2009;25:2078–2079 [CrossRef][PubMed]
    [Google Scholar]
  26. George S, Pankhurst L, Hubbard A, Votintseva A, Stoesser N et al. Resolving plasmid structures in Enterobacteriaceae using the MinION nanopore sequencer: assessment of MinION and MinION/Illumina hybrid data assembly approaches. Microb Genom 2017;1–8 [CrossRef]
    [Google Scholar]
  27. White R, Pellefigues C, Ronchese F, Lamiable O, Eccles D. Investigation of chimeric reads using the MinION. F1000Res 2017;6:631 [CrossRef]
    [Google Scholar]
  28. Yang F, Yang J, Zhang X, Chen L, Jiang Y et al. Genome dynamics and diversity of Shigella species, the etiologic agents of bacillary dysentery. Nucleic Acids Res 2005;33:6445–6458 [CrossRef][PubMed]
    [Google Scholar]
  29. Goh HM, Beatson SA, Totsika M, Moriel DG, Phan MD et al. Molecular analysis of the Acinetobacter baumannii biofilm-associated protein. Appl Environ Microbiol 2013;79:6535–6543 [CrossRef][PubMed]
    [Google Scholar]
  30. Treangen TJ, Salzberg SL. Repetitive DNA and next-generation sequencing: computational challenges and solutions. Nat Rev Genet 2011;13:36–46 [CrossRef][PubMed]
    [Google Scholar]
  31. Rastogi R, Wu M, Dasgupta I, Fox GE. Visualization of ribosomal RNA operon copy number distribution. BMC Microbiol 2009;9:208 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/mgen/10.1099/mgen.0.000132
Loading
/content/journal/mgen/10.1099/mgen.0.000132
Loading

Data & Media loading...

Supplementary File 1

PDF

Supplementary File 2

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error