title: 16S rRNA amplicon sequencing of wastewater effluent treated by varying treatment methods
design: A total of 900 mL of each wastewater sample was filtered through a 0.2-micron pore size polycarbonate filter, aseptically transferred to a sterile microcentrifuge tube, and extracted using Qiagen DNeasy PowerWater Kit according to manufacturers instructions. DNA concentrations were measured with a Quantus fluorometer (Promega; Madison, Wisconsin, USA).Genomic DNA was delivered frozen to the Michigan State University Research Support Technology Facility Genomics Core (MSU RTSF; East Lansing, MI) for 16S rRNA amplicon sequencing on the Illumina MiSeq sequencing platform (Illumina; San Diego, California, USA) for library preparation and sequencing. 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), except that the index sequences were revised to unique dual index pairs. Following PCR, all amplified products were batch normalized using an Invitrogen (Carlsbad, California, USA) SequalPrep DNA Normalization plate, and the products recovered from this plate pooled. Individual pools were concentrated and cleaned using a QIAquick Spin column (Qiagen) and AMPure XP magnetic beads (Beckman Coulter; Brea, California, USA). The pools were then verified and quantified using a combination of the Qubit dsDNA HS (ThermoFisher Scientific; Waltham, Massachusetts, USA), Agilent 4200 TapeStation High Sensitivity DNA1000 (Agilent; Santa Clara, California, USA), and Invitrogen Collibri Illumina Library Quantification qPCR assays (ThermoFisher Scientific; Waltham, Massachusetts, USA). Each 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 the output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.20.0.