Snail specimen identification
All PCR set up was performed in a clean ‘PCR room’ within a UV sterilisable cabinet and in a separate laboratory to DNA extraction using dedicated equipment and PPE. To ensure the unidirectional workflow DNA extracts are collected from the DNA extraction laboratory and transferred to the PCR set-up laboratory. Laboratory personnel do not return to the DNA extraction laboratory during that same day thus maintaining the unidirectional workflow.
PCR was performed to confirm the identity of the provided snail specimens using the mICOIintF/jgHCO2198 primer combination (Leray et al. 2013). These primers amplify a fragment of the Cytochrome c Oxidase subunit I gene (COI) and have been shown to perform well in invertebrate metabarcoding studies (Leray et al. 2013; Geller et al. 2013). PCRs were set up in a total volume of 25 µL consisting of: 2 µL of extracted template DNA, 2.5 µL of each primer (0.4 µmol/L), 12.5 µL of Itaq (BioRad) Sybr Green mastermix, and 5.5 µL ddH2O. Each sample was run in duplicate on a Bio-Rad CFX Connect real-time PCR machine as follows: an initial incubation for 1 minute at 95⁰C; followed by 35 cycles with a melting temperature of 95°C for 1 minute; an annealing temperature of 40⁰C and a final extension step at 72⁰C for 90 seconds before holding at 4⁰C until collection of PCR products for analysis. After PCR and amplicon clean-up, PCR products were Sanger sequenced using mICOIintF and returned sequences identified using BLAST.