MW-GA
Microbial communities were characterized using Illumina 16S rRNA gene iTag sequencing. Groundwater samples for microbial community analyses were collected by pumping a minimum of 1 liter of water through a 0.22 M Sterivex-GP filter in the field. The filters were frozen on dry ice then shipped overnight to the USGS Reston Microbiology Laboratory, Reston, Virginia for DNA extractions using the DNeasy PowerWater Sterivex kit (Qiagen, Inc., Hilden, Germany) according to the manufacturers instructions. Extracted DNA was quantified using Quant-iT PicoGreen dsDNA assay kit (Invitrogen, Carlsbad, CA), following the procedure described in Blotta et al. (2005) with slight modifications. DNA extracts were sent to Michigan State Universitys Research Technology Support Facility (RTSF, East Lansing, MI, USA) for Illumina 16S iTag sequencing. Amplicon libraries of the V4 hypervariable region of the 16S rRNA gene were prepared using dual indexed, Illumina compatible primers 515f and 806r (Apprill et al. 2015, Parada et al. 2016, Walters et al. 2016) following the protocol developed by the Patrick Schloss lab (Kozich et al. 2013). Following polymerase chain reaction (PCR), all products were batch normalized using Invitrogen SequalPrep DNA Normalization (Invitrogen, Carlsbad, California) plates and product recovered from the plate pooled. The pool was quality controlled and quantified using a combination of Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA), Agilent 4200 TapeStation System HS DNA1000 (Agilent Technologies, Santa Clara, CA) and Kapa Library Quantification Kit qPCR assays (Roche Holding AG, Basel, Switzerland).  This pool was loaded onto an Illumina MiSeq Standard v2 flow cell and sequencing was performed in a 2x250bp paired end format using a 500 cycle v2 reagent cartridge. Custom sequencing primers were added to appropriate wells of the reagent cartridge as described in Kozich et al. 2013. Base calling was done by Illumina Real Time Analysis (RTA) v1.18.54 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.19.1.