library_construction_protocol: Amplicons of 16S rRNA genes were generated using a two-step PCR approach which was adapted and modified from Berry et al. (2011). The first PCR was performed with primers 515-530f, 5'-GTGCCAGCMGCNGCGG-3' and 1406-1392r, 5'-ACGGGCGGTGWGTRC-3' in 20 mL reactions with 0.2 mM dNTPs (Thermo Scientific, USA), 0.5 mM primers, 0.1 mg/mL of BSA and 0.4 U of Phusion Hot Start II DNA Polymerase with the corresponding HF buffer (Thermo Scientific, USA). Thermocycling conditions were set as follows: 98°C for 3 min, followed by 35 cycles of 98°C for 10 s, 61°C for 30 s, and 72°C for 30s, with the final elongation at 72°C for 10 min. The resulting PCR products were used as templates for a second PCR run, in which the primers 515-530f and 1406-1392r were fused with sequencing adapters (454 Sequencing Application Brief No. 001-2009, Roche) at 5'-ends. The forward primers contained different tags (454 Sequencing Technical Bulletin No. 005-2009, Roche) which allowed for multiplexing. PCR was performed in 25 mL reactions with 0.5 mL of the template, 0.2 mM dNTPs (Thermo Scientific, USA), 1 mM primers, 0.1 mg/mL of BSA and 0.5 U of Phusion Hot Start II DNA Polymerase with the corresponding HF buffer (Thermo Scientific, USA). The cycling program was set to 98°C for 3 min, followed by 10 cycles at 98°C for 10 s, 61°C for 30 s, and 72°C for 30s, with the final elongation at 72°C for 10 min. Finally, the PCR products were pooled and purified using AMPure XP Beads (Agencourt, Beckman Coulter, USA) to remove residual primer-dimers according to the manufacturer’s instructions. Amplicons were unidirectionally sequenced from the forward primer using GS FLX+ chemistry followed by standard analysis of signal processing (Roche).
identifiers.namespace: SUB1102379
identifiers.id: r5a.20day07a_CTCGCGTGTC.GTGCCAGCMGCNGCGG.sff.20day07a_CTCGCGTGTC.GTGCCAGCMGCNGCGG