title: Fecal microbiome of Odocoileus virginianus
design: Scat (fecal material) was collected from hunter-harvested deer in sterile specimen cups, and mailed on ice overnight to the USGS Columbia Environmental Research Center (CERC; Columbia, Missiouri) where they were frozen upon receipt.   Frozen fecal samples were shipped to the USGS Michigan Bacteriological Research Laboratory (MI-BaRL) in Lansing, Michigan. Genomic DNA (gDNA) was extracted in triplicate from fecal samples using the DNeasy PowerSoil Pro kit (Qiagen, Inc., Hilden, Germany) according to the manufacturers instructions. DNA from the triplicate extractions were combined with ethanol precipitation. Field blanks and extraction blanks were extracted and processed with environmental samples.  Extracted gDNA was quantified using a Quantus fluorometer (Promega; Madison, WI) in preparation for amplicon sequencing. Field blanks and extraction blanks were extracted and processed with environmental samples. Blanks did not yield quantifiable DNA or did not successfully amplify during PCR and were removed from downstream processing.  DNA extracts were sent to Michigan State Universitys Research Technology Support Facility (MSU RTSF, East Lansing, MI) for Illumina 16S rRNA amplicon 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 described in Kozich et al. (2013). Following PCR, all products were batch normalized using Invitrogen SequalPrep DNA Normalization Plate Kit (Invitrogen, Carlsbad, California) and product recovered from the plate pooled. The pool was concentrated and cleaned up using a QIAquick PCR Purification Kit (Qiagen, Inc., Hilden, Germany) and AMPure XP magnetic beads (Beckman Coulter, San Jose, California). 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 Collibri Illumina Library Quantification qPCR assays (Invitrogen, Waltham, MA). This pool was loaded onto an Illumina MiSeq Standard v2 flow cell and sequencing was performed in a 2x250bp paired end format using a MiSeq 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 v2.20.0.
library_name: DS_33_1-10_S62