title: NTC - Powersoil Kit
design: DNA was extracted with MoBio Powersoil 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 QIAquick PCR Purification Kit (Qiagen) and sequenced using the Illumina MiSeq Nano platform and v2 (2x250bp, paired end reads) chemistry 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.