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Volume 5, Issue 2

Evaluation of five commercial DNA extraction kits using as a model for implementation of rapid Nanopore sequencing in routine diagnostic laboratories Open Access

Abstract

Oxford Nanopore long-read sequencing offers advantages over Illumina short reads for the identification and characterization of bacterial pathogens for outbreak detection and surveillance activities within a diagnostic public health laboratory context. Compared to Illumina, Nanopore is more cost-effective for small batches, has a lower capital cost and has a faster turnaround time, in addition to the ability to assemble complete bacterial genomes. The quantity and quality of DNA required for Nanopore sequencing are greater than for Illumina, and the DNA extraction methods recommended for obtaining high-molecular-weight DNA are different from those typically used in diagnostic laboratories. Using a isolate with a previously closed PacBio genome as a model Enterobacteriaceae organism, we evaluated the quantity, quality and fragmentation of five commercial DNA extraction kits. Nanopore sequencing performance was evaluated for the top three methods: Qiagen EZ1 DNA Tissue, Qiagen DNeasy Blood and Tissue, and a modified, in-house version of the MasterPure Complete DNA and RNA purification. To evaluate the effect of post-extraction DNA purification methods, we subjected extracted DNA from the three selected extraction methods to purification by AMPure beads or ethanol precipitation and compared these outputs with untreated DNA as a control. All methods are suitable for routine whole-genome sequencing (WGS), since all 60 replicates had very high genome recovery rates, with ≥98 % of the reference genome covered by mapped Nanopore reads. For 85 % of the replicates, assembly was able to produce a complete, circular chromosome using either Flye or Canu. In most cases, it is recommended to move directly from extraction to sequencing, as untreated DNA had the highest rates of genome closure regardless of extraction method. Using our evaluation criteria, the Qiagen DNeasy Blood and Tissue kit was found to be the best overall method due to its low cost, ability to scale from single tubes to 96-well plates, and high consistency in yield and sequencing performance.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): bacterial genomics , DNA extractions , long-read sequencing , Oxford Nanopore sequencing , public health and whole-genome sequencing
Funding
This study was supported by the:
  • Genomics Research and Development Initiative Phase VI Shared Priority Project Management Plan on Antimicrobial Resistance
    • Principal Award Recipient: JohnH.E. Nash
  • Public Health Agency of Canada
    • Principal Award Recipient: NotApplicable
© 2023 © His Majesty the King in Right of Canada, as represented by the Minister of Health (2023)
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2023-02-21
2026-03-12

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References

  1. Genome assembly. Oxf Nanopore Technol 2022. n.d http://nanoporetech.com/applications/investigation/assembly accessed 26 April 2022
  2. Koren S, Phillippy AM. One chromosome, one contig: complete microbial genomes from long-read sequencing and assembly. Curr Opin Microbiol 2015; 23:110–20 https://doi.org/10.1016/j.mib.2014.11.014
     [Google Scholar]
  3. Valiente-Mullor C, Beamud B, Ansari I, Francés-Cuesta C, García-González N et al. One is not enough: On the effects of reference genome for the mapping and subsequent analyses of short-reads. PLOS Comput Biol 2021; 17:e1008678 [View Article]
     [Google Scholar]
  4. Antipov D, Hartwick N, Shen M, Raiko M, Lapidus A et al. plasmidSPAdes: assembling plasmids from whole genome sequencing data. Bioinformatics 2016; 32:3380–3387 [View Article]
     [Google Scholar]
  5. Carattoli A, Zankari E, García-Fernández A, Voldby Larsen M, Lund O et al. In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother 2014; 58:3895–3903 [View Article]
     [Google Scholar]
  6. Robertson J, Nash JHE. MOB-suite: software tools for clustering, reconstruction and typing of plasmids from draft assemblies. Microb Genom 2018; 4:e000206 [View Article] [PubMed]
     [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 [View Article] [PubMed]
     [Google Scholar]
  8. Prjibelski A, Antipov D, Meleshko D, Lapidus A, Korobeynikov A. Using SPAdes De Novo Assembler. Curr Protoc Bioinformatics 2020; 70:e102 [View Article] [PubMed]
     [Google Scholar]
  9. Wick RR, Holt KE. Benchmarking of long-read assemblers for prokaryote whole genome sequencing. F1000Res 2020; 8:2138 [View Article]
     [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. Goldstein S, Beka L, Graf J, Klassen JL. Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing. BMC Genomics 2019; 20:23 [View Article] [PubMed]
     [Google Scholar]
  14. Wick RR, Judd LM, Gorrie CL, Holt KE. Completing bacterial genome assemblies with multiplex MinION sequencing. Microb Genom 2017; 3:e000132 [View Article] [PubMed]
     [Google Scholar]
  15. Dohm JC, Peters P, Stralis-Pavese N, Himmelbauer H. Benchmarking of long-read correction methods. NAR Genom Bioinform 2020; 2:lqaa037 [View Article] [PubMed]
     [Google Scholar]
  16. 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]
  17. 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] [PubMed]
     [Google Scholar]
  18. World Health Organization WHO estimates of the global burden of foodborne diseases: foodborne disease burden epidemiology reference group -2015 2015; 2007
  19. CDC Antibiotic resistance threats in the United States, 2019. Centers for Disease Control and Prevention (US) 2019 [View Article]
     [Google Scholar]
  20. Labbé G, Edirmanasinghe R, Ziebell K, Nash JHE, Bekal S et al. Complete Genome and Plasmid Sequences of Three Canadian Isolates of Salmonella enterica subsp. enterica Serovar Heidelberg from Human and Food Sources. Genome Announc 2016; 4:e01526-15 [View Article] [PubMed]
     [Google Scholar]
  21. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012; 9:671–675 [View Article] [PubMed]
     [Google Scholar]
  22. Green MR, Sambrook J. Molecular Cloning: a laboratory manual, 4th ed. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 2012
     [Google Scholar]
  23. De Coster W, D’Hert S, Schultz DT, Cruts M, Van Broeckhoven C. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 2018; 34:2666–2669 [View Article] [PubMed]
     [Google Scholar]
  24. Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V et al. Twelve years of SAMtools and BCFtools. Gigascience 2021; 10:giab008 [View Article] [PubMed]
     [Google Scholar]
  25. Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 2010; 26:841–842 [View Article] [PubMed]
     [Google Scholar]
  26. Oxford Nanopore Technologies Rapid Barcoding Sequencing (SQK-RBK004). Nanopore Community 2019. n.d https://community.nanoporetech.com/protocols/rapid-barcoding-sequencing-sqk-rbk004/v/rbk_9054_v2_revs_14aug2019 accessed 19 August 2021
  27. Oxford Nanopore Technologies Input DNA/RNA QC. Nanopore Community 2016 https://community.nanoporetech.com/protocols/input-dna-rna622qc/v/idi_s1006_v1_revb_18apr2016
     [Google Scholar]
  28. Timme RE, Lafon PC, Balkey M, Adams JK, Wagner D et al. Gen-FS coordinated proficiency test data for genomic foodborne pathogen surveillance, 2017 and 2018 exercises. Sci Data 2020; 7:402 [View Article] [PubMed]
     [Google Scholar]
  29. Xian Z, Li S, Mann DA, Huang Y, Xu F et al. Subtyping Evaluation of Salmonella Enteritidis Using Single Nucleotide Polymorphism and Core Genome Multilocus Sequence Typing with Nanopore Reads. Appl Environ Microbiol 2022; 88:e0078522 [View Article] [PubMed]
     [Google Scholar]
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Evaluation of five commercial DNA extraction kits using Salmonella as a model for implementation of rapid Nanopore sequencing in routine diagnostic laboratories
Access Microbiology 5, 000468.v3 (2023); https://doi.org/10.1099/acmi.0.000468.v3
/content/journal/acmi/10.1099/acmi.0.000468.v3
/content/journal/acmi/10.1099/acmi.0.000468.v3
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