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.