qiime_experiment_1026:380614:166
Saliva samples were collected for genotyping study. Community DNA was extracted, prepared and used for 16S rRNA pyrosequencing (454 Life Sciences).
S08496:166
Mouthwash-saliva mixture was centrifuged and genomic DNA was isolated from pellet with Puregene (Gentra, now Qiagen) kit. The remaining steps were performed as directed by the manufacturer.16S rRNA genes were amplified by PCR with a forward primer containing the 454 Life Sciences primer B sequence and the broadly conserved bacterial primer 27F,and a reverse primer containing the 454 Life Sciences primer A sequence, a unique 12-nucleotide error-correcting barcode used to tag each PCR product, and the broad-range bacterial primer 338R. PCR reactions were carried out in  triplicate. 25 ul reactions with 0.6 uM forward and reverse primers, with 1 ul template DNA, and 1X of Invitrogen Platinum PCR Super mix. Thermal cycling consisted of initial denaturation at 94C for 5 minutes followed by 32 cycles of denaturation at 94C for 45 seconds, annealing at 50C for 30 seconds, and extension at 72C for 90 seconds. Replicate amplicons were pooled, visualized by electrophoresis using 1/0% agarose gels and  purified with Mobio Ultraclean-htp. Negative extraction control samples were treated similarly except that no template DNA was added to the PCR reactions: these failed to produce visible PCR products, but were still sequenced.  Amplicons  were quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen). Amplicons were combined in equimolar ratios into a single tube with a final concentration of 80 ng/ul. Pyrosequencing was carried on a 454 Life Sciences Genome Sequencer Titanium instrument at Memorial Sloan-Kettering Cancer Center, NY.