title: Groundwater microbiome well 4 day 4
design: DNA was extracted from groundwater with MoBio Powerwater DNA Isolation kit (MO BIO Laboratories, Inc.). Bacteria-specific PCR primers were used to amplify the 16S rRNA gene V4 region. Primer design: (Illumina linker)-(barcode)-(pad)-(linker)-(16S primer).  The two primers used were 515f (AATGATACGGCGACCACCGAGATCTACAC-NNNNNNNN-TATGGTAATT-GT-GTGCCAGCMGCCGCGGTAA) and 806r (CAAGCAGAAGACGGCATACGAGAT-NNNNNNNN-AGTCAGTCAG-CC-GGACTACHVGGGTWTCTAAT). Amplicons were purified and pooled in normalized masses using SequalPrep plates (Qiagen) and sequenced on an Illumina MiSeq sequencer and v2 chemistry (2x250bp, paired end reads) at the Center for Genome Research and Biocomputing at Oregon State University (http://cgrb.oregonstate.edu).  Unique pairs of 8 bp barcodes associated with PCR primers were used for each sample, and these barcodes were used to demultiplex the output data from MiSeq sequencing.