title: "Site 27, 48 colonies, 18 primers"
design: Eighteen microsatellite loci developed by Wang, Zhang, and Matz (2009) were selected for this study. We designed short tags according to Faircloth & Glenn (2012) to create 48 unique colony IDs on the forward primer for each microsatellite locus. Polymerase chain reactions (PCRs) for each colony were performed in a 25 µl reaction volume containing 12.5 µl of MyTaq™ Red Mix (Bioline, Taunton, MA, USA), 1 µl of each forward and reverse primer at 5µM, 1 µL of genomic DNA (5 to 10 ng/µl) and 9.5 µl of water. Thermal cycling followed a touch-down protocol with an initial denaturation at 95° C for 3 min followed by 20 cycles of 95° C for 15 sec, 63 - 55° C for 15 sec (annealing temperature reduced 0.4° C each cycle), 72° C for 30 sec followed by 20 cycles of 95° C for 15 sec, 55° C for 15 sec, 72° C for 30 sec with a final elongation at 72° C for 3 min.   Two µl of each uniquely barcoded amplicon were pooled by site for subsequent library construction and sequencing. The pooled samples were concentrated by reducing the total volume using Amicon Ultra 0.5 mL centrifugal filters (EMD Millipore, Darmstadt, Germany) and cleaned using Agentcourt Ampure XP (Beckman Coulter Inc., Brea, CA, USA) to eliminate traces of dye and unincorporated dNTPs and primers. Genomic libraries were generated using the KAPA Hyper Prep kit (Kapa Biosystems, Wilmington, MA, USA). A unique Illumina adaptor (Illumina Inc., Hayward, CA, USA) was ligated to each pool of individually barcoded amplicon samples, creating a site-specific tag (ID) and generating the following unique structure:  siteID-colonyID-FWDprimer-flankingregion-tandemrepeats-flankingregion-RVSprimer.   Libraries were sequenced on an Illumina MiSeq at the Hawai‘i Institute of Marine Biology (HIMB) Evolutionary Genetic core facility.