MiSeq of environmental samples
Samples were collected using a long pole sampler which held one sterile sample bottle per sampling location, and the bottle was filled to its maximum volume to minimize air exposure. Upon arrival in the lab, 50% the samples were filtered using a Millipore SterivexTM 0.22 m pressure filters attached to a peristaltic pump as specified in the user manual. DNA extraction from filtered field samples was performed using DNeasy PowerWater Sterivex Kit from Qiagen following the manufacturers instructions. Extracted total DNA was amplified for the V4/V5 region of the 16S rDNA gene and sequencing was performed on an Illumina MiSeq sequencer. Dual indexed barcode adapters were added to the ends of the 16S rDNA amplicons by limited cycle PCR. Sequencing libraries were validated by automated electrophoresis using Agilent Tapestation 4150 and quantified by Thermo-Fisher Scientific Qubit and Applied Biosystem 7500 Fast real-time PCR. DNA libraries were multiplexed and loaded on an Illumina MiSeq instrument according to manufacturer's protocol. Base calling was conducted by the MiSeq Control Software on the MiSeq instrument.