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Rapid in situ identification of biological specimens via DNA amplicon sequencing using miniaturized laboratory equipment

Abstract

In many parts of the world, human-mediated environmental change is depleting biodiversity faster than it can be characterized, while invasive species cause agricultural damage, threaten human health and disrupt native habitats. Consequently, the application of effective approaches for rapid surveillance and identification of biological specimens is increasingly important to inform conservation and biosurveillance efforts. Taxonomic assignments have been greatly advanced using sequence-based applications, such as DNA barcoding, a diagnostic technique that utilizes PCR and DNA sequence analysis of standardized genetic regions. However, in many biodiversity hotspots, endeavors are often hindered by a lack of laboratory infrastructure, funding for biodiversity research and restrictions on the transport of biological samples. A promising development is the advent of low-cost, miniaturized scientific equipment. Such tools can be assembled into functional laboratories to carry out genetic analyses in situ, at local institutions, field stations or classrooms. Here, we outline the steps required to perform amplicon sequencing applications, from DNA isolation to nanopore sequencing and downstream data analysis, all of which can be conducted outside of a conventional laboratory environment using miniaturized scientific equipment, without reliance on Internet connectivity. Depending on sample type, the protocol (from DNA extraction to full bioinformatic analyses) can be completed within 10 h, and with appropriate quality controls can be used for diagnostic identification of samples independent of core genomic facilities that are required for alternative methods.

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Fig. 1: Examples of equipment used to carry out field-deployed DNA amplicon sequencing.
Fig. 2: Illustration of the steps involved in the laboratory part of the protocol.
Fig. 3: Illustration of the steps involved in the bioinformatic part of the protocol.

Data availability

Example data files can be found in the Supplementary Information.

Code availability

The code used can be found on the NGSpeciesID GitHub page: https://github.com/ksahlin/NGSpeciesID. The code in this protocol has been peer reviewed.

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Acknowledgements

We thank L. Comai for the adjustment of the barcode_generator tool to work for our MinION indexing protocol. We thank ONT for providing technical support, and for making the offline MinKNOW software available to us. We also thank H. Asahara and the UC Berkeley DNA Sequencing Facility for technical support. We thank Tropical Herping, Rainforest Expeditions, Field Projects International and the Inkaterra Guides Field Station for support during fieldwork.

Author information

Authors and Affiliations

Authors

Contributions

A.P., N.V., A.S., E.H., S.K., H.K., S.W., R.O. and S.P. optimized and further developed the laboratory protocols. A.P. and S.P. tested and validated the approach and equipment in the field. K.S., M.L. and S.P. developed and validated the bioinformatics processing pipeline. R.O. and S.P. conceived the project. All authors wrote the manuscript and approved the contents of the manuscript.

Corresponding author

Correspondence to Stefan Prost.

Ethics declarations

Competing interests

A.P. became an employee of ONT PLC after the completion of the research described in the paper. The other authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks Simon Creer, Sujeevan Ratnasingham and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Pomerantz, A. et al. Gigascience 7, giy033 (2018): https://doi.org/10.1093/gigascience/giy033

Sahlin, K. et al. Ecol. Evol. 11, 1392–1398 (2021): https://doi.org/10.1002/ece3.7146

Vasiljevic, N. et al. Forensic Sci. Int. Genet. 53, 102493 (2021): https://doi.org/10.1016/j.fsigen.2021.102493

Supplementary information

Reporting Summary

Supplementary Data 1

An example MinION reads dataset generated using the outlined protocol (3,000 reads). The file contains reads of three fish species: the Atlantic cod (Gadus morhua), the Haddock (Melanogrammus aeglefinus) and the Whiting (Merlangius merlangus), sequenced on a Flongle flow cell. This is previously unpublished data.

Supplementary Data 2

An example index file for demultiplexing with minibar (can be used to demultiplex the example read data in Supplementary Data 1).

Supplementary Data 3

An example file of the primer sequences in fasta format (can be used for primer removal using NGSpeciesID).

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Pomerantz, A., Sahlin, K., Vasiljevic, N. et al. Rapid in situ identification of biological specimens via DNA amplicon sequencing using miniaturized laboratory equipment. Nat Protoc 17, 1415–1443 (2022). https://doi.org/10.1038/s41596-022-00682-x

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