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Open AccessArticle

A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field

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Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy
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Department of Environmental and Life Sciences, Faculty of Science, Universiti Brunei Darussalam, Jalan Tungku Link, Gadong BE1410, Bandar Seri Begawan, Brunei
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Museum für Naturkunde, Leibniz Institute for Evolution and Biodiversity Science, Invalidenstraße 43, 10115 Berlin, Germany
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Department of Biology, School of Science & Engineering, Ateneo de Manila University, Loyola Heights, Quezon City 1101, Philippines
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Taxon Expeditions B.V., Rembrandtstraat 20, 2311 VW Leiden, The Netherlands
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Naturalis Biodiversity Center, Darwinweg 2, 2333 CR Leiden, The Netherlands
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Institute for Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands
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Division of Biology & Conservation Ecology, Manchester M15 6BH, UK
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PAMS Foundation, P.O. Box 16556, Arusha, Tanzania
*
Author to whom correspondence should be addressed.
S.M. and E.C. contributed equally.
Genes 2019, 10(6), 468; https://doi.org/10.3390/genes10060468
Received: 23 April 2019 / Revised: 11 June 2019 / Accepted: 18 June 2019 / Published: 20 June 2019
(This article belongs to the Special Issue MetaGenomics Sequencing In Situ)
Genetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION sequencer. However, to achieve a broader application, current proof-of-principle workflows for on-site barcoding analysis must be standardized to ensure a reliable and robust performance under suboptimal field conditions without increasing costs. Here, we demonstrate the implementation of a new on-site workflow for DNA extraction, PCR-based barcoding, and the generation of consensus sequences. The portable laboratory features inexpensive instruments that can be carried as hand luggage and uses standard molecular biology protocols and reagents that tolerate adverse environmental conditions. Barcodes are sequenced using MinION technology and analyzed with ONTrack, an original de novo assembly pipeline that requires as few as 1000 reads per sample. ONTrack-derived consensus barcodes have a high accuracy, ranging from 99.8 to 100%, despite the presence of homopolymer runs. The ONTrack pipeline has a user-friendly interface and returns consensus sequences in minutes. The remarkable accuracy and low computational demand of the ONTrack pipeline, together with the inexpensive equipment and simple protocols, make the proposed workflow particularly suitable for tracking species under field conditions. View Full-Text
Keywords: nanopore sequencing; long reads; field ecology; barcoding; portable lab; biodiversity nanopore sequencing; long reads; field ecology; barcoding; portable lab; biodiversity
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Maestri, S.; Cosentino, E.; Paterno, M.; Freitag, H.; Garces, J.M.; Marcolungo, L.; Alfano, M.; Njunjić, I.; Schilthuizen, M.; Slik, F.; Menegon, M.; Rossato, M.; Delledonne, M. A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field. Genes 2019, 10, 468.

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