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

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...

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Autores principales: Maestri, Simone, Cosentino, Emanuela, Paterno, Marta, Freitag, Hendrik, Garces, Jhoana M., Marcolungo, Luca, Alfano, Massimiliano, Njunjić, Iva, Schilthuizen, Menno, Slik, Ferry, Menegon, Michele, Rossato, Marzia, Delledonne, Massimo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627956/
https://www.ncbi.nlm.nih.gov/pubmed/31226847
http://dx.doi.org/10.3390/genes10060468
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author Maestri, Simone
Cosentino, Emanuela
Paterno, Marta
Freitag, Hendrik
Garces, Jhoana M.
Marcolungo, Luca
Alfano, Massimiliano
Njunjić, Iva
Schilthuizen, Menno
Slik, Ferry
Menegon, Michele
Rossato, Marzia
Delledonne, Massimo
author_facet Maestri, Simone
Cosentino, Emanuela
Paterno, Marta
Freitag, Hendrik
Garces, Jhoana M.
Marcolungo, Luca
Alfano, Massimiliano
Njunjić, Iva
Schilthuizen, Menno
Slik, Ferry
Menegon, Michele
Rossato, Marzia
Delledonne, Massimo
author_sort Maestri, Simone
collection PubMed
description 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.
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spelling pubmed-66279562019-07-23 A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field Maestri, Simone Cosentino, Emanuela Paterno, Marta Freitag, Hendrik Garces, Jhoana M. Marcolungo, Luca Alfano, Massimiliano Njunjić, Iva Schilthuizen, Menno Slik, Ferry Menegon, Michele Rossato, Marzia Delledonne, Massimo Genes (Basel) Article 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. MDPI 2019-06-20 /pmc/articles/PMC6627956/ /pubmed/31226847 http://dx.doi.org/10.3390/genes10060468 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Maestri, Simone
Cosentino, Emanuela
Paterno, Marta
Freitag, Hendrik
Garces, Jhoana M.
Marcolungo, Luca
Alfano, Massimiliano
Njunjić, Iva
Schilthuizen, Menno
Slik, Ferry
Menegon, Michele
Rossato, Marzia
Delledonne, Massimo
A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field
title A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field
title_full A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field
title_fullStr A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field
title_full_unstemmed A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field
title_short A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field
title_sort rapid and accurate minion-based workflow for tracking species biodiversity in the field
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627956/
https://www.ncbi.nlm.nih.gov/pubmed/31226847
http://dx.doi.org/10.3390/genes10060468
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