title: 16S rRNA gene pyrosequencing based microbial community analysis of FranzJosef chronosequence
design:  The nine ages were: 5y, 60y, 130y, 280y, 500y, 1000y, 5000y, 60000y, and 120000y. Each age comprised of 5 independant replicates collected from a transect of five independent 5-m radius plots using a 6.5cm diameter corer (0 to 10-cm depth). Total community DNA was extracted from 0.5 g of soil using the ZR Soil-Microbe DNA™ kit (Zymo Research).   For pyrosequencing, partial bacterial 16S rRNA gene fragments were PCR-amplified in 20-cycle reactions using primer 515R-NK linked to the 454 Life Science’s FLX-A sequencing adaptor, and a sevenfold-degenerate primer 27F-YM+3 linked to the FLX-B sequencing adaptor. An 8-nt sample-specific barcode was added between the A-adaptor and primer 515R-NK and was used to bioinformatically separate amplicons from the multiplexed reactions post-sequencing.    Library construction protocol: Each DNA was separately amplified in a 25 µl PCR reaction that contained 1.25 µl (20-50 ng) of DNA, 12.5 pmol of each primer, and 22.5 µl of Platinum® PCR SuperMix High Fidelity (Invitrogen). Reaction conditions included an initial denaturation at 95°C for 3 min followed by 20 or 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 68°C for 1 min; and a final elongation at 68°C for 4 min on a Veriti® 96-well Thermal Cycler (Applied Biosystems). The PCR products were electrophoresed on 1% agarose gel in 0.5x TBE, stained with SYBR® Green I nucleic acid gel stain (Invitrogen) and scanned using Typhoon Trio+ Variable mode imager (GE Healthcare). The resultant images were quantified using Image Quant 5.2 (Molecular Dynamics). PCR products from replicates of a soil age were then pooled in equimolar concentrations, followed by gel elution using Zymoclean™ Gel DNA Recovery Kit (Zymo Research). The eluted amplicons were quantified on the Experion System (Bio-Rad), followed by mixing of amplicons from all soil ages in equimolar concentration. The final mixed amplicon pool was further purified using the Agencourt AMPure XP system (Beckman Coulter Genomics) and submitted to the Environmental Genomics Core Facility at the University of South Carolina for pyrosequencing. Emlusion PCRs were carried out as per their protocols. Design description: Forward Sequecing primer: CCGCNGCKGCTGGCAC  The table below describes the barcodes for the samples.    Sample Library ID Barcode 5y, rep 1 FA1 ACGAGAAC; 5y, rep 2 FA2 ACGAGTTC; 5y, rep 3 FA3 ACGATGGA; 5y, rep 4 FA4 ACTCACTC; 5y, rep 5 FA5 ACTCCTCA; 60y, rep 1 FB1 ACTCCTGT; 60y, rep 2 FB2 ACTCGTCT; 60y, rep 3 FB3 ACTCTCAC; 60y, rep 4 FB4 ACTCTGTC; 60y, rep 5 FB5 ACTGACAC; 130y, rep 1 FC1 ACTGGAGA; 130y, rep 2 FC2 ACTGTCTC; 130y, rep 3 FC3 AGACACTC; 130y, rep 4 FC4 AGACCTCA; 130y, rep 5 FC5 AGACGACA; 280y, rep 1 FD1 AGACTCAC; 280y, rep 2 FD2 AGAGACAC; 280y, rep 3 FD3 AGAGAGTC; 280y, rep 4 FD4 AGAGGAGA; 280y, rep 5 FD5 AGGAAGGA; 500y, rep 1 FE1 AGGACAAC; 500y, rep 2 FE2 AGGTAGGT; 500y, rep 3 FE3 AGGTCATC; 500y, rep 4 FE4 AGGTTCGT; 500y, rep 5 FE5 AGTCACAC; 1k, rep 1 FF1 AGTCCTCT; 1k, rep 2 FF2 AGTCGTCA; 1k, rep 3 FF3 AGTCTCTC; 1k, rep 4 FF4 ATCCGCAA; 1k, rep 5 FF5 ATCCGCTT; 5k, rep 1 FG1 ATCCTACC; 5k, rep 2 FG2 ATCGTTCC; 5k, rep 3 FG3 CAACTCCT; 5k, rep 4 FG4 CAACTGGT; 5k, rep 5 FG5 CAAGACCT; 60k, rep 1 FH1 CAAGACGA; 60k, rep 2 FH2 CAAGAGGT; 60k, rep 3 FH3 CAAGGAAC; 60k, rep 4 FH4 CAAGGTTC; 60k, rep 5 FH5 CAAGTCGT; 120k, rep 1 FI1 CACAACAC; 120k, rep 2 FI2 CACAAGTC; 120k, rep 3 FI3 CACACACA; 120k, rep 4 FI4 CACACTCT; 120k, rep 5 FI5 CACACTGA 