BMW77_1 groundwater well from northern area
"Microbial communities were characterized using Illumina 16S rRNA gene iTag sequencing from DNA extracted using the DNeasy PowerSoil or PowerWater Kits (Qiagen) according 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. Sequence data was processed for quality control and alignment, and taxonomic assignment using mothur v1.42.3 and 1.43.0 (Schloss et al. 2009, Kozich et al. 2013, Schloss 2015) using the USGS Advanced Research Computing (ARC) Yeti high-performance computing facility. Operational taxonomic units were assigned based on a 97% similarity cutoff, with taxonomy assigned based on similarity to the Silva nr99 v132 database (Pruesse et al. 2007, Quast et al. 2012). The mothur batch script used for sequence processing is presented in the Supplementary Material. Data analysis was performed in R, using CORE components and the VEGAN, PHYLOSEQ, and DPLYR packages (McMurdie and Holmes 2013, Oksanen 2017, R Core Team 2017, Oksanen et al. 2018, Wickham et al. 2018); the code used for processing is presented in the Supplementary Material. Maximum likelihood trees for weighted UniFrac (Lozupone et al. 2011) analysis were constructed using the packages RAxML (Stamatakis 2014) and ExaML (Kozlov et al. 2015). Differential abundance analysis was performed using the package DESeq2 (Love et al. 2014)."