bac.co14.5
Bacterial libraries were generated using 515-Y and 926R primer pair that encompasses V4 and V5 hypervariable regions of the 16S rDNA (Parada, Needham, &amp; Fuhrman, 2016. Amplicons were produced in a two-step protocol as suggested by Berry et al. (2011). First-round PCR was carried out in 25  uL reactions containing 1X Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific, Inc., Waltham, MA), an additional 1.5 mM MgCl2 (3.0 mM total), 200 nM each primer, and 2  uL each DNA sample. Cycling conditions were 95oC for 2 min, then 25 cycles of 95oC for 30 sec, 55oC for 30 sec, and 60oC for 4 min. PCR products were checked on 1% agarose gel, purified by bead-prep. Indexing and flowcell sequences were added in second round amplification under the same reaction conditions and using 2uL of purified, first-round product as template, but for only 15 cycles. Indexed PCR products were purified using carboxylated magnetic beads as described in Rohland & Reich (2012), quantified by PicoGreen (Life Technologies, Inc, Carlsbad, CA) fluorescence, and an equal mass of each sample was combined into a final sample pool. The pool was then purified and subsequently quantified by qPCR against Illumina DNA Standards (Kapa Biosystems, Wilmington, MA).