title: Metbarcoding: MiFish WAB 3
design: We employed a custom two-step library preparation (prep) protocol. This protocol involved the use of spacer inserts, serving as indices for the first PCR step. Four spacer inserts and four primer sets effectively results in 16 unique first step indices per pooled Illumina (Illumina, San Diego, CA, USA) sequencing library (see below). First step PCR reactions were 25 or 50l (with the latter used for inhibited samples, assessed according to the presence of primer dimer, see below) containing 1X Qiagen AllTaq PCR buffer, 0.3mM dNTPs, 0.5M of each primer, an additional 1.5mM MgCl2, 5U AllTaq (per 25l total reaction volume), 18l template DNA, and ddH20 to total volume. PCR conditions were: 2min initial denaturation at 95C, followed by 40 cycles of 95C for 5sec, primer specific annealing temp for 15sec (Table 1), and 72C for 10sec. Reactions were assessed by gel electrophoresis using 1% agarose gels stained with GelRed (Biotium, Fremont, CA, USA). Successful reactions were cleaned with MagBio HighPrepTM beads (MagBio Genomics, Gaithersburg, MD, USA) at a 0.7X ratio following the manufacturers instructions. Libraries were indexed using 2.5 l of unique combinations of 5 and 3 Nextera 96 indices (eight unique i5 and 12 unique i7), using the lowest numbers of shared forward and reverse indices per marker and spacer inserts, following the manufacturers protocol for 12 thermal cycles. Indexed samples were visualized on 1% agarose gels stained with GelRed. Successful reactions were bead cleaned (see above) at 1X ratio, sized, and quantified on an Agilent Bioanalyzer (Santa Clara, CA, USA), and pooled in equimolar concentrations. Pools then were bead cleaned at 0.8X ratio to remove any remaining primer dimer.  Pippen Prep (Sage Science, Beverly, MA) removed any amplicons >600bp, which might be undesirable PCR multimers potentially affecting run quality. Resulting libraries were sequenced in five separate Illumina MiSeq runs at Ohio State Woosters Molecular and Cellular Imaging Center (https://mcic.osu.edu/home), targeting a 40% PhiX spike in.