Metabarcoding 18S V4 rRNA of eukaryotes: ocean water
A two-step nested PCR protocol was used to produce dual-indexed amplicons of the V4 region of the 18S ribosomal RNA gene (18S V4). The first PCR targeted the V4 region by oligonucleotides designed to amplify eukaryotes but exclude metazoans (Bower et al., 2004; del Campo et al., 2019). The second PCR added the necessary adaptors for Illumina MiSeq sequencing.