Library preparation
After the initial PCR, we cleaned all amplicons using PCRClean DX solid-phase reversible immobilization magnetic beads (Aline Biosciences, Woburn, MA). Each PCR reaction was quantified using Accublue High Sensitivity dsDNA Quantitation kit (Biotium, Fremont, CA) and normalized to 6 ng/ul. For 12S preparation, each group of 384 PCR products was then pooled into a single library for a total of four libraries. For COI preparation, each group of 192 PCR products was pooled into a single library for a total of eight libraries. Individual libraries were then tagged with an additional 6 base pair index using the NEBnext Ultra II DNA Library Prep kit (New England Biolabs, Ipswich, MA). Pooled samples were analyzed on a Bioanalyzer to confirm fragment size. The libraries were then sequenced using the Illumina HiSeq 3000 2 x 150 bp PE at the Center for Genome Research and Biocomputing at Oregon State University.