COI amplicon metabarcoding of seawater for marine metazoa: Pillar Point, Half Moon Bay, CA (USA)
Water samples were gravity filtered onto Sterivex 0.22 m filter cartridges (dx.doi.org/10.17504/protocols.io.bp2l69y7klqe/v2). DNA was extracted using a modified version of the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD, USA), in methods described here (dx.doi.org/10.17504/protocols.io.ewov1qyyygr2/v1). Samples were PCR amplified using the mlCOIintF/ jgHCO2198 primer set targeting a 313 bp fragment of the mitochondrial COI region optimized by Leray et al. (2013) (Forward: GGWACWGGWTGAACWGTWTAYCCYCC; Reverse: TAIACYTCIGGRTGICCRAARAAYCA) with Nextera modifications. Full PCR methods can be found here (dx.doi.org/10.17504/protocols.io.dm6gp3wpdvzp/v1). PCR products were sent for library preparation and sequencing at the Georgia Genomics and Bioinformatics Core (GGBC, UG Athens, GA, RRID:SCR_010994). In short, provided PCR amplicons were cleaned using AMPure XP magnetic beads (Beckman Coulter, Indianapolis, IN, USA), barcoded with Nextera adapters (Illumina, San Diego, CA, USA) during a second PCR (3 min at 95 C; 15 cycles of 30 sec at 95 C, 30 sec at 67 C, and 30 sec at 72 C; and 4 min at 72 C), cleaned again using AMPureXP magnetic beads, and pooled in equimolar ratio. The resulting library was sequenced on a MiSeq PE 2x250bp (500 cycles) using Reagent Kit V2 with 25% PhiX spike-in (Illumina, San Diego, CA, USA).