alias: SB09.16S
title: Metabarcoding of AMD Stream Biofilm: WB51_Moderately Impaired
design: DNA was extracted using the DNeasy PowerBiofilm kit (QIAGEN). The V4 region of 16S rDNA was amplified using primers 515F and 806RB (Caporaso et al. 2011, Apprill et al. 2015). 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
identifiers.namespace: SUB4238231
identifiers.id: SB09.16S