16S rRNA gene pyrosequences from Columbia River coastal margin across a river to ocean gradient from 2007 and 2008
384 samples were PCR-amplified using primers targeting bacterial 16S ribosomal RNA genes. Each sample was assigned a uniquely barcoded reverse primer and amplified in four replicate 20 µl reactions (Hamady et al 2008). Primers used for amplification were bacteria-specific primers focusing on the V2 region, 27F with 454B FLX linker (GCCTTGCCAGCCCGCTCAG TC AGRGTTTGATYMTGGCTCAG) and 338R with 454A linker and unique 8 basepair barcode, denoted by N in primer sequence (GCCTCCCTCGCGCCATCAG NNNNNNN CA TGCWGCCWCCCGTAGGWGT) (Modified from Hamady et al 2008). Replicate amplifications were combined, purified, and normalized using Invitrogen SequelPrep normalization plates (Invitrogen, Carlsbad, CA, USA). Five µl from each sample were combined into a single tube and sent for pyrosequencing on a Roche-454 FLX pyrosequencer at Engencore at the University of South Carolina (http://engencore.sc.edu/).