title: 16S rRNA amplicon sequencing of surface water from Lake Superior and Lake Superior tribuitaries, 2021-2057
design: Runoff events were sampled with a combination of manual grab sampling and automated sampling with ISCO samplers at locations with stream gages and manually at the Bois Brule Rivermouth (Station ID040260078) and Siskiwit Rivermouth (Station ID 465130091060701). Near monthly, manual base-flow samples also were collected at the sites. Samples were collected during the open water season of March through October in 2021 and 2022. Nearshore water quality transect sampling was conducted along the southwestern shore of Lake Superior following a storm event on September 22-23, 2021 and during a wind-driven sediment resuspension event on August 23-26, 2022. Transects ran from the tributary mouths out into the nearshore. A total of 700 mL of surface water was filtered through a 0.22 m filter. The filter was then placed in a sterile microcentrifuge tube and frozen. Frozen filters were thawed and aseptically placed into a PowerWater Bead Tube (Qiagen) and then DNA was extracted using the Qiagen DNeasy PowerWater Kit according to manufacturers instructions. DNA was subsequently extracted from the sediment in triplicate using the Qiagen DNeasy PowerSoil Pro Kit according to manufacturers instructions. DNA from the triplicate extractions were combined with ethanol precipitation. All DNA concentrations were quantitated with a Quantus fluorometer (Promega; Madison, WI) using 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 16S rDNA amplicon sequencing on the Illumina MiSeq sequencing platform (Illumina; San Diego, CA) for library preparation and sequencing. The Bacteria 16S rRNA V4 hypervariable region was amplified with dual indexed, Illumina compatible primers (515f/806r) 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. Pools were concentrated and cleaned using a QIAquick Spin column and AMPure XP magnetic beads (Beckman Coulter; Indianapolis, IN). The pool was then checked for quality and quantified using a combination of the Qubit dsDNA HS (Invitrogen; Carlsbad, CA), Agilent 4200 TapeStation HS DNA1000 (Agilent; Santa Clara, CA) and Invitrogen Collibri Illumina Library Quantification qPCR assays (ThermoFisher; Waltham, MA). Each pool was sequenced using an Illumina MiSeq v2 standard flow cell in a 2x250 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. Raw sequencing data were deposited into the NCBI Short Read Archive database.