AO_32.1_18S_RNA
For metabarcoding analyses, the V4 region of the 18S rRNA was amplified for general eukaryotes, while two 16S rRNA gene regions were aimed at detecting particular taxa of interest, one for fish (16S rRNA fish) and another for marine mammals (16S rRNA mammal). The primers used for the 18S rRNA gene (~500 bp) 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). For the 16S rRNA fish gene (~200 bp), the primers were Fish16sF/D: 5-GACCCTATGGAGCTTTAGAC-3 (Berry et al., 2017) and 16s2R:5-CGCTGTTATCCCTADRGTAACT-3(Deagle et al., 2007). The primers for the 16S rRNA marine mammal gene (~245 bp) were MarVer3F: 5-AGACGAGAAGACCCTRTG-3 and MarVer3R: 5- GGATTGCGCTGTTATCCC-3(Valsecchi et al., 2020). Illumina overhang adaptors were appended to the primers for dual indexing, as described in Kozich et al. (2013). The 18S rRNA, 16S rRNA fish, and marine vertebrate gene amplified products underwent cleaning and normalization using the SequalPrep Normalization kit (Thermo Fisher Scientific, USA), resulting in a concentration of approximately 1 ng/L. Subsequently, the amplicons were sent to Sequench Limited (Nelson, Aotearoa-New Zealand) for indexing with the Nextera XT kit (Illumina, USA) and high-throughput sequencing on an Illumina MiSeq platform. The library pool was diluted to a final loading concentration of 6 pM with a 15% PhiX spike and sequenced using the MiSeq Reagent Kit v3-600 cycle (2 x 301 bp) (Illumina, USA) for the 18S rRNA gene region and v2-500 cycle (2 x 251 bp) for the 16S rRNA fish and marine vertebrate gene regions. NOTE FOR 16S FISH DATA WAS PAIRED, HOWEVER, THE ONLY AVAILABLE DATA WAS AFTER PROCESSING WITH DADA2, WHICH INCLUDES MERGING READS