HPVpos5
Amplicons of ~456pb containing V3-V4 variable regions from 16S rRNA gene were obtained for DNA libraries preparation. Primers 347F Forward 5'-GGAGGCAGCAGTRRGGAAT-3' and 803R Reverse 5'-CTACCRGGGTATCTAATCC-3', described by Nossa et. al 2010, were used. A first PCR was performed with the following conditions: 50ng  of template from each DNA sample were added to a final reaction mixture of 30?l containing 1X reaction buffer, 2.5 mM MgSO4, 1mM dNTP, 0.2U Platinum Taq DNA Polymerase High Fidelity (Invitrogen) and 5pmol/?L of the primers. The amplification program was: 94şC for 3 minutes, then 30 cycles at 94şC for 30 seconds, 55şC for 30 seconds, 68şC for 30 seconds; followed by 68şC for 5 minutes. Amplicons were visualized in 1% agarose gel electrophoresis. DNA bands obtained were purified by MinElute from Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany).  Amplicons were re-amplified and processed with the same primers linked to adapter A of the 454 sequencing protocol followed by 6-mer multiplex identifier and followed by the Primer 347F. The reaction conditions were: 0.0625 ng of each DNA sample to a final reaction mixture of 30?l containing 1X reaction buffer, 2.5 mM MgSO4, 1 mM dNTP, 0.2 U Platinum Taq DNA Polymerase High Fidelity (Invitrogen) and 5pmol/?L of the primers 347F and 803R. Amplification cycles were: 94şC for 3 minutes, then 15 cycles at 94şC for 30 seconds, 55şC for 30 seconds, 68şC for 30 seconds; followed by 68şC for 5 minutes. Gel electrophoresis was performed in 1.5% agarose, to visualize the integrity of the amplified products. DNA fragments were purified by MinElute from Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany) and were quantified and assessed.