eDNA freshwater metabarcoding: 24SAR-223
eDNA was extracted from Smith-Root Self-Preserving filters using Qiagen DNeasy Tissue Kit with the following modifications: used 720 ul ATL with 10 min vortexing and 30 min rotisserie incubation at 56C twice prior to addition of 80ul Proteinase K followed by a 2 hour 56C incubation. Metabarcoding markers used were: 12S and 16S. The twin-tagging TagSteady protocol was used. PCR product from triplicate PCRS were pooled and visualized on 2% agarose gels. PCR products were pooled at approximate equimolar ratios determined by gel band strength. Amplicon pools were purified using a 1.8X SPRI bead solution and quantified using a Qubit Fluorometer. 2 pmol of each amplicon pool was end-repaired in 30 L using 0.1875 U/L Klenow fragment, 0.5 U/L T4 PNK, 0.125 mM dATP, 1x T4 DNA ligase buffer, and reaction booster (1.25% PEG, 20mM NaCl, 0.1 mg/mL BSA) at 37C for 30 min then 65C for 30 min. Amplicon pools were ligated with Y-adapters with 3 dT overhangs (xGen UDI-UMI Adapters) that had the Illumina TruSeq backbone and dual matching indices as follows: 3 L of adapter solution was added and ligated using: 1x T4 DNA ligase buffer, 6.25% PEG, 8 U/L T4 DNA ligase and incubated for 20C for 30 min then 65C for 10 min. Each library was purified using 2x volume SPRI beads and quantified using the NEBNext Library Quant Kit for Illumina. Equimolar amounts of each library were pooled to a final molarity of 5 nM and sequenced on two Illumina NovaSeq 6000 runs.