16S sequencing of microbial metagenome: ditch soil
~0.25 g soil extracted with DNeasy PowerSoil kit (Qiagen); Extracts were quantified on a Qubit Fluorometer; DNA amplified in duplicate using 515FB and 806RB with 60  annealing temperature. PCR reactions contained 20  5Prime master mix (Quanta), ~20 ng DNA, primers, and nuclease-free H2O to 50 . Duplicate amplicons were pooled, visualized on an agarose gel, and purified (Agencourt AMpure XP, Beckman Coulter).