These data accompany the paper 'Long-distance Southern Ocean environmental DNA (eDNA) transect provides insights into spatial marine biota and invasion pathways for non-native species'.
Samples (n=138) were collected aboard the RSV Aurora Australis on a resupply voyage between Hobart, Tasmania (42°52’54.84”S 147°20’29.76”E) and Davis Station, Antarctica (66°26’14.28”S 77°28’24.6”E) in November 2019 (Figure 1; Table A1). Two combinations of water volume and filter pore sizes were tested at each sampling location: 12 L with 20 μm, and 2 L with 0.45 μm, herein referred to as “LargeVF” and “SmallVF” respectively. Water samples were collected and filtered approximately every 4 hours (4.52 ± 0.23) via the ship’s uncontaminated seawater intake line (4 ± 2 m depth). The seawater intake line was run for 3 – 5 minutes prior to sample collection to ensure the sample collected represented the seawater surrounding the ship at the time. SmallVF water samples (2 L, n=69) were filtered using a Sentino microbiology peristaltic pump (Pall Life Sciences) through 47 mm diameter, 0.45 μm pore size polyethersulfone filter membranes (Pall Life Sciences). Simultaneously, LargeVF water samples (12 L, n=69) were filtered using a Masterflex L/S console pump system (Cole-Parmer) through 25 mm diameter, 20 μm pore size nylon filter membranes (Merck). SmallVF filter membranes (47 mm diameter) were cut in half and immediately preserved at -80°C, with one half to be analysed and one to be stored as a reserve and a form of eDNA biobanking (Jarman et al., 2018). LargeVF filter membranes were not cut in half due to their smaller diameter (25 mm) and were stored whole at -80°C. Filtration equipment was rinsed with a 10% bleach solution and freshwater from the laboratory in between every sample, and soaked for 15 minutes with 10% bleach every tenth sample. Field controls consisted of 500 mL samples (n=10) of laboratory freshwater and the 10% bleach solution used for sterilisation, taken approximately every 10 samples.
DNA was extracted from the filter membrane using a DNeasy Blood and Tissue Kit (Qiagen) in an automated QIAcube (Qiagen) DNA extraction system with the following modifications: 540 μl of ATL lysis buffer, 60 μl of Proteinase K, and a 3-hour digestion at 56°C. Extraction controls were processed in parallel with all samples to detect any laboratory or between sample contamination. Final DNA extracts were eluted in 100 μl of AE buffer.
DNA was amplified to target Animalia taxa using mitochondrial cytochrome c oxidase subunit I (COI) markers: m1COIintF (Leray et al., 2013) and jgHCO2198 (Geller et al., 2013), herein referred to as Leray-COI. Samples were serially diluted (1/5, 1/10 and 1/100) to optimise DNA input levels for quantitative PCR (qPCR) and remove potential PCR inhibitors. Samples were found to perform optimally with no dilution. No-template controls were included on each qPCR plate. Metabarcoding was performed using fusion-tagged primers consisting of Illumina compatible sequencing adapters, a unique 6-8 bp multiple identifier tag (MID-tag), and the Leray-COI primer. Each sample and control were processed in duplicate using the same MID tag, to reduce stochasticity for species with low amounts of template DNA. qPCR reactions (25 μl) consisted of the following concentrations: 2 mM MgCl2, 1× AmpliTaq Gold PCR buffer, 1 U AmpliTaq Gold DNA polymerase (Applied Biosystems), 0.4 μM dNTPs (Astral Scientific), 0.1 mg BSA (Fisher Biotec), 0.6 μL of 5X SYBR Green dye (Life Technologies), 0.4 μM forward and reverse primer, 4 μL of eDNA template, made to volume with Ultrapure Distilled Water (Life Technologies).
qPCR amplifications were performed using a StepOnePlus Real-Time PCR System (Applied Biosystems) in a single-step process using an adjusted touchdown thermocycler protocol with conditions: 94°C for 10 min, 16 cycles of 95°C for 10 s, 62°C (-1°C per cycle) for 30 s, and 72°C for 45 s, followed by 25 cycles of 46°C for 30 s, with a final extension of 72°C for 5 minutes. qPCRs were prepared in dedicated clean facilities within the TrEnD Laboratory (Curtin University). Amplicons were visualised on 1.5% agarose gels and duplicate reactions from the same eDNA template were combined and then pooled into a library at equimolar ratios. The resulting library was size selected using a Pippin Prep (Sage Science), purified using a Qiaquick PCR Purification Kit (Qiagen), and quantified using a Qubit 4.0 Fluorometer (Invitrogen) and a Qiaxcel Advanced System (Qiagen). The final library was diluted to 2 nM and sequenced on an Illumina MiSeq platform (Illumina) using a 500 cycle (2 x 250 bp) MiSeq V2 Reagent Kit for paired-end sequencing.
