Metabarcoding of AMD Stream Biofilm: MC0300_Recovered
DNA was extracted using the DNeasy PowerBiofilm kit (QIAGEN). The Universal Plastid Amplicon (UPA), which targets a portion of the 23S rDNA chloroplast gene in algae, was amplified using primers p23SrV_f1 and p23SrV_r1 (Sherwood and Presting 2007). The primary PCR amplification was completed using Kapa HiFi HotStart ReadyMix (Kapa Biosystems, Wilmington, MA, USA). One master mix per gene was prepared and aliquoted for all samples along with a negative control (autoclaved Nanopure H2O) and amplified simultaneously. Samples were purified using Agencourt AmPure XP beads (Beckman Coulter Inc., Indianapolis, IN, USA) and quantified using a Qubit Fluorometer 3.0. Indexing was completed using Nextera Index 1 and 2 primers and enriched with the Kapa Hifi HotStart 2x PCR Master Mix (Kapa Biosystems, Wilmington, MA, USA). Amplified pooled samples were analyzed on an Agilent 2100 Bioanalyzer High Sensitivity DNA Chip (Agilent Technologies, Santa Clara, CA, USA) and quantified on the Qubit Fluorometer 3.0. Libraries from all samples were pooled in equimolar ratios, denatured using 0.2 N NaOH, diluted to 18 pM and loaded with 10% PhiX clustering control on the Illumina MiSeq with the 600 cycle (2 x 300 bp paired end) Sequencing Reagents V3.