Preparation of amplicon libraries for metabarcoding of marine eukaryotes using illumina MiSeq: The dual-PCR method

Abstract

This protocol details the preparation of multiplexed amplicon libraries for metabarcoding (amplicon-based) studies of microscopic marine eukaryotes. Metabarcoding studies, based on the amplifi cation of a taxonomically informative marker from a collection of organisms or an environmental sample, can be performed to analyze biodiversity patterns or predator-prey interactions. For Metazoa, we use the mitochondrial cytochrome oxidase 1 (CO1) or the small ribosomal subunit (SSU) markers. Here, we describe a strategy for the preparation of multiplexed Illumina MiSeq libraries using a dual-PCR approach for the addition of index and adaptor sequences.