title: Cone Pool Raw 16S reads, 2015
design: Extracted DNA was amplified using 16S rRNA V4 primers 519F and 802R. The forward primer (M13L-519F: 5'- GTA AAA CGA CGG CCA GCA CMG CCG CGG TAA -3') contains the M13 forward primer, followed by the 16S rRNA gene-specific sequence to allow for barcoding of each sample in a separate reaction (Stamps et al., 2016). The reverse primer (785R: 5'-TAC NVG GGT ATC TAA TCC-3') was taken directly from the reverse primer ńS-D-Bact07850b-A-18î in Klindworth et al. (2013). Each 50 ľL reaction mixture consisted of: 1X 5 PRIME HOT master mix, 0.2 ľM of each primer, and molecular grade water. A volume of 4 ľL of extracted template DNA was added to each reaction. PCR reactions were then purified using AmpureXP paramagnetic beads at a final concentration of 0.8 x v/v. A second, six cycle PCR was used to add a unique 12 bp barcode (Hamady et al., 2008) to each previously amplified sample using a forward primer containing the barcode+M13 forward sequence (5'-3') and the 785R primer. The final barcoded PCR products were again cleaned using AmpureXP paramagnetic beads at a final concentration of 0.8X, quantified, pooled in equimolar amounts, and concentrated to a final volume of 80 ľL using two Amicon Ultra-0.5 mL 30K Centrifugal Filters. The final pooled library was run on the Illumina MiSeq platform using PE250 V2 chemistry.