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.