SpringerLink
Log in

Scalable, Cost-Effective, and Decentralized DNA Barcoding with Oxford Nanopore Sequencing

  • Protocol
  • First Online:
DNA Barcoding

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2744))

Abstract

DNA barcodes are useful in biodiversity research, but sequencing barcodes with dye termination methods (“Sanger sequencing”) has been so time-consuming and expensive that DNA barcodes are not as widely used as they should be. Fortunately, MinION sequencers from Oxford Nanopore Technologies have recently emerged as a cost-effective and efficient alternative for barcoding. MinION barcodes are now suitable for large-scale species discovery and enable specimen identification when the target species are represented in barcode databases. With a MinION, it is possible to obtain 10,000 barcodes from a single flow cell at a cost of less than 0.10 USD per specimen. Additionally, a Flongle flow cell can be used for small projects requiring up to 300 barcodes (0.50 USD per specimen). We here describe a cost-effective laboratory workflow for obtaining tagged amplicons, preparing ONT libraries, sequencing amplicon pools, and analyzing the MinION reads with the software ONTbarcoder. This workflow has been shown to yield highly accurate barcodes that are 99.99% identical to Sanger barcodes. Overall, we propose that the use of MinION for DNA barcoding is an attractive option for all researchers in need of a cost-effective and efficient solution for large-scale species discovery and specimen identification.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 219.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 279.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Hartop E, Srivathsan A, Ronquist F, Meier R (2022) Towards Large-Scale Integrative Taxonomy (LIT): resolving the data conundrum for dark taxa. Syst Biol 71:1404–1422. https://doi.org/10.1093/sysbio/syac033

    Article  PubMed  PubMed Central  Google Scholar 

  2. Yeo D, Puniamoorthy J, Ngiam RWJ, Meier R (2018) Towards holomorphology in entomology: rapid and cost-effective adult-larva matching using NGS barcodes. Syst Entomol 43:678–691. https://doi.org/10.1111/syen.12296

    Article  Google Scholar 

  3. Yeo D, Srivathsan A, Puniamoorthy J et al (2021) Mangroves are an overlooked hotspot of insect diversity despite low plant diversity. BMC Biol 19:202. https://doi.org/10.1186/s12915-021-01088-z

    Article  PubMed  PubMed Central  Google Scholar 

  4. Hebert PDN, Ratnasingham S, Zakharov EV et al (2016) Counting animal species with DNA barcodes: Canadian insects. Philos Trans R Soc B Biol Sci 371:20150333. https://doi.org/10.1098/rstb.2015.0333

    Article  Google Scholar 

  5. Morinière J, Balke M, Doczkal D et al (2019) A DNA barcode library for 5,200 German flies and midges (Insecta: Diptera) and its implications for metabarcoding-based biomonitoring. Mol Ecol Resour 19:900–928. https://doi.org/10.1111/1755-0998.13022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hebert PDN, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes. Proc R Soc Lond B Biol Sci 270:313–321. https://doi.org/10.1098/rspb.2002.2218

    Article  CAS  Google Scholar 

  7. Karlsson D, Hartop E, Forshage M et al (2020) The Swedish Malaise Trap Project: a 15 year retrospective on a countrywide insect inventory. Biodivers Data J 8:e47255. https://doi.org/10.3897/BDJ.8.e47255

    Article  PubMed  PubMed Central  Google Scholar 

  8. Srivathsan A, Ang Y, Heraty JM et al (2022) Global convergence of dominance and neglect in flying insect diversity. bioRxiv. https://doi.org/10.1101/2022.08.02.502512

  9. Meier R, Wong W, Srivathsan A, Foo M (2016) $1 DNA barcodes for reconstructing complex phenomes and finding rare species in specimen-rich samples. Cladistics 32:100–110. https://doi.org/10.1111/cla.12115

    Article  PubMed  Google Scholar 

  10. Geiger MF, Moriniere J, Hausmann A et al (2016) Testing the Global Malaise Trap Program - how well does the current barcode reference library identify flying insects in Germany? Biodivers Data J 4:e10671. https://doi.org/10.3897/BDJ.4.e10671

    Article  Google Scholar 

  11. Srivathsan A, Lee L, Katoh K et al (2021) ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone. BMC Biol 19:217. https://doi.org/10.1186/s12915-021-01141-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Pomerantz A, Peñafiel N, Arteaga A et al (2018) Real-time DNA barcoding in a rainforest using nanopore sequencing: opportunities for rapid biodiversity assessments and local capacity building. Gigascience 7:giy033. https://doi.org/10.1093/gigascience/giy033

  13. Menegon M, Cantaloni C, Rodriguez-Prieto A et al (2017) On site DNA barcoding by nanopore sequencing. PLoS One 12:e0184741. https://doi.org/10.1371/journal.pone.0184741

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Srivathsan A, Loh RK, Ong EJ et al (2022) Network analysis with either Illumina or MinION reveals that detecting vertebrate species requires metabarcoding of iDNA from a diverse fly community. Mol Ecol. https://doi.org/10.1111/mec.16767

  15. Vasilita C, Feng V, Hansen AK, Hartop E, Srivathsan A, Struijk R, Meier R (2023) Express barcoding with NextGenPCR and MinION for species-level sorting of ecological samples. Molecular Ecology Resources. https://doi.org/10.1111/1755-0998.13922

  16. Silvestre-Ryan J, Holmes I (2021) Pair consensus decoding improves accuracy of neural network basecallers for nanopore sequencing. Genome Biol 22:38. https://doi.org/10.1186/s13059-020-02255-1

    Article  PubMed  PubMed Central  Google Scholar 

  17. Srivathsan A, Hartop E, Puniamoorthy J et al (2019) Rapid, large-scale species discovery in hyperdiverse taxa using 1D MinION sequencing. BMC Biol 17:96. https://doi.org/10.1186/s12915-019-0706-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Srivathsan A, Baloğlu B, Wang W et al (2018) A MinION™-based pipeline for fast and cost-effective DNA barcoding. Mol Ecol Resour 18:1035–1049. https://doi.org/10.1111/1755-0998.12890

    Article  CAS  Google Scholar 

  19. Shokralla S, Porter TM, Gibson JF et al (2015) Massively parallel multiplex DNA sequencing for specimen identification using an Illumina MiSeq platform. Sci Rep 5:9687. https://doi.org/10.1038/srep09687

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wang WY, Srivathsan A, Foo M et al (2018) Sorting specimen-rich invertebrate samples with cost-effective NGS barcodes: validating a reverse workflow for specimen processing. Mol Ecol Resour 18:490–501. https://doi.org/10.1111/1755-0998.12751

    Article  CAS  PubMed  Google Scholar 

  21. Ho JKI, Puniamoorthy J, Srivathsan A, Meier R (2020) MinION sequencing of seafood in Singapore reveals creatively labelled flatfishes, confused roe, pig DNA in squid balls, and phantom crustaceans. Food Control 112:107144. https://doi.org/10.1016/j.foodcont.2020.107144

    Article  CAS  Google Scholar 

  22. Srivathsan A, Feng V, Suárez D, Emerson B, Meier, R (2024) ONTbarcoder 2.0: rapid species discovery and identification with real‐time barcoding facilitated by Oxford Nanopore R10.4. Cladistics 40(2):192–203. https://doi.org/10.1111/cla.12566

  23. Truett GE, Heeger P, Mynatt RL et al (2000) Preparation of PCR-quality mouse genomic DNA with hot Sodium Hydroxide and Tris (HotSHOT). BioTechniques 29:52–54. https://doi.org/10.2144/00291bm09

    Article  CAS  PubMed  Google Scholar 

  24. Thongjued K, Chotigeat W, Bumrungsri S et al (2019) A new cost-effective and fast direct PCR protocol for insects based on PBS buffer. Mol Ecol Resour 19:691–701. https://doi.org/10.1111/1755-0998.13005

    Article  CAS  PubMed  Google Scholar 

  25. Srivathsan A, Meier R Supplementary_Table1-3.xlsx. figshare. https://doi.org/10.6084/m9.figshare.22630741

  26. Folmer O, Black M, Hoeh W et al (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294–299

    CAS  PubMed  Google Scholar 

  27. Rohland N, Reich D (2012) Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res 22:939–946. https://doi.org/10.1101/gr.128124.111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Benton M (2021) GPU musings (with an eye on genomics). In: https://doi.org/10.5281/zenodo.5005787

  29. Srivathsan A (2021) ONTbarcoder. In: https://github.com/asrivathsan/ONTbarcoder

  30. Katoh K, Standley DM (2013) MAFFT Multiple Sequence Alignment Software Version 7: improvements in performance and usability. Mol Biol Evol 30:772–780. https://doi.org/10.1093/molbev/mst010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hamada M, Ono Y, Asai K, Frith MC (2017) Training alignment parameters for arbitrary sequencers with LAST-TRAIN. Bioinformatics 33:926–928. https://doi.org/10.1093/bioinformatics/btw742

    Article  CAS  PubMed  Google Scholar 

  32. Frith MC, Mitsuhashi S, Katoh K (2021) Lamassemble: multiple alignment and consensus sequence of long reads, pp 135–145

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Center for Integrative Biodiversity Discovery, Leibniz Institute for Evolution and Biodiversity Science, Museum für Naturkunde, Berlin, Germany

    Amrita Srivathsan & Rudolf Meier

  2. Institute for Biology, Humboldt University, Berlin, Germany

    Rudolf Meier

Authors
  1. Amrita Srivathsan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rudolf Meier
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rudolf Meier .

Editor information

Editors and Affiliations

  1. Division of Invertebrate Zoology, American Museum of Natural History, New York, NY, USA

    Robert DeSalle

Rights and permissions

Reprints and permissions

Copyright information

© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Srivathsan, A., Meier, R. (2024). Scalable, Cost-Effective, and Decentralized DNA Barcoding with Oxford Nanopore Sequencing. In: DeSalle, R. (eds) DNA Barcoding. Methods in Molecular Biology, vol 2744. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3581-0_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3581-0_14

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3580-3

  • Online ISBN: 978-1-0716-3581-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation