FOG Bacteria Day 0 - Rep3
Two-step nested PCR was conducted prior to sequencing to improve PCR sensitivity. The primers used in the initial PCR were a modified 341F (CCTAYGGGRBGCASCAG) and a modified 806R (GGACTACNNGGGTATCTAAT) (Sundberg et al.2013), which targeted a 465 bp DNA fragment flanking the V3 and V4 regions of the 16S rRNA gene of Bacteria and Archaea. The initial PCR was performed in a total volume of 20  L containing 5  L of sample DNA extract (diluted 1:10), 500 nM of each of the primers 341F and 806R, 0.2 mM dNTPs mixture, 0.4 U of Phusion High-Fidelity DNA Polymerase (Finnzymes, Finland), 1X Phusion High-Fidelity Buffer (Finnzymes) and PCR grade water. The PCR amplification was performed with an initial incubation at 98 C for 30 s, followed by 35 cycles of 98 C for 5 s, annealing at 60 C for 20 s, extension at 72 C for 20 s and a final extension step at 72 C for 5 min. PCR was run in duplicate for each DNA sample and then subsequently pooled. PCR products were purified using the High-Pure PCR Cleanup Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. DNA concentrations of the purified PCR product were subsequently quantified using the Quant-IT dsDNA High-Sensitivity Assay Kit and a Qubit fluorometer, and amplicon size was verified by gel electrophoresis. Addition of adapters and indices to DNA fragments was done in a second PCR using the modified primers 515F (GTGCCAGCMGCCGCGGTAA) and 806R. The second PCR was performed in a total volume of 20  L containing 2  L AccuPrime PCR Buffer (10X, Invitrogen), 0.24 U AccuPrime Taq DNA Polymerase High-Fidelity (Invitrogen), 500 nM each of the fusion primers 515F and 806R with indexes, 2  L of 1:10 diluted PCR product from the first PCR and PCR grade water. The second PCR was run with the following conditions: 94 C for 2 min, followed by 15 cycles of 94 C for 20 s, 56 C for 30 s and 68 C for 40 s, and a final extension at 68 C for 5 min. PCR amplicons were purified using Agencourt AMPure XP (Agencourt Bioscience Corporation, MA, USA), and the concentration was measured using PicoGreen staining (Invitrogen), according to the manufacturer's protocols. The sample amplicons were pooled at equimolar concentrations, and subjected to high-throughput sequencing on the Illumina MiSeq platform at the University of Copenhagen Molecular Microbial Ecology Lab according to MiSeq Reagent Kit Preparation Guide (Illumina, Inc., San Diego, CA, USA).