metabarcoding of pelagic zooplankton
DNA metabarcoding was performed on bulk zooplankton samples from two size fractions (0.21.0 mm, >1.0 mm) per net using a modified OMEGA E.Z.N.A. Blood Maxi extraction kit. DNA was eluted into 2 mL elution buffer, quantified with the Qubit dsDNA BR Assay (ThermoFisher Scientific), and normalized to 20 ng/L before PCR. Samples below 5 ng/L were cleaned and concentrated using Zymo Research DNA Clean and Concentrator-5 (D4003) before PCR. Zooplankton communities were characterized by amplicon sequencing of a COI gene fragment (~313 bp) using forward primer mlCOIintF [5- GGW ACWGGWTGAACWGTWTAYCCYCC-3] and reverse primer jgHCO2198 [5-TAIACYTCIGGRTGICC RAARAAYCA-3] (Geller et al., 2013; Leray et al., 2013), with Illumina overhang adaptors.  PCR reactions included 1 L of each primer (10 M), 0.05 L of bovine serum albumin (BSA, Sigma,  5 ng/L), 12.5 L of MyFi Mix (Bioline), 1-3 L of template DNA, and ddH2O to reach a total reaction volume of 25 L. Thermal cycling included an initial denaturation at 95C for 4 minutes, followed by 35 cycles of 95C for 30 s, 48C for 30 s, 72C for 45 s, and a final extension at 72C for 5 minutes. PCR amplification was run in duplicate for each sample, pooled, purified with AMPure XP beads (Beckman Coulter), and quantified using the Qubit (ThermoFisher Scientific). Products were normalized to 2ng/uL and pooled across markers at a 1.5:1 ratio (18S:COI). Samples were processed at the Advanced Studies in Genomics Proteomics and Bioinformatics (ASGPB) at the University of Hawaii at Manoa (HI, USA), indexed with the NexteraTM DNA library Prep Kit (Illumina, California, USA), pooled into libraries, and sequenced on MiSeq Illumina using V3 chemistry and paired end sequencing (2x300 bp). Extraction and PCR negative controls (N=19) were added undiluted to the library at 25uL to assess contamination.
SUB15272453
=A323 & ":COI"