1887

Abstract

Oxford Nanopore Technologies (ONT) sequencing platforms currently offer two approaches to whole-genome native-DNA library preparation: ligation and rapid. In this study, we compared these two approaches for bacterial whole-genome sequencing, with a specific aim of assessing their ability to recover small plasmid sequences. To do so, we sequenced DNA from seven plasmid-rich bacterial isolates in three different ways: ONT ligation, ONT rapid and Illumina. Using the Illumina read depths to approximate true plasmid abundance, we found that small plasmids (<20 kbp) were underrepresented in ONT ligation read sets (by a mean factor of ~4) but were not underrepresented in ONT rapid read sets. This effect correlated with plasmid size, with the smallest plasmids being the most underrepresented in ONT ligation read sets. We also found lower rates of chimaeric reads in the rapid read sets relative to ligation read sets. These results show that when small plasmid recovery is important, ONT rapid library preparations are preferable to ligation-based protocols.

Funding
This study was supported by the:
  • Australian Government Research Training Program
    • Principle Award Recipient: RyanR Wick
  • Sylvia and Charles Viertel Charitable Foundation
    • Principle Award Recipient: KathrynE Holt
  • Bill and Melinda Gates Foundation (Award OPP1175797)
    • Principle Award Recipient: KathrynE Holt
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Loading

Article metrics loading...

/content/journal/mgen/10.1099/mgen.0.000631
2021-08-25
2021-12-01
Loading full text...

Full text loading...

/deliver/fulltext/mgen/7/8/mgen000631.html?itemId=/content/journal/mgen/10.1099/mgen.0.000631&mimeType=html&fmt=ahah

References

  1. Land M, Hauser L, Jun S-R, Nookaew I, Leuze MR et al. Insights from 20 years of bacterial genome sequencing. Funct Integr Genomics 2015; 15:141–161 [View Article] [PubMed]
    [Google Scholar]
  2. Bobay LM, Ochman H. The evolution of bacterial genome architecture. Front Genet 2017; 8:1–6 [View Article] [PubMed]
    [Google Scholar]
  3. Baker S, Hardy J, Sanderson KE, Quail M, Goodhead I et al. A novel linear plasmid mediates flagellar variation in Salmonella Typhi. PLoS Pathog 2007; 3:0605–0610 [View Article] [PubMed]
    [Google Scholar]
  4. Arredondo-Alonso S, Top J, McNally A, Puranen S, Pesonen M et al. Plasmids shaped the recent emergence of the major nosocomial pathogen Enterococcus faecium. mBio 2020; 11:1–17 [View Article] [PubMed]
    [Google Scholar]
  5. Ciok A, Dziewit L, Grzesiak J, Budzik K, Gorniak D et al. Identification of miniature plasmids in psychrophilic Arctic bacteria of the genus Variovorax. FEMS Microbiology Ecology 2016; 92:1–9 [View Article]
    [Google Scholar]
  6. Rozwandowicz M, Brouwer MSM, Fischer J, Wagenaar JA, Gonzalez-Zorn B et al. Plasmids carrying antimicrobial resistance genes in Enterobacteriaceae. J Antimicrob Chemother 2018; 73:1121–1137 [View Article]
    [Google Scholar]
  7. Shintani M, Sanchez ZK, Kimbara K. Genomics of microbial plasmids: Classification and identification based on replication and transfer systems and host taxonomy. Front Microbiol 2015; 6:1–16 [View Article] [PubMed]
    [Google Scholar]
  8. Smalla K, Jechalke S, Top EM. Plasmid detection, characterization, and ecology. Plasmids 2015; 3:445–458 [View Article]
    [Google Scholar]
  9. San Millan A, Escudero JA, Catalan A, Nieto S, Farelo F et al. β-Lactam resistance in Haemophilus parasuis is mediated by plasmid pB1000 bearing blaROB-1. Antimicrob Agents Chemother 2007; 51:2260–2264 [View Article] [PubMed]
    [Google Scholar]
  10. Anantham S, Hall RM. pCERC1, a Small, Globally Disseminated Plasmid Carrying the dfrA14 Cassette in the strA Gene of the sul2-strA-strB Gene Cluster. Microb Drug Resist 2012; 18:364–371 [View Article] [PubMed]
    [Google Scholar]
  11. Lanza VF, de Toro M, Garcillán-Barcia MP, Mora A, Blanco J et al. Plasmid Flux in Escherichia coli ST131 Sublineages, Analyzed by Plasmid Constellation Network (PLACNET), a New Method for Plasmid Reconstruction from Whole Genome Sequences. PLoS Genet 2014; 10:12 [View Article] [PubMed]
    [Google Scholar]
  12. 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 [View Article] [PubMed]
    [Google Scholar]
  13. Arredondo-Alonso S, Willems RJ, van Schaik W, Schürch AC. On the (im)possibility of reconstructing plasmids from whole-genome short-read sequencing data. Microb Genom 2017; 3:10 [View Article] [PubMed]
    [Google Scholar]
  14. 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 [View Article]
    [Google Scholar]
  15. Weingarten RA, Johnson RC, Conlan S, Ramsburg AM, Dekker JP et al. Genomic analysis of hospital plumbing reveals diverse reservoir of bacterial plasmids conferring carbapenem resistance. mBio 2018; 9:1–16 [View Article] [PubMed]
    [Google Scholar]
  16. 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] [PubMed]
    [Google Scholar]
  17. Koren S, Phillippy AM. One chromosome, one contig: Complete microbial genomes from long-read sequencing and assembly. Curr Opin Microbiol 2015; 23:110–120 [View Article] [PubMed]
    [Google Scholar]
  18. Wick RR, Judd LM, Gorrie CL, Holt KE. Completing bacterial genome assemblies with multiplex MinION sequencing. Microb Genom 2017; 3:1–7 [View Article] [PubMed]
    [Google Scholar]
  19. Taylor TL, Volkening JD, DeJesus E, Simmons M, Dimitrov KM et al. Rapid, multiplexed, whole genome and plasmid sequencing of foodborne pathogens using long-read nanopore technology. Sci Rep 2019; 9:1–11 [View Article] [PubMed]
    [Google Scholar]
  20. Elliott I, Batty EM, Ming D, Robinson MT, Nawtaisong P et al. Oxford nanopore MinION sequencing enables rapid whole genome assembly of rickettsia typhi in a resource-limited setting. Am J Trop Med Hyg 2020; 102:408–414 [View Article] [PubMed]
    [Google Scholar]
  21. Antipov D, Korobeynikov A, McLean JS, Pevzner PA. HybridSPAdes: An algorithm for hybrid assembly of short and long reads. Bioinformatics 2016; 32:1009–1015 [View Article] [PubMed]
    [Google Scholar]
  22. Koren S, Walenz BP, Berlin K, Miller JR, Phillippy AM. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res 2017; 27:722–736 [View Article] [PubMed]
    [Google Scholar]
  23. Kolmogorov M, Yuan J, Lin Y, Pevzner PA. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol 2019; 37:540–546 [View Article] [PubMed]
    [Google Scholar]
  24. Wick RR, Holt KE. Benchmarking of long-read assemblers for prokaryote whole genome sequencing. F1000Res 2019; 8:2138 [View Article] [PubMed]
    [Google Scholar]
  25. Lam MMC, Wick RR, Wyres KL, Gorrie CL, Judd LM et al. Genetic diversity, mobilisation and spread of the yersiniabactin-encoding mobile element ICEKp in Klebsiella pneumoniae populations. Microb Genom 2018; 4: [View Article] [PubMed]
    [Google Scholar]
  26. Lam MMC, Wyres KL, Judd LM, Wick RR, Jenney A et al. Tracking key virulence loci encoding aerobactin and salmochelin siderophore synthesis in Klebsiella pneumoniae. Genome Med 2018; 10:1–15 [View Article] [PubMed]
    [Google Scholar]
  27. Wyres KL, Hawkey J, Hetland MAK, Fostervold A, Wick RR et al. Emergence and rapid global dissemination of CTX-M-15-associated Klebsiella pneumoniae strain ST307. J Antimicrob Chemother 2019; 74:577–581 [View Article] [PubMed]
    [Google Scholar]
  28. Lam MMC, Wyres KL, Wick RR, Judd LM, Fostervold A et al. Convergence of virulence and MDR in a single plasmid vector in MDR Klebsiella pneumoniae ST15. J Antimicrob Chemother 2019; 74:1218–1222 [View Article] [PubMed]
    [Google Scholar]
  29. Wyres KL, Wick RR, Judd LM, Froumine R, Tokolyi A et al. Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae. PLoS Genet 2019; 15:1–25 [View Article] [PubMed]
    [Google Scholar]
  30. Wyres KL, Nguyen TNT, Lam MMC, Judd LM, van Vinh Chau N et al. Genomic surveillance for hypervirulence and multi-drug resistance in invasive Klebsiella pneumoniae from South and Southeast Asia. Genome Med 2020; 12:11 [View Article] [PubMed]
    [Google Scholar]
  31. Jorgensen JH, Ferraro MJ. Antimicrobial susceptibility testing: A review of general principles and contemporary practices. Clin Infect Dis 2009; 49:1749–1755 [View Article] [PubMed]
    [Google Scholar]
  32. Klingström T, Bongcam-Rudloff E, Pettersson OV. A comprehensive model of DNA fragmentation for the preservation of High Molecular Weight DNA. bioRxiv 2018 [View Article]
    [Google Scholar]
  33. Ou S, Liu J, Chougule KM, Fungtammasan A, Seetharam AS et al. Effect of sequence depth and length in long-read assembly of the maize inbred NC358. Nat Commun 2020; 11:1–10 [View Article] [PubMed]
    [Google Scholar]
  34. Hamidian M, Ambrose SJ, Blackwell GA, Nigro SJ, Hall RM. An outbreak of multiply antibiotic-resistant ST49:ST128:KL11:OCL8 Acinetobacter baumannii isolates at a Sydney hospital. J Antimicrob Chemother 2021; 76:893–900 [View Article]
    [Google Scholar]
  35. Watts SC, Judd LM, Carzino R, Ranganathan S, Holt KE. Genomic diversity and antimicrobial resistance of Haemophilus colonising the airways of young children with cystic fibrosis. bioRxiv 2020; 2020.11.23.388074: [View Article]
    [Google Scholar]
  36. Wyres KL, Hawkey J, Mirčeta M, Judd LM, Wick RR et al. Genomic surveillance of antimicrobial resistant bacterial colonisation and infection in intensive care patients. medRxiv 2020; 2020.11.03.20224881: [View Article]
    [Google Scholar]
  37. Wick RR, Judd LM, Holt KE. Performance of neural network basecalling tools for Oxford Nanopore sequencing. Genome Biol 2019; 20:129 [View Article] [PubMed]
    [Google Scholar]
  38. Chaumeil PA, Mussig AJ, Hugenholtz P, Parks DH. GTDB-Tk: A toolkit to classify genomes with the genome taxonomy database. Bioinformatics 2020; 36:1925–1927 [View Article] [PubMed]
    [Google Scholar]
  39. Parks DH, Chuvochina M, Chaumeil PA, Rinke C, Mussig AJ et al. A complete domain-to-species taxonomy for Bacteria and Archaea. Nat Biotechnol 2020; 38:1098 [View Article] [PubMed]
    [Google Scholar]
  40. Chen S, Zhou Y, Chen Y, Gu J. Fastp: An ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 2018; 34:i884–90 [View Article] [PubMed]
    [Google Scholar]
  41. Wick RR, Holt KE. Trycycler [Internet]. GitHub 2020
    [Google Scholar]
  42. Li H. Minimap and miniasm: Fast mapping and de novo assembly for noisy long sequences. Bioinformatics 2016; 32:2103–2110 [View Article] [PubMed]
    [Google Scholar]
  43. Vaser R, Š M, Šikić M. Raven: a de novo genome assembler for long reads. bioRxiv 2020 [View Article]
    [Google Scholar]
  44. Wright C, Wykes M. Medaka [Internet]. GitHub 2020
    [Google Scholar]
  45. 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; 9:11 [View Article] [PubMed]
    [Google Scholar]
  46. Šošić M, Šikić M. Edlib: A C/C ++ library for fast, exact sequence alignment using edit distance. Bioinformatics 2017; 33:1394–1395 [View Article] [PubMed]
    [Google Scholar]
  47. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES et al. Integrative genomics viewer. Nat Biotechnol 2011; 29:24–26 [View Article] [PubMed]
    [Google Scholar]
  48. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods 2012; 9:357–359 [View Article] [PubMed]
    [Google Scholar]
  49. Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 2018; 34:3094–3100 [View Article] [PubMed]
    [Google Scholar]
  50. Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M. ISfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Res 2006; 34:D32–6 [View Article] [PubMed]
    [Google Scholar]
  51. Alcock BP, Raphenya AR, Lau TTY, Tsang KK, Bouchard M et al. CARD 2020: Antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acids Res 2020; 48:D517–25 [View Article] [PubMed]
    [Google Scholar]
  52. Chen L, Yang J, Yu J, Yao Z, Sun L et al. VFDB: A reference database for bacterial virulence factors. Nucleic Acids Res 2005; 33:325–328 [View Article] [PubMed]
    [Google Scholar]
  53. Aird D, Ross MG, Chen WS, Danielsson M, Fennell T et al. Characterizing and measuring bias in sequence data. Genome Biol 2012; 02:1 [View Article]
    [Google Scholar]
  54. Jain M, Koren S, Quick J, Rand AC, Sasani TA et al. Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat Biotechnol 2018; 36:338–345 [View Article] [PubMed]
    [Google Scholar]
  55. Amarasinghe SL, Su S, Dong X, Zappia L, Ritchie ME et al. Opportunities and challenges in long-read sequencing data analysis. Genome Biol 2020; 21:1–16 [View Article] [PubMed]
    [Google Scholar]
  56. Wick RR, Judd LM, Holt KE. Deepbinner: Demultiplexing barcoded Oxford Nanopore reads with deep convolutional neural networks. PLoS Comput Biol 2018; 14:11 [View Article] [PubMed]
    [Google Scholar]
  57. White R, Pellefigues C, Ronchese F, Lamiable O, Eccles D. Investigation of chimeric reads using the MinION. F1000Res 2017; 6:631 [View Article] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/mgen/10.1099/mgen.0.000631
Loading
/content/journal/mgen/10.1099/mgen.0.000631
Loading

Data & Media loading...

Supplements

Supplementary material 1

EXCEL

Supplementary material 2

PDF

Most cited this month Most Cited RSS feed

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