title: S14-V6-Z-B17-calc
design: "Library construction Z for S14 extract of B17. V6 primers; primers are NOT trimmed. General information: Three sets of universal PCR primers were designed to amplify the 16S rRNA gene V3 (351-507), V5 (800-900), and V6 (958-1044) regions, respectively. Because this study focuses on highly fragmented ancient DNA, the PCR target regions must be very short (<250 bp). The primers used to amplify these regions are: V3F 5?-ACTCCTACGGGAGGCAGCAGT-3?, V3R 5?-GTATTACCGCGGCTGCTGGCAC-3?; V5F 5?-CAGGATTAGATACCCTGGTAGTCC-3?, V5R 5?-CCCGTCAATTCCTTTGAGTT-3?; V6F 5?-GGTGGAGCATGTGGTTTAATTCGA-3?, V6R 5?-GAGCTGACGACAGCCATGCA-3?. Expected amplicon sizes for these primer pairs are: V3 (~200 bp), V5 (~145 bp), and V6 (~130 bp). The Fusion Adaptor sequences were: forward, 5?-CGTATCGCCTCCCTCGCGCCATCAG-3?; and reverse, 5?- CTATGCGCCTTGCCAGCCCGCTCAGA-3?. A unique, 9-base MID was also added to each reverse Fusion Primer sequence, and a total of 10 MIDs were designed so that multiple samples could be pooled in a single 454 run. These MIDs also served to prevent cross-contamination in the 454 sequencing facility as only sequences with a valid MID from our study were accepted for further analysis. 454 libraries were prepared for the calculus, root, carious root, and alveolar bone abscess extracts using V3, V5, and V6 454 Fusion primer sets. Duplicate extracts for the root and bone samples were pooled before amplification. Each 25ml PCR reaction was set up as follows: 0.25ml Phusion Hot Start II polymerase, 5ml 5X Phusion HF buffer, 2.5ml of 2mM dNTPs, 1ml of 2.5mg/mL BSA, 1ml of 10 mM forward primer, and 1ml of reverse primer, plus a sample-dependent volume of extract and water (~2-5ng DNA per reaction). Cycling conditions were performed as follows: enzyme activation at 98?C for 30s, followed by 30 cycles of denaturation at 98?C for 15s, annealing at 65?C for 20s, and extension at 72?C for 20s, and ending with 72?C for 5 min. The V3 region was amplified for an additional 5 cycles (total of 35 cycles) to compensate for the fact that the V3 454 Fusion primers were found to form primer dimers. Slowing the temperature ramp speed of the thermocycler to ?2?C/s was found to reduce primer dimer formation. No amplification was observed in the negative extraction controls or reagent blanks. A minimum of three amplifications were performed for each sample, and the resulting PCR products were pooled and concentrated using a QiaQuick PCR Purification Kit (Qiagen). DNA concentration was measured with a Qubit DNA High-Sensitivity Assay Kit (Invitrogen), and additional PCR amplifications were performed, if necessary, until the total DNA yield for the pooled and cleaned PCR products was ?300ng. A 30ml aliquot with a concentration of 10ng/ml was submitted to Omya A.G. (Switzerland) for sequencing. Amplicon libraries were titrated to equimolar concentrations and sequenced on a 454 GS Junior System using 454 Titanium Chemistry (Roche) by Omya AG. A second set of G12 root libraries for the V3 and V5 regions were sequenced again in a second run as quality control duplicates. Sequenced reads were quality filtered using the Roche GS RunProcessor amplicon pipeline with default settings, and reads without a valid MID were discarded."