Microbial DNA on the Sterivex filters was retrieved using a ChargeSwitch Forensic DNA Purification Kit (Invitrogen) according to the supplier's protocol with one exception: the filters were directly suspended in the extraction solution from the kit during the cell lysis process. The V5-V6 region of the prokaryotic 16S rRNA gene was amplified using a standard PCR protocol with TaKaRa Ex Taq (TaKaRa) and the following high-performance liquid chromatography-purified primers: 784F (5′- RGGATTAGATACCC -3′) and 1064R (5′- CGACRRCCATGCANCACCT -3′) (Wang and Qian, 2009 (link); Claesson et al., 2010 (link)). Amplified DNA was concatenated to multiplex identifier tags that were unique to each sample, and a mixture of 10 samples on average was sequenced in one run on a 454 GS Junior System (Roche) after size selection (350 ± 50 bp). Pre-packaged sterile water for injection (in lieu of water from a laboratory water purification system) was used throughout the DNA extraction, PCR amplification, and DNA library preparation steps to avoid water-mediated contamination.
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