HLGB_44_2008Bact
After centrifugation of the mat/sediment/water slurry sample at 3,900*g for 20 min, approximately 5 g was used for extraction via a protocol modified from Zhou et al. (1996; PMID 8593035) that has been described previously (De Le*n et al., 2012; PMID 22476815).    Bacterial small subunit (16S) rRNA gene amplification was performed using universal bacterial primers 530F (5′-GTGCCAGCMGCNGCGG-3′) and 1100R (5′-GGGTTNCGNTCGTTR-3′) under conditions described in Hwang et al. (2009; PMID 18769457).    All primers had 10-nt barcodes on the 5′ end for sample identification purposes.  Cycle numbers were optimized for each sample to minimize PCR-induced artifacts with 10-12 ng DNA per reaction  Amplicons were sequenced on a 454 GS-FLX Titanium Pyrosequencer at SeqWright, Incorporated (Houston, Texas, USA).