Ecological assessment of the Scott Reef system using environmental DNA metabarcoding (WEL)

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In October 2021, a total of 94 seawater samples were collected approximately 1m below the surface from the Scott reef system (Figure 1). Samples were collected at 10 sites spread across the three atolls (South Scott, North Scott, and Seringapatam). At each site, a minimum of five replicate 1L samples were collected in sterile Nalgene bottles sites by snorkellers. We also included for comparison seawater samples collected from the Rowley Shoals (n=13), a neighbouring oceanic atoll system with pristine coral reef communities, and from inside the Darwin Harbour (n=6), a tidally-dominated turbid inshore environment. Samples inside Darwin Harbour were collected using the RV Solander’s Niskin array. Water samples were stored on ice until filtration within 6 hours of sample collection.


A series of control samples that consisted of the 10-20% bleach solution used to rinse collection bottles were also collected. All samples were filtered using a 250 mL funnel EZ-Fit filtration unit with 0.48 μm mixed cellulose ester membranes and an EZ-Fit Manifold base (3-place) from Merck Millipore (Merck Group, Burlington, USA). Gloves were changed and the manifold base and cup were sterilized with a 10% bleach solution between the filtering of samples from different sites. Following filtration, the filter papers were rolled and kept in cryogenic tubes at -20°C until transport and storage at -80°C.

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Statement: DNA extraction and sequencing All laboratory work was carried out by eDNA Frontiers at Curtin University using state-of-the-art clean room technology designed for ancient DNA research (http://www.ednafrontiers.com). DNA was extracted from half of each 0.48 μm mixed cellulose ester membranes using a Qiagen DNeasy blood and tissue kit, following the eDNA Frontiers SOPs using robotic extraction (refs). Samples were assigned a unique combination of index tags using fusion tagged primers (FTP) and amplified by PCR using three different metabarcoding assays: (1) a universal nuclear rRNA assay (18S), (2) a broad mitochondrial assay cytochrome-oxidase Cytochrome c oxidase subunit I (CO1), and a more specific ITS2 ribosomal DNA assay targeting basal metazoans (sponges and corals). Libraries were generated and sequenced on an Illumina MiSeq using paired-end sequencing (500 cycles), with appropriate laboratory extraction and PCR controls included to test for contamination. Bioinformatic pipelines and data analysis Three eDNA assays were amplified and analysed independently using a suite of metabarcoding tools. Sequence reads were quality filtered and clustered into amplicon sequence variants (ASVs) using DADA2 (10), which removes the arbitrary sequence similarity threshold component of OTU-based clustering. The ASVs were then curated using the LULU (11) algorithm with default parameters. The resulting ASVs were queried against NCBI’s GenBank nucleotide databases (downloaded on 28/07/2021) using basic local alignment search tool (BLAST). Taxonomic assignments were processed using the lowest common ancestor (LCA) approach. All taxonomic assignments required a 100% query coverage and over 95% match for family-level classification. Taxonomic hits that were detected in the field and/or laboratory controls were deemed to be potential contaminants and were excluded from the datasets. We transformed all counts matrices into binary (presence/absence) to avoid confounding our data with PCR biases associated with preferential amplification of certain taxa DNA. The alien invasive species (AIS) analyses were carried out using a slightly different bioinformatic pipeline developed by eDNA Frontiers (12) that uses USEARCH (13) instead of DADA2 to cluster reads into ASVs. Species level-assignments to an alien invasive species (AIS) required a 99% match. All downstream analyses were carried out using the free software R (2020).

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Credit
Funding Body: Woodside Energy Ltd