title: Pyrosequencing controls for 16S rRNA gene sequence based analysis of microbial communities in FJ-HA-GA chronosequences
design:  To optimize the sequence analysis pipeline, a set of six controls prepared from cloned 16S rRNA gene fragments representing different G+C content were used in the sequencing run: Control 1 (EF662369; Actinobacteria), Control2 (EF665883; Gammaproteobacteria), control3 (EF664802: Firmicutes), control 4 (C1:C2:C3::1:1:1), control 5 (C1:C2:C3::1:2:3) and control6 (C1:C2:C3::3:2:1). While controls 1 to 3 used pure cloned plasmids as templates for PCR amplification, controls 4 to 6 used a mixture of these cloned plasmids in the ratios mentioned.  For pyrosequencing, the cloned 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 Sequencing primer: CCGCNGCKGCTGGCAC.  The table below describes the barcodes for the samples.    Sample Library ID Barcode Control 1 C1 CCTTGGTT; Control 2 C2 CGAACCTA; Control 3 C3 CGAACGAA; Control 4 C4 CGAATACC; Control 5 C5 CGATAACC; Control 6 C6 CGATGGAA 
identifiers.namespace: University of Georgia
identifiers.id: FJ-HA-GA Controls