title: SSU rRNA Gene Hypervariable Region Tag Sequencing (Bacterial V6)
design: DNA Extraction. DNA was extracted according to the method described in Huber, J. A., et al. (2002, Appl . Environ. Microbiol . 68, 1585–1594) with minor modifications. These modifications included the addition of phenol to the chloroform:isoamyl alcohol step (25:24:1), the addition of lysozyme (200 microL; 50 mg/ml) once along with SDS and proteinase K, and resuspension of the DNA in 95 microL of TE buffer (10 mM Tris/1 mM EDTA, pH 8.0). DNA was visualized by using pulse field gel electrophoresis and quantified both spectrophotometrically and with PicoGreen (Molecular Probes, Carlsbad, CA).         PCR Amplicon Library Construction and Sequencing of Environmental Samples. We used PCR primers that flank the V6 hypervariable region of bacterial 16S rRNAs (Escherichia coli positions 967-1046). The oligonucleotide design included 454 Life Science’s A or B sequencing adapter (shown in lowercase in the following) fused to the 5' end of primer A-967F, 5'-gcctccctcgcgccatcag-CAACGCGAAGAACCTTACC-3', and B-1046R, 5'-gccttgccagcccgctcag-CGACAGCCATGCANCACCT-3'. We generated PCR amplicon libraries for each environmental DNA sample. The amplification mix contained 5 units of Pfu Turbo polymerase (Stratagene, La Jolla, CA), 1X Pfu reaction buffer, 200 microM dNTPs (Pierce Nucleic Acid Technologies, Milwaukee, WI), and a 0.2 microM concentration of each primer in a volume of 100 microL. Genomic DNA (3–10 ng) was added to three separate 30-microL amplification mixes. Cycling conditions were an initial denaturation at 94°C for 3 min; 30 cycles of 94°C 30 s, 57°C for 45 s, and 72°C for 1 min; and a final 2-min extension at 72°C. The products were pooled after cycling and cleaned by using the MinElute PCR purification kit (Qiagen, Valencia, CA). The quality of the product was assessed on a Bioanalyzer 2100 (Agilent, Palo Alto, CA) using a DNA1000 LabChip. Only sharp, distinct amplification products with a total yield of >200 ng were used for 454 sequencing. The fragments in the amplicon libraries were bound to beads under conditions that favor one fragment per bead. The beads were emulsified in a PCR mixture in oil, and PCR amplification occurred in each droplet, generating ~10 million copies of a unique DNA template. After breaking the emulsion, the DNA strands were denatured, and beads carrying single-stranded DNA clones were deposited into wells on a PicoTiter-Plate (454 Life Sciences) for pyrosequencing on a Genome Sequencer FLX system (Roche, Basel, Switzerland).