Environmental DNA metabarcoding is being applied as a biodiversity monitoring tool in marine conservation areas by the department of Fisheries and Oceans. Between 2021 to 2023, offshore surface and benthic eDNA samplers were collected in the Eastern Shore Islands Area of Interest in Nova Scotia to establish baseline fish and other eukaryote diversity data for monitoring potential changes in species richness and community structure over time. In each of the three years of this project, we collected water samples by a 6-bottle CTD rosette sampler deployed from the Canadian Coast Guard ship M. Perley along four transects with four stations in each transect (n=16 sites). These four transects extended from inshore to offshore and were spread across the AOI boundary to cover a longitudinal gradient.
Water samples were filtered onto a 0.22um Sterivex filter (2021 and 2022) or a 1.2um Smith-Root filter (2023) to collect eDNA. eDNA was then extracted in a laboratory, and libraries were created to target fish (12S MiFishU marker in all 3 years, 16S in 2021) and general biodiversity (COI using Leray or Leray-XT primers) in 2022 and 2023. Samples from 2021 and 2022 were sequenced on an Illumina NovaSeq platform at the Centre for Environmental Genomics Applications (CEGA; St. John's, Newfoundland), while samples from 2023 were sequenced on an Illumina MiSeq at the Aquatic Biotechnology Lab (ABL) in Dartmouth, Nova Scotia.
Samples were analyzed through the QIIME2 pipeline and denoised with DADA2. Amplicon sequence variant taxonomy was assigned using BLAST for 12S and 16S markers, while taxonomy for COI was assigned using an RDP Bayesian classifier.
The overall objective for this study was to create baseline species richness and community data entirely from eDNA samples, and being a time series for detecting change or stability over time. Since our methods in the field and lab changed between 2021 to 2023, any temporal trends detected between years should be interpreted with caution.
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Between 2021 to 2023, offshore surface and benthic eDNA samplers were collected in the Eastern Shore Islands Area of Interest in Nova Scotia to establish baseline fish and other eukaryote diversity data for monitoring potential changes in species richness and community structure over time. In each of the three years of this project, we collected water samples by a 6-bottle CTD rosette sampler deployed from the Canadian Coast Guard ship M. Perley along four transects with four stations in each transect (n=16 sites). These four transects extended from inshore to offshore and were spread across the AOI boundary to cover a longitudinal gradient. \n\nWater samples were filtered onto a 0.22um Sterivex filter (2021 and 2022) or a 1.2um Smith-Root filter (2023) to collect eDNA. eDNA was then extracted in a laboratory, and libraries were created to target fish (12S MiFishU marker in all 3 years, 16S in 2021) and general biodiversity (COI using Leray or Leray-XT primers) in 2022 and 2023. Samples from 2021 and 2022 were sequenced on an Illumina NovaSeq platform at the Centre for Environmental Genomics Applications (CEGA; St. John's, Newfoundland), while samples from 2023 were sequenced on an Illumina MiSeq at the Aquatic Biotechnology Lab (ABL) in Dartmouth, Nova Scotia. \n\nSamples were analyzed through the QIIME2 pipeline and denoised with DADA2. Amplicon sequence variant taxonomy was assigned using BLAST for 12S and 16S markers, while taxonomy for COI was assigned using an RDP Bayesian classifier. \n\nThe overall objective for this study was to create baseline species richness and community data entirely from eDNA samples, and being a time series for detecting change or stability over time. Since our methods in the field and lab changed between 2021 to 2023, any temporal trends detected between years should be interpreted with caution."},"samplingDescription":{"studyExtent":"The study area occurs within the Eastern Shore Islands Area of Interest study area, a coastal marine area of about 2000 squared kilometres in Nova Scotia, Canada. Samples are collected from four transects each consisting of four sampling stations, which extend from inshore to offshore within the study area. \nThe study includes samples and data collected in summer 2021 and 2022 from all four transects. In 2023, only one transect was sampled extending offshore from Sheet Harbour due to limited vessel availability. However, the transect was sampled in spring (April), summer (August), and fall (November) in this year, representing the only seasonal samples collected.\nSampling along these transects has not occurred since 2023.","sampling":"From August 9 to 14, 2021, a total of 88 seawater samples were collected using 4 L Niskin bottles on an SBE 19plus CTD and SBE 55 rosette (Sea-Bird Scientific), closed at prescribed depths at regular intervals aboard the Canadian Coast Guard Ship M. Perley along 4 transects starting near the coast out to 20 km offshore (Jeddore (ESI-13 to ESI-16), Ship Harbour (ESI-9 to ESI-12), Sheet Harbour (ESI-00 and ESI-5 to ESI-8) and Moser River (ESI-1 to ESI-4)) in the AOI. Measured parameters from water sampled at these locations included total inorganic carbon (TIC), total alkalinity (TA), pCO2 and pCH4 (partial pressure of carbon dioxide and methane gases), salinity, dissolved oxygen, dissolved inorganic nutrients (nitrate, nitrite, ammonium, silicate and phosphate) and oxygen isotope composition (δ18O) (along the Sheet Harbour transect and the three rivers).\nNiskin bottles dedicated to eDNA filtration were also collected at the bottom and surface of each CTD rosette profile. In 2021 and 2022, 1 L water samples were frozen in Nalgene bottles until later thawing and filtration in a laboratory setting. In 2023, eDNA was collected from filtering 3 L of seawater directly from the Niskin bottle while aboard the vessel. The CTD also profiled depth, temperature, and conductivity at each sampling station. \n\nFour offshore eDNA transects were conducted within the AOI in 2021 and 2022 using the CCGS M. Perley to gain baseline and time series information on fish species in the AOI. These transects were evenly distributed from west to east within the AOI boundaries and spanning from inshore to offshore (depths of approximately 15 – 100 m). In 2021 and 2022 we collected triplicate 1 L water samples from surface and bottom water at each station, and captured eDNA on 0.22 µm Sterivex filters using a peristaltic pump. This sampling involved freezing collected water samples in Nalgene bottles, thawing the samples later, and filtering in the lab at BIO, which was labour-intensive and may cause DNA damage and breakage with the freeze-thaw cycle.\nIn 2023, we updated our sampling strategy, instead filtering triplicate 3 L water samples using 1.2 µm Smith-Root filters using an on-board vacuum pump. The larger pore size selected allowed some microbes to pass through the filter, reducing non-target eDNA amplification, and allowed for efficient filtering of a larger water volume to capture more eukaryotic eDNA. However, we were only able to sample one transect (four stations directly off Sheet Harbour, ESI-05 to ESI-08) in spring, summer, and fall due to vessel priorities in St. Anns Bank MPA. No offshore transects were sampled in 2024 or 2025 due to a lack of dedicated research vessel time.","qualityControl":"During sampling, field blanks consisting of 1-2 litres of distilled water are filtered at the same time as the eDNA water samples to detect potential contaminants originating from the filtering equipment or personnel. In the laboratory, extraction and PCR negatives are included with all samples to detect potential contamination in the laboratory reagents or equipment. Any DNA detected in the various blanks are removed from the samples that the contamination is found in.","methodSteps":["The eDNA filters from 2021 and 2022 were shipped on ice to the Centre for Environmental Genomics Applications (St. John’s, Newfoundland) for DNA extraction and sequencing. Briefly, each Sterivex filter was removed from its housing and digested in lysis buffer. Captured DNA is extracted from each filter, after which the DNA undergoes PCR amplification for the specific marker region being targeted. Nucleotide barcodes are added to the DNA from each filter to identify the sample during bioinformatic processing, and Illumina adapters are also added to enable next-generation sequencing on an Illumina platform. Amplified, barcoded DNA from each sample is then pooled into a sequencing library and sequenced on an Illumina instrument. In 2021 and 2022, libraries were sequenced at CEGA on an Illumina NovaSeq 6000 machine with a sequencing depth target of 1 million reads per sample.\nThe eDNA samples collected in 2023 were processed and sequenced by the Aquatic Biotechnology Laboratory (Dartmouth, Nova Scotia). DNA was extracted from Smith-Root filters using the Qiagen DNeasy Blood & Tissue Kit and QIAcube Connect robotic workstation (Qiagen). Extracted DNA was amplified at a region of cytochrome c oxidase I (COI) and 12S ribosomal RNA (12S) with custom Ultramer DNA primers (IDT) and simultaneously indexed with a unique combination of nucleotide barcodes using PCR. The PCR products were evaluated for quality using the Agilent 4200 TapeStation electrophoresis system with D1000 ScreenTapes and Reagents (Agilent). Successful PCR-amplified samples were purified using the AxyPrep Mag PCR Clean-up Reagent (Axygen), then quantified using the AccuClear Ultra High Sensitivity dsDNA Quantitation kit (Biotium). Equimolar amounts of each purified PCR sample were pooled to create a library (either COI or 12S). Target input amounts of DNA were 50ng/sample for the COI libraries and 25ng/sample for the 12S libraries. The completed libraries were purified again using the AxyPrep Mag PCR Clean-up Reagent, evaluated using the 4200 TapeStation with the HS D1000 assay kit, and quantified using the NEBNext Library Quant Kit for Illumina (New England Biolabs). Libraries were sequenced using the Illumina MiSeq System with either v3 600-cycle paired-end chemistry (COI libraries) or v2 500-cycle paired-end chemistry (12S libraries). \nRaw sequence reads were converted to fastq format and demultiplexed. Forward and reverse primers were trimmed from all sequences using cutadapt v.4.0 (Martin 2011) with an error rate of 0.11, minimum quality score of 20, and minimum length set to 20. Demultiplexed reads were processed using the QIIME2-2024.10 pipeline (Boyen et al. 2019). Paired-end reads were imported for each marker separately using the import tool, and denoised using the DADA2 plugin. For 16S, –p-trunc-len-f and –p-trunc-len-r were both set to 125, but were set to 116 and 108 respectively for 12S. A consensus method was used to detect chimeras, and the –p-min-fold-parent-over-abundance parameter was set to 2 to improve the number of reads passing the chimera filter. Following denoising, the results were summarized as feature-tables including the filtered amplicon sequence variant (ASV) feature table, representative fasta sequences, and denoising statistics using the feature-table plugin. The taxonomy of each ASV sequence was assigned using BLASTn against the eukaryotic nucleotide database for 12S and 16S markers, while a Ribosomal Database Project (RDP) naive Bayesian classifier v5.1.0 was used to assign the taxonomy for the COI marker (Porter et al. 2014; Porter and Hajibabaei 2018). \nASV tables for each eDNA marker consisting of species names and read counts per species per eDNA sample were filtered for taxonomic identifications with a percent match <98%, and for non-aquatic species (e.g., terrestrial mammals, insects, fungi). Following data filtration, barplots of confirmed species and their read counts were created for each marker and each year of data. Species accumulation curves and non-metric multidimensional scaling (nMDS) plots were conducted in the vegan R package (Oksanen et al. 2025) to show species diversity and sample community similarities. Jaccard dissimilarities were used to create the distance matrix for the nMDS of the eDNA communities. Finally, for the 2023 dataset, alluvial plots were generated using the ggalluvial package (Brunson and Read 2023) to visualize the species found at each station in the Sheet Harbour transect (ESI-5 to ESI-8) in different seasons (spring, summer, and fall), and whether they were found in surface or bottom samples."]},"countryCoverage":[],"collections":[],"dataDescriptions":[],"dataLanguage":"eng","acknowledgements":"This work was funded by the Fisheries and Oceans Canada's Marine Conservation Targets program. We thank the captain and crew of the Canadian Coast Guard ship M. Perley for their time at sea and assistance with operating equipment. ","pubDate":"2026-04-28T00:00:00.000+00:00","maintenanceUpdateFrequency":"IRREGULAR","maintenanceDescription":"Offshore eDNA sampling took place in 2021, 2022, and 2023 in the Eastern Shore Islands. As of February 2026, the data in this resource only includes eDNA data from those particular years.","license":"http://creativecommons.org/publicdomain/zero/1.0/legalcode","dwca":{"coreType":"http://rs.tdwg.org/dwc/terms/Occurrence","extensions":["http://rs.iobis.org/obis/terms/ExtendedMeasurementOrFact","http://rs.gbif.org/terms/1.0/DNADerivedData"],"modified":"2026-04-30T10:41:47.512+00:00"},"category":["eDNA"],"approximateCounts":{"occurrenceCount":1208771}}