A200040
For metabarcoding analyses, polymerase chain reactions (PCRs) were performed to amplify the V4 region of the nuclear small ribosomal subunit (18S rRNA) and mitochondrial cytochrome c oxidase I (COI) genes. The primers for the 18S rRNA gene were Uni18SF: 5-AGG GCA AKY CTG GTG CCA GC-3 and Uni18SR: 5-GRC GGT ATC TRA TCG YCT T-3 (Zhan et al., 2013) and for COI: mlCOIintF: 5-GGW ACW GGW TGA ACW GTW TAY CCY CC-3 and jgHCO2198: 5-TAN ACY TCN GGR TGN CCR AAR AAY CA-3 (Leray et al., 2013). IlluminaTM overhang adaptors were attached to the primers to allow dual indexing as described in Kozich et al. 2013. PCR amplification used an Eppendorf Mastercycler (Eppendorf, Germany), with 50 L reactions containing 25 L MyFi Mix (Bioline, UK), 1 L of each 10 M primer, 20 L DNA-free water, and 3 L template DNA. Cycling conditions were: initial denaturation at 95 C for 2 min, followed by 39 cycles of 95 C for 20 s, 52 C for 20 s, 72 C for 20 s, and a final extension of 72 C for 10 min. The amplification products were purified and normalized using the SequalPrep Normalization Kit (Thermo Fisher Scientific, USA) to ~1 ng/L. The purified amplicons were sent to Sequench Ltd (Whakat/Nelson, Aotearoa-New Zealand) for indexing using the Nextera XT kit (Illumina, USA) and sequenced on an Illumina MiSeq platform. To monitor potential contamination during library preparation, a water blank was included as a sequencing control. The final library pool was adjusted to a loading concentration of 6 pM, with a 15% PhiX spike, before sequencing with the MiSeq Reagent Kit v3-600 cycle (2  301 bp) (Illumina, USA).