DNA metabarcode amplification from eDNA

One-step quantitative polymerase chain reactions (qPCR) were performed in duplicate for each sample using 2  µL of extracted DNA and a mitochondrial DNA 16S ribosomal DNA universal primer set targeting fish taxa (16SF/D, 5ʹ-GACCCTATGGAGCTTTAGAC-3ʹ and 16S2R-degenerate, 5ʹ-CGCTGTTATCCCTADRGTAACT-3ʹ)^44,45, with the addition of fusion tag primers unique to each sample that included Illumina P5 and P7 adaptors. We chose to analyse fish eDNA because this is one of the most common assessments made in aquatic eDNA metabarcoding studies^6,8–11 and is associated with a substantial reference database for taxonomic identification. A single round of qPCR was performed in a dedicated PCR laboratory. qPCR reagents included 5 μL AllTaq PCR Buffer (Qiagen; Venlo, The Netherlands), 0.5 μL AllTaq DNA polymerase, 0.5 μL dNTPs (10 mM), 1.0 μL Ultra BSA (500 μg/μL), SYBR Green I (10 U/μL), 0.5 μL forward primer (20 μM) and 5.0 μL reverse primer (20 μM), 2 μL of DNA and Ultrapure™Distilled Water (Life Technologies) made up to 25 μL total volume. Mastermix was dispensed manually and qPCR was performed on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, California, USA) using the following conditions: initial denaturation at 95 °C for 5 min, followed by 40 cycles of 30 s at 95 °C, 30 s at the primer annealing temperature 54°C and 45 s at 72 °C, with a final extension for 10 min at 72 °C. All duplicate qPCR products from the same subsample were combined prior to library pooling. The mean Cq value from qPCR duplicates was used as an indication of initial DNA copy number. Two sequencing libraries were made by pooling amplicons into equimolar ratios based on qPCR Ct values and sequenced on an Illumina Miseq platform (Illumina, San Diego, USA); one for Daw Island samples and the other for Ashmore Reef samples. The libraries were size selected using a Pippin Prep (Sage Science, Beverly, USA) and purified using the Qiaquick PCR Purification Kit (Qiagen, Venlo, The Netherlands). The volume of purified library added to the sequencing run was determined by quantifying the concentration⁴⁶ using a Qubit 4 fluorometer (Thermo Fisher Scientific). The library was unidirectionally sequenced using a 300 cycle MiSeq® V2 Reagent Kit and standard flow cell.

PCR plates included blank laboratory extraction controls (extraction reagents used with no DNA template), PCR-negative controls (2 μL of deionized water used rather than DNA template) and positive controls (dhufish; Glaucosoma hebraicum). Dhufish were an appropriate positive control for the tropical Ashmore samples since they are a subtropical fish species that do not occur at Ashmore reef. Dhufish do naturally occur in the vicinity of Daw Island at low densities, although no Dhufish were detected in any Daw Island samples other than positive controls. In addition, negative field controls were also conducted at Daw Island by filtering 500 mL of deionized water in situ. No negative control (extraction, PCR or field control) contained more than five reads for any fish species, except for one PCR-negative control which contained 11 reads. All positive controls amplified multiple reads identifying dhufish with 100% identity. However, 36 reads of the positive control showed up in two Ashmore Reef samples. Therefore, to ensure a conservative approach to detection efficiency, we required a minimum of 40 reads to count a fish species as present.