Agencourt ampure xp purification system

To amplify the variable V3-V4 regions of the 16S rRNA gene, the primers 341 F (5′-CCTACGGGNGGCWGCAG-3′) and 805 R (5′-GACTACHVGGGTATCTAATCC-3′) were used. MiSeq sequencing adaptor sequences were added to the 5′ ends of forward and reverse primers. Approximately 12.5 ng of purified DNA from each sample was used as a template for PCR amplification in a 25-μL reaction mixture by using 2× KAPA HiFi Hot Start ready mix (Kapa Biosystems, MA, USA). For PCR amplification, the following conditions were followed: denaturation at 95°C for 3 min, followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, with a final extension at 72°C for 5 min. Amplified PCR products were purified with the Agencourt AMPure XP purification system (Beckman Coulter), and Nextera PCR was performed by using sample-specific barcodes. The constructed Nextera libraries were then sequenced by the Illumina MiSeq platform using MiSeq reagent kit v2 chemistry.

The V5-V6 hypervariable region (~ 280 bp) of the 16S ribosomal RNA (rRNA) gene was amplified using the degenerate universal primers 802F: 5'-GGATTAGATACCCBNGTA-3' (originally designed as reverse primer by Claesson et al. (2009) (link)) and 1027R: 5'-CGACRRCCATGCANCACCT-3' (Claesson et al. 2009 (link)) that target Bacteria and Archaea. In order to pool samples prior to sequencing, the primers contained a 10 bp length of multiplex identifiers (MIDs) between the 454 adaptor A (forward primer) and B (reverse primer), according to Roche. PCR reactions were performed in a total volume of 25 ul containing 0.3 uM of each primer, 0.3 mM dNTPs, 0.5 U KAPA high-fidelity HotStart DNA polymerase (Kapa Biosystems), 2.0 mM MgCl₂ and approximately 20 ng of template DNA. PCR reactions were carried out in a thermal cycler (Bio-Rad, MyCycler) under the following conditions: initial denaturation at 95°C for 5 min; followed by 25 cycles of denaturation at 98°C for 20 s, annealing at 55°C for 15 s and extension at 72°C for 60 s, followed by a final extension step at 72°C for 5 min. PCR amplicons were checked by electrophoresis in 1% agarose gel. Amplicons were purified by Agencourt AMPure XP purification system (Beckman Coulter Genomics) and quantified with Quantit PicoGreen dsDNA (Invitrogen) in a Quantiflour fluorometer (Promega). Equal concentrations of amplicons were pooled prior to sequencing.