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Optimising the detection of marine taxonomic richness using environmental DNA metabarcoding: the effects of filter material, pore size and extraction method


Deiner, Kristy; Lopez, Jacqueline; Bourne, Steve; Holman, Luke E; Seymour, Mathew; Grey, Erin K; Lacoursière-Roussel, Anaïs; Li, Yiyuan; Renshaw, Mark A; Pfrender, Michael E; Rius, Marc; Bernatchez, Louis; Lodge, David M (2018). Optimising the detection of marine taxonomic richness using environmental DNA metabarcoding: the effects of filter material, pore size and extraction method. Metabarcoding and Metagenomics, 2:e28963.

Abstract

The analysis of environmental DNA (eDNA) using metabarcoding has increased in use as a method for tracking biodiversity of ecosystems. Little is known about eDNA in marine human-modified environments, such as commercial ports, which are key sites to monitor for anthropogenic impacts on coastal ecosystems. To optimise an eDNA metabarcoding protocol in these environments, seawater samples were collected in a commercial port and methodologies for concentrating and purifying eDNA were tested for their effect on eukaryotic DNA yield and subsequent richness of Operational Taxonomic Units (OTUs). Different filter materials [Cellulose Nitrate (CN) and Glass Fibre (GF)], with different pore sizes (0.5 μm, 0.7 μm and 1.2 μm) and three previously pub- lished liquid phase extraction methods were tested. The number of eukaryotic OTUs detected differed by a factor of three amongst the method combinations. The combination of CN filters with phenol-chloroform-isoamyl alcohol extractions recovered a higher amount of eukaryotic DNA and OTUs compared to GF filters and the chloroform-isoamyl alcohol extraction method. Pore size was not independent of filter material but did affect the yield of eukaryotic DNA. For the OTUs assigned to a highly successful non-in- digenous species, Styela clava, the two extraction methods with phenol significantly outperformed the extraction method without phenol; other experimental treatments did not contribute significantly to detection. These results highlight that careful consideration of methods is warranted because choice of filter material and extraction method create false negative detections of marine eukaryotic OTUs and underestimate taxonomic richness from environmental samples.

Abstract

The analysis of environmental DNA (eDNA) using metabarcoding has increased in use as a method for tracking biodiversity of ecosystems. Little is known about eDNA in marine human-modified environments, such as commercial ports, which are key sites to monitor for anthropogenic impacts on coastal ecosystems. To optimise an eDNA metabarcoding protocol in these environments, seawater samples were collected in a commercial port and methodologies for concentrating and purifying eDNA were tested for their effect on eukaryotic DNA yield and subsequent richness of Operational Taxonomic Units (OTUs). Different filter materials [Cellulose Nitrate (CN) and Glass Fibre (GF)], with different pore sizes (0.5 μm, 0.7 μm and 1.2 μm) and three previously pub- lished liquid phase extraction methods were tested. The number of eukaryotic OTUs detected differed by a factor of three amongst the method combinations. The combination of CN filters with phenol-chloroform-isoamyl alcohol extractions recovered a higher amount of eukaryotic DNA and OTUs compared to GF filters and the chloroform-isoamyl alcohol extraction method. Pore size was not independent of filter material but did affect the yield of eukaryotic DNA. For the OTUs assigned to a highly successful non-in- digenous species, Styela clava, the two extraction methods with phenol significantly outperformed the extraction method without phenol; other experimental treatments did not contribute significantly to detection. These results highlight that careful consideration of methods is warranted because choice of filter material and extraction method create false negative detections of marine eukaryotic OTUs and underestimate taxonomic richness from environmental samples.

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Additional indexing

Item Type:Journal Article, not_refereed, original work
Communities & Collections:07 Faculty of Science > Institute of Evolutionary Biology and Environmental Studies
Dewey Decimal Classification:570 Life sciences; biology
590 Animals (Zoology)
Uncontrolled Keywords:eDNA, 18S ribosomal, seawater, high-throughput-sequencing, metazoan eukaryotes, non-indigenous species
Language:English
Date:2 November 2018
Deposited On:14 Nov 2018 15:56
Last Modified:14 Nov 2018 16:07
Publisher:Pensoft Net
ISSN:2534-9708
OA Status:Green
Free access at:Official URL. An embargo period may apply.
Publisher DOI:https://doi.org/10.3897/mbmg.2.28963

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