Preparation of amplicon libraries for metabarcoding of marine eukaryotes using Illumina MiSeq: the adaptor ligation method

Amplicon-based studies of marine microscopic eukaryotes, also referred to as metabarcoding studies, can be performed to analyze patterns of biodiversity or predator–prey interactions targeting the mitochondrial cytochrome oxidase 1 (CO1) or the small ribosomal subunit (SSU) markers. Because high-throughput sequencing (HTS) Illumina platforms provide millions of reads per run, hundreds of samples may be sequenced simultaneously. This protocol details the preparation of multiplexed amplicon libraries for Illumina MiSeq sequencing. We describe a strategy for sample multiplexing using a combination of tailed PCR primers and ligation of indexed adapters.