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Volume 9, Issue 1

Comparison of R9.4.1/Kit10 and R10/Kit12 Oxford Nanopore flowcells and chemistries in bacterial genome reconstruction Open Access

An Erratum was published on 28 November 2023

Abstract

Complete, accurate, cost-effective, and high-throughput reconstruction of bacterial genomes for large-scale genomic epidemiological studies is currently only possible with hybrid assembly, combining long- (typically using nanopore sequencing) and short-read (Illumina) datasets. Being able to use nanopore-only data would be a significant advance. Oxford Nanopore Technologies (ONT) have recently released a new flowcell (R10.4) and chemistry (Kit12), which reportedly generate per-read accuracies rivalling those of Illumina data. To evaluate this, we sequenced DNA extracts from four commonly studied bacterial pathogens, namely , , and , using Illumina and ONT’s R9.4.1/Kit10, R10.3/Kit12, R10.4/Kit12 flowcells/chemistries. We compared raw read accuracy and assembly accuracy for each modality, considering the impact of different nanopore basecalling models, commonly used assemblers, sequencing depth, and the use of duplex versus simplex reads. ‘Super accuracy’ (sup) basecalled R10.4 reads - in particular duplex reads - have high per-read accuracies and could be used to robustly reconstruct bacterial genomes without the use of Illumina data. However, the per-run yield of duplex reads generated in our hands with standard sequencing protocols was low (typically <10 %), with substantial implications for cost and throughput if relying on nanopore data only to enable bacterial genome reconstruction. In addition, recovery of small plasmids with the best-performing long-read assembler (Flye) was inconsistent. R10.4/Kit12 combined with sup basecalling holds promise as a singular sequencing technology in the reconstruction of commonly studied bacterial genomes, but hybrid assembly (Illumina+R9.4.1 hac) currently remains the highest throughput, most robust, and cost-effective approach to fully reconstruct these bacterial genomes.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): Genome sequencing , hybrid assembly and long-read assembly
Funding
This study was supported by the:
  • NIHR Oxford Biomedical Research Centre
    • Principle Award Recipient: NotApplicable
  • National Institute for Health and Care Research (Award 203141/Z/16/Z)
    • Principle Award Recipient: NotApplicable
  • National Institute for Health and Care Research (Award NIHR200915)
    • Principle Award Recipient: NotApplicable
© 2023 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.

Erratum

An erratum has been published for this content:
Erratum: Comparison of R9.4.1/Kit10 and R10/Kit12 Oxford Nanopore flowcells and chemistries in bacterial genome reconstruction
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References

  1. Van Goethem N, Descamps T, Devleesschauwer B, Roosens NHC, Boon NAM et al. Status and potential of bacterial genomics for public health practice: a scoping review. Implement Sci 2019; 14:79 [View Article]
     [Google Scholar]
  2. Shaw LP, Chau KK, Kavanagh J, AbuOun M, Stubberfield E et al. Niche and local geography shape the pangenome of wastewater- and livestock-associated Enterobacteriaceae. Sci Adv 2021; 7:15 [View Article] [PubMed]
     [Google Scholar]
  3. Arredondo-Alonso S, Pöntinen AK, Cléon F, Gladstone RA, Schürch AC et al. A high-throughput multiplexing and selection strategy to complete bacterial genomes. Gigascience 2021; 10:giab079 [View Article] [PubMed]
     [Google Scholar]
  4. Lipworth S, Pickford H, Sanderson N, Chau KK, Kavanagh J et al. Optimized use of Oxford Nanopore flowcells for hybrid assemblies. Microb Genom 2020; 6:11 [View Article] [PubMed]
     [Google Scholar]
  5. Oxford Nanopore Technologies. n.d https://nanoporetech.com/about-us/news/r103-newest-nanopore-high-accuracy-nanopore-sequencing-now-available-store
  6. Wick RR, Judd LM, Wyres KL, Holt KE. Recovery of small plasmid sequences via Oxford Nanopore sequencing. Microb Genom 2021; 7: [View Article] [PubMed]
     [Google Scholar]
  7. Benton M. Nanopore Guppy GPU basecalling on Windows using WSL2; 2021 https://hackmd.io/@Miles/rkYKDHPsO
  8. Shen W, Le S, Li Y, Hu F. SeqKit: a cross-platform and ultrafast toolkit for FASTA/Q file manipulation. PLoS One 2016; 11:e0163962 [View Article]
     [Google Scholar]
  9. Hall MB. n.d. Rasusa: randomly subsample sequencing reads to a specified coverage. JOSS 7:3941 [View Article]
     [Google Scholar]
  10. 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 [View Article] [PubMed]
     [Google Scholar]
  11. 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]
  12. Wick RR, Holt KE. Benchmarking of long-read assemblers for prokaryote whole genome sequencing. F1000Res 2019; 8:2138 [View Article] [PubMed]
     [Google Scholar]
  13. 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]
     [Google Scholar]
  14. Prjibelski A, Antipov D, Meleshko D, Lapidus A, Korobeynikov A. Using SPAdes de novo assembler. Curr Protoc Bioinformatics 2020; 70:e102 [View Article]
     [Google Scholar]
  15. De Maio N, Shaw LP, Hubbard A, George S, Sanderson ND et al. Comparison of long-read sequencing technologies in the hybrid assembly of complex bacterial genomes. Microb Genom 2019; 5: [View Article]
     [Google Scholar]
  16. Klockgether J, Munder A, Neugebauer J, Davenport CF, Stanke F et al. Genome diversity of Pseudomonas aeruginosa PAO1 laboratory strains. J Bacteriol 2010; 192:1113–1121 [View Article] [PubMed]
     [Google Scholar]
  17. Chandler CE, Horspool AM, Hill PJ, Wozniak DJ, Schertzer JW et al. Genomic and phenotypic diversity among ten laboratory isolates of Pseudomonas aeruginosa PAO1. J Bacteriol 2019; 201:e00595-18 [View Article]
     [Google Scholar]
  18. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 2014; 30:2068–2069 [View Article] [PubMed]
     [Google Scholar]
  19. 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 [View Article]
     [Google Scholar]
  20. Sereika M, Kirkegaard RH, Karst SM, Michaelsen TY, Sørensen EA et al. Oxford Nanopore R10.4 long-read sequencing enables the generation of near-finished bacterial genomes from pure cultures and metagenomes without short-read or reference polishing. Nat Methods 2022; 19:823–826 [View Article] [PubMed]
     [Google Scholar]
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Comparison of R9.4.1/Kit10 and R10/Kit12 Oxford Nanopore flowcells and chemistries in bacterial genome reconstruction
M Gen 9, 000910 (2023); https://doi.org/10.1099/mgen.0.000910
/content/journal/mgen/10.1099/mgen.0.000910
/content/journal/mgen/10.1099/mgen.0.000910
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