Microbiome of biofilm associated with a nitrate sensor
All DNA concentrations were determined using a Quantus fluorometer (Promega; Madison, WI) and the Promega QuantiFluor dsDNA system.  Genomic DNA was delivered frozen to the Michigan State University Research Support Technology Facility Genomics Core (MSU RTSF; East Lansing, MI) for library preparation and 16S rRNA amplicon sequencing on the Illumina MiSeq sequencing platform (Illumina; San Diego, CA). The V4 hypervariable region of the bacterial 16S rRNA gene was amplified with dual-indexed, Illumina compatible primers (515F: GTGCCAGCMGCCGCGGTAA /806R:  GGACTACHVGGGTWTCTAAT) following the protocol described by Kozich et al. 2013.  Following PCR, all amplified products were batch normalized using an Invitrogen (Carlsbad, CA) SequalPrep DNA Normalization plate and the products recovered from this plate pooled. The pool was verified and quantified using a combination of the Qubit dsDNA HS (Invitrogen; Carlsbad, CA), Agilent 4200 TapeStation High Sensitivity DNA1000 (Agilent; Santa Clara, CA), Invitrogen Collibri Illumina Library Quantification qPCR assays(). The pool was sequenced using an Illumina MiSeq v2 standard flow cell in a 2 x 250 base pair (bp) paired end format using a v2 500 cycle reagent cartridge. Custom sequencing and index primers complementary to the 515F/806R target specific sequences were added to appropriate wells of the reagent cartridge.  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.