454 16S rRNA V1-V2 amplicon from Human microbiome sample
Triplicate PCR reactions were performed for metagenomic DNA extracted from each sample using a primer set         described by Costello et. al. (2009) and Turnbauggh et. al. (2009) respectively. The forward primer         (5'-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG- 3') contained the 454 Life Sciences primer B sequence, the broadly conserved         bacterial primer 27F, and a two-base linker sequence ('TC'). The reverse primer (5'-         GCCTCCCTCGCGCCATCAGNNNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3') contained the 454 Life Sciences primer A sequence, a unique 10-nt barcode         used to tag each PCR product (designated by NNNNNNNNNN and provided by Roche), the broad-range bacterial primer 338R, and a 'CA'         linker sequence inserted between the barcode and the rRNA primer. Each 25 ul reaction contained 0.2 uM forward and reverse primers,         3 ul template DNA, and 2.5 ul 10X PCR buffer plus Mg2+ (TaKaRa), 2.0 ul dNTP (2.5 mM each) (TaKaRa), 0.75 ul DMSO (100%%%%), 0.25 ul         TaKaRa TaqTM (5 U/ul). All dilutions were carried out using sterile ddH2O. PCR reactions were assembled within a PCR hood in which         all surfaces and pipettes had been decontaminated with 30 min autoclaving at 120c; and UV irradiated for 30 minutes. Thermal cycling         was 95c for 10 minutes, followed by 30 cycles of 95c for 30 seconds, 55c for 30 seconds, and 72c for 90 seconds, with a final         extension of 10 minutes at 72c. Replicate amplicons were pooled and visualized on 1.0%%%% agarose gels using EB stain in 0.5X TE.         Amplicons were cleaned using PCR Products Purification Kit (Spin-column) (BioTeke Corporation, Beijing, China) according to the         manufacturer's instructions. Amplicon DNA concentrations were determined using the Quant-iT PicoGreen dsDNA reagent and kit         (Invitrogen).