See Varaljay et al. 2010 for detailed methods, which are paraphrased below.

"Surface water was collected between October 2000 and April 2005 at two sampling sites at the Sapelo Island Microbial Observatory (SIMO) in coastal Georgia: the Dean Creek site (a salt marsh tidal creek) and the Doboy Sound (a coastal ocean inlet). Approximately 20 liters of water was filtered sequentially through 8.0-um, 1.0-um, and 0.2-um pore size polycarbonate membrane filters, with two replicate samples obtained at each site. Cells captured on the 1.0-um (particle associated) and 0.2-um filter (free-living) were stored at -20 degrees C until DNA extraction using a PowerMax Soil DNA Isolation kit (MO BIO Laboratories, Inc.). 76 DNA extracts were used in the study, representing 38 samples of each size fraction. Samples were separately pooled by size fraction in equal amounts to produce composite free-living and particle-associated DNA samples.

Primer pairs giving single amplicons of the correct size from the composite SIMO DNA were chosen for analysis by sequencing. Amplicons suitable for 454 sequencing were prepared by modifying each primer pair with an adaptor sequence at the 5 end of the forward primer according to the method of Huber et al. Additional four-base key sequences in between the adaptor and primer sequence were used to distinguish inosine and degenerate primer sequences. PCRs were carried out in duplicate using 24 ng template DNA and then pooled before sequencing. Amplicons were cleaned using the AMPure purification method (Agencourt Bioscience Corp., Beverly, MA) according to the 454 Life Sciences protocol (Roche Diagnostics Corp., Branford, CT), with modifications to the volume of purified PCR products (30.0 l) and AMPure beads (50.4 l). Products were quantified spectrophotometrically and combined in equal concentrations in four separate pools based on primer and size fraction. Four-region 454 FLX LR70 sequencing was carried out at the University of South Carolina EnGenCore facility. Amplicon sequences were annotated by BLASTx analysis. This analysis was used to distinguish correct target sequences from closely related paralogous sequences and to classify amplicons by clade."