SSU rRNA Gene Bacteria V5-V6 amplification of sample: w_11_buccal
Genomic Bacteria DNA of the SSU rRNA gene V5-V6 hypervariable region was amplified using the forward primer 785F (GGATTAGATACCCBRGTAGTC) and the reverse primer 1061R (TCACGRCACGAGCTGACGAC). The primers included the 454 Life Sciences Adapter A (for forward primers) and B (for reverse primers) fused to the 5' end of the 16S rRNA bacterial primer sequence and a unique trinucleotide sample identification key. The PCR was carried out in a coctail containing 2 units of Goldstar DNA polymerase (Eurogentec, Liege, Belgium), 1x Goldstar polymerase buffer (Eurogentec), 2.5 mM MgCl2 , 200 uM dNTP PurePeak DNA polymerase Mix (Pierce Nucleic Acid Technologies, Milwaukee, WI), 1.5 mM MgSO4 and 0.2 uM of each primer. After denaturation (94 degreesC; 2 min), 30 cycles were performed that consisted of denaturation (94 degreesC; 30 sec), annealing (50 degreesC; 40 sec), and extension (72 degreesC; 80 sec). DNA was isolated by means of the MinElute kit (Qiagen, Hilden, Germany). The quality and size of the amplicon was analzed on a Agilent 2100 Bioanalyser, using a DNA 1000 Lab Chip. DNA was quantified on a Nanodrop ND-1000 spectrophotomter.          The amplicon libraries were pooled in equimolar amounts in two separate pools. Each pool was sequenced unidirectionally in the reverse direction (B-adaptor) by means of the Genome Sequencer FLX (GS-FLX) system (Roche, Basel, Switzerland).