Amplicon and Cas9-targeted nanopore sequencing of Varroa destructor at the onset of an outbreak in Australia

McFarlane, GR; Robinson, KL; Whitaker, K; Webster, J; Drysdale, L; Brancalion, L; Webster, A; O’Rourke, B; Bogema, DR

Amplicon and Cas9-targeted nanopore sequencing of Varroa destructor at the onset of an outbreak in Australia

McFarlane, GR Robinson, KL Whitaker, K Webster, J Drysdale, L Brancalion, L Webster, A O’Rourke, B Bogema, DR

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Publisher:: Frontiers
Publication Type:: Journal Article
Citation:: Frontiers in Bee Science, 2, pp. 1334543

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Field	Value	Language
dc.contributor.author	McFarlane, GR
dc.contributor.author	Robinson, KL
dc.contributor.author	Whitaker, K
dc.contributor.author	Webster, J
dc.contributor.author	Drysdale, L
dc.contributor.author	Brancalion, L
dc.contributor.author	Webster, A
dc.contributor.author	O’Rourke, B
dc.contributor.author	Bogema, DR
dc.date.accessioned	2024-12-17T06:05:56Z
dc.date.available	2024-12-17T06:05:56Z
dc.identifier.citation	Frontiers in Bee Science, 2, pp. 1334543
dc.identifier.issn	2813-5911
dc.identifier.issn	2813-5911
dc.identifier.uri	http://hdl.handle.net/10453/182663
dc.description.abstract	<jats:p><jats:italic>Varroa destructor</jats:italic> is a major pest of European honey bees (<jats:italic>Apis mellifera</jats:italic>), causing significant economic and welfare impacts. Australia remained the last continent free from <jats:italic>V. destructor</jats:italic>. In June 2022, a detection of <jats:italic>V. destructor</jats:italic> was reported in sentinel colonies at the Port of Newcastle, Australia. Rapid and accurate identification of the species was critical for timely response and management. In this case study, two Nanopore DNA sequencing methods, PCR amplicon sequencing and Cas9-targeted sequencing, were used to rapidly diagnose the species and mitochondrial haplotype of <jats:italic>Varroa</jats:italic> mites in parasitized colonies. Nanopore PCR amplicon sequencing provided molecular identification of the species and halogroup determination within 24 hours based on a 458 bp amplicon of the mitochondrial Cytochrome c oxidase subunit I (<jats:italic>COXI)</jats:italic> gene. We also developed and applied a Cas9-targeted Nanopore sequencing technique that used eight guide RNAs to enrich for 5240 bp of the mitochondrial genome. This method delivered richer data for identification within the same timeframe. Our results underscore the efficacy of Nanopore amplicon sequencing and represent the first reported application of Cas9-targeted Nanopore sequencing within a biosecurity framework. These findings enhance the repertoire of diagnostic tools available for biosecurity applications.</jats:p>
dc.language	en
dc.publisher	Frontiers
dc.relation.ispartof	Frontiers in Bee Science
dc.relation.isbasedon	10.3389/frbee.2024.1334543
dc.rights	info:eu-repo/semantics/openAccess
dc.title	Amplicon and Cas9-targeted nanopore sequencing of Varroa destructor at the onset of an outbreak in Australia
dc.type	Journal Article
utslib.citation.volume	2
pubs.organisational-group	University of Technology Sydney
pubs.organisational-group	University of Technology Sydney/Faculty of Science
pubs.organisational-group	University of Technology Sydney/UTS Groups
pubs.organisational-group	University of Technology Sydney/UTS Groups/Australian Institute for Microbiology & Infection (AIMI)
pubs.organisational-group	University of Technology Sydney/UTS Groups/Australian Institute for Microbiology & Infection (AIMI)/Australian Institute for Microbiology & Infection (AIMI) Associate Members
utslib.copyright.status	open_access	*
dc.rights.license	This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0). To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/
dc.date.updated	2024-12-17T06:05:55Z
pubs.publication-status	Published online
pubs.volume	2

Abstract:

Varroa destructor is a major pest of European honey bees (Apis mellifera), causing significant economic and welfare impacts. Australia remained the last continent free from V. destructor. In June 2022, a detection of V. destructor was reported in sentinel colonies at the Port of Newcastle, Australia. Rapid and accurate identification of the species was critical for timely response and management. In this case study, two Nanopore DNA sequencing methods, PCR amplicon sequencing and Cas9-targeted sequencing, were used to rapidly diagnose the species and mitochondrial haplotype of Varroa mites in parasitized colonies. Nanopore PCR amplicon sequencing provided molecular identification of the species and halogroup determination within 24 hours based on a 458 bp amplicon of the mitochondrial Cytochrome c oxidase subunit I (COXI) gene. We also developed and applied a Cas9-targeted Nanopore sequencing technique that used eight guide RNAs to enrich for 5240 bp of the mitochondrial genome. This method delivered richer data for identification within the same timeframe. Our results underscore the efficacy of Nanopore amplicon sequencing and represent the first reported application of Cas9-targeted Nanopore sequencing within a biosecurity framework. These findings enhance the repertoire of diagnostic tools available for biosecurity applications.

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http://hdl.handle.net/10453/182663