Amplicon Sequencing of Bacterial Isolates

We inoculated 5 ml of filter-sterilized nutrient broth with a single colony of E. coli K12 ATCC 10798 or S. epidermidis ATCC 14990, and grew these cultures overnight at 37°C to an OD₆₀₀ of ∼1 [nutrient broth is 8 g Difco nutrient broth powder (Invitrogen) in Milli-Q+ ultrapure water (Millipore) to 1 l; all loops, pipets, flasks, etc. were disposable sterile polycarbonate or polypropylene]. For each species, we inoculated three 250 ml flasks, each containing 50 ml nutrient broth, with 100 µl of the overnight culture. We incubated the flasks at 37°C with vigorous shaking and harvested the cultures at late stationary phase (OD₆₀₀ of ∼1.25). We then divided each culture into three aliquots, extracted genomic DNA and used each DNA extraction in a separate amplification of the V6 region. We split one amplicon library from each species into two emPCR amplification prior to pyrosequencing for a total of 10 subsamples of single clones of E. coli and of S. epidermidis. We also generated an amplicon library from a previously prepared pool of plasmid DNA from 43 different cloned 16S rRNA genes from deep-sea vent organisms (Huse et al., 2007 (link); Huber et al., 2009 (link)).
We generated amplicon libraries using primer pools designed to span the V6 or V4-V5 hypervariable regions of as many known bacteria or archaea as possible (Tables S5 and S6). We used Invitrogen Platinum HiFi Taq polymerase for amplification, as we have found that the fidelity of standard Taq is insufficient for the level of deep sequencing provided by the GS FLX. We sequenced from the A adapter on a Roche GS FLX using standard Roche protocols and supplies and the amplicon base-calling pipeline. Sequences are available at the NCBI Short Read Archive (SRP001610).
Data from environmental samples were collected as previously described (Sogin et al., 2006 (link); Huber et al., 2007 (link); Gilbert et al., 2009 (link); Turnbaugh et al., 2009 (link); McLellan et al., 2010 (link)) using the amplification and processing methods outlined above.