Metabarcoding of environmental DNA
A two-step qPCR indexing approach in which barcode markers were first amplified in round one qPCR with a mix of primers with and without Nextera tails, and then indexed in round two qPCR with Illumina Unique Dual Index primers. Pools of 5L of round two qPCR product from each sample were created and cleaned with 1.5x volume of HighPrep PCR (MagBio, Cat. No. AC-60050) magnetic beads to pooled DNA and two 750 L 70% ethanol washes, and eluted in 43 L of AE buffer (QIAGEN). A Blue Pippin run was conducted to target fragment of size set to 515 bp +/- 150 bp. Quantification of 1L of the size selected product was done with the BioAnalyzer (Agilent, Santa Clara, USA).