DNA extraction and amplification.
Cheesecloth filters were transferred to sterile 50 ml centrifuge tubes with 10 ml of molecular grade PBS (Gibco) and were rocked gently using a platform shaker for 30 mins at room temperature to dislodge particulate matter. The cheesecloth filters were then removed and the PBS solution centrifuged at 8,000 xg for 10 mins. The supernatant was removed, and the pellet resuspended in 180 µl of Buffer ATL before transfer to a new clean microfuge tube. For lysis, 20 µl of Proteinase K was added and vortexed briefly to mix before being heated at 56oC for 1 hour.  DNA was purified using a DNeasy Blood & Tissue kit (Qiagen) following manufacturer’s protocol from step 3. DNA was eluted in a total of 100 µl by passing 2 x 50 µl of Buffer AE through the spin column and then stored at -20oC.
Primers selected for this study targeting mitochondrial DNA were previously employed by two recent studies and were validated for use in similar applications (Clare et al. 2022, Lynggaard et al. 2022). DNA was amplified with the primers 16Smam1 forward (5’-CGGTTGGGGTGACCTCGGA-3’) and 16Smam2 reverse (5’-GCTGTTATCCC-TAGGGTAACT-3’) (Taylor 1996) to produce a fragment of approximately 100 base pairs long (bp). PCR was initially performed in 20 µL reactions consisting of, 10 µl AmpliTaq 360 mastermix (Thermo Scientific); 0.6 µM each of forward and reverse primer and 2 µL DNA template. To reduce the presence of human contamination, the human blocker (5’–3’ GCGACCTCGGAGCAGAACCC–spacerC3) as reported by (Vestheim and Jarman 2008a), was included in each reaction. The thermal cycling profile was 95oC for 10 min, followed by 40 cycles of 95oC for 12 s, 59oC for 30 s, and 70oC for 25 s, with a final extension of 72oC for 7 mins. The PCR reactions were visualised on a 1.5% agarose gel alongside non-template controls and koala DNA positive controls. Successfully amplified DNA fragments were selected and subjected to a second round of PCR using primers that included a sequencing tag to enable longer product for sequencing. TSP1_16Smam_forward 5’-TCTACACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCGGTTG GGGTGACCTCGGA-3’ and TSP2_16Smam_reverse: 5’-GTCTCGTGGGCTCG GAGATGTGTATAAGAGACAGGCTGTTATCCCTAGGGTAACT-3’. For the second-step PCR, conditions were identical to above but for using 1 µl of the PCR product from the first reaction and a lower annealing temperature of 55oC. Samples were sent for amplicon sequencing using the Illumina MiSeq Platform at the Australian Genome Research Facility.
An addition sample of DNA isolated from tissue collected from a skin biopsy an Indo-Pacific bottlenose dolphin (Tursiops aduncus) was used as a positive control to check for cross-contamination from demultiplexing and laboratory processing for high-throughput sequencing.