Materials and Methods
Sediment Collection
Samples were collected in two-day windows in January 2016 and February 2018 (see Figure 2.1 for locations of collection). Each sediment sample was taken from at least three different locations within a small area in the creek or river, then homogenized in sterile 50mL tubes for subsequent DNA extraction and quantification of metals. Biofilm samples were collected when observed in the creek or river bed. Sediment and biofilm samples were stored on dry ice until arriving at the lab, where they were kept at -80C until extraction. Temperature, salinity, pH, and redox potential were measured with a YSI Pro Plus multiparameter meter at the time of sampling. Samples collected in 2016 were also sent to the Earth Microbiome Project 500 (EMP500) for 16S, 18S, and ITS amplicon sequencing, shotgun metagenomic library sequencing, and non-targeted LC-MS/MS metabolomic analysis (data not included herein).
DNA Extraction
Genomic DNA was extracted from re-homogenized samples using the PowerSoil DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad, CA, USA) following manufacturer's protocol, with the following changes: solutions C1 and C6 were heated to 65C. Additionally, we allowed solution C6 to sit on the filter membrane for at least one minute before centrifugation and repeated the C6 step.
16S rRNA Amplicon Diversity Sequencing
Samples were amplified for bacterial 16S DNA amplicon sequencing at RTLGenomics (Lubbock, TX). In the first round of amplification for all samples (2016 & 2018), the V1-V3 of the 16S gene was targeted for sequencing using a combination of the 28F and Illumina i5 sequencing primers and the Illumina i7 sequencing primer with the 519R primer. Amplifications were performed in 25 ul reactions with Qiagen HotStarTaq master mix (Qiagen Inc, Valencia, CA, USA), 1ul of each 5uM primer, and 1ul of template. Reactions were performed on ABI Veriti thermocyclers (Applied Biosytems, Carlsbad, CA, USA) under the following thermal profile: 95C for 5 min, then 25 cycles of 94C for 30 sec, 54C for 40 sec, 72C for 1 min, followed by one cycle of 72C for 10 min and 4C hold. Amplicons from the first stage of PCR reactions were added to a second amplification step based on qualitatively determine concentrations using ethidium bromide stained gels. Primers for the second PCR were designed based on the Illumina Nextera PCR primers. The second stage amplification utilized the same cycling parameters as the first round, except it only ran for 10 cycles. Products were then pooled in equimolar concentrations and each pool was size selected in two rounds using SPRIselect (BeckmanCoulter, Indianapolis, IN, USA) in a 0.7 ratio for both rounds. Size selected pools were then quantified using the Quibit 2.0 fluorometer (Life Technologies) and loaded on an Illumina MiSeq (Illumina, Inc. San Diego, CA, USA) 2x300 flow cell at 10pM and sequenced at RTLGenomics. Less...