title: gadid metabarcoding of mock communities and aquarium metagenome
design: One liter water samples were collected and filtered with single-use Nalgene funnels onto 0.45 um filters.  Filters were transferred to a 5mL vials containing 3mL of Longmires buffer and stored at room temperature. DNA was extracted 400uL of Longmire's buffer using the Macherey-Nagel NucleoSpin Tissue Kit.  A two-step PCR approach was used, amplicon PCR was performed with gadid-specific primers (ND1_528F: AGTAYAACTCARGARGCAACATGRC; ND1_789R: ATGTAAAYTCSGGCATTGTRGTRGA). Primers were constructed with Illumina adapters fused to their 5' ends. Secondary barcode PCR used Illumina-compatible primers. Libraries were normalized to 5 ng/uL and pooled.  Pooled libraries were cleaned using AMPure XP Beads at 0.5x and 0.65x ratios for double-sided bead size selection. Sequencing runs were performed on an Illumina MiSeq using 2x150 bp paired-end sequencing with a 20% PhiX spike-in. Sequencing was performed at NOAA Alaska Fisheries Science Center Auke Bay Laboratories.