DOI: 10.5061/dryad.2z34tmpp7
title: Saw Kill river (NY, USA) metagenomics and environmental variables

description: Microbial community structure and diversity in waterways are altered by
 wastewater treatment plant (WWTP) discharge which can introduce
 human-associated microbes, antimicrobial resistance (AMR), and
 significantly change environmental variables. To better understand these
 interactions, we investigated the impact of the Bard College WWTP on
 microbial communities collected from the surface water and sediment from
 four sites, two sites above and two sites below the Bard College outflow,
 as well as the outflow itself, this was performed over a period of five
 months. We measured physico-chemical parameters such as temperature,
 turbidity, conductivity, dissolved oxygen, and salinity as well as the
 bioindicators Escherichia coli, total coliforms, and Enterococcus sp.
 concentration, endotoxins, the intI1 gene marker, and the total bacterial
 abundance through 16S rRNA gene.
description: <strong>Study
 site</strong> The Saw Kill
 river is a 23.0 km tributary of the Hudson River that drains 57
 km<sup>2</sup> area of northwestern Dutchess County, New York,
 USA. The Saw Kill flows predominantly through forests and farmland at a
 mean rate of 19 cubic feet/sec (ranging from 0.4 – 323 cubic
 feet/sec). Bard College uses a
 wastewater treatment plant that operates by filtration, sedimentation,
 fermentation in a bioreactor network, and chlorination to treat waste and
 gray water. The treated wastewater is then sent down for aeration followed
 by de-chlorination before being released into the Saw Kill via a single
 outflow pipe at a site approved by the New York State Department of
 Environment Conservation (SPDES Permit # NY0031925). The treated
 wastewater is released in an area located near the mouth of the river
 (42.017226, -73.915546). Upstream
 (5.2 km) of Bard’s campus is Red Hook, a village (pop. ~1900) with a small
 WWTP (0.05 MGD flow, NY SPDES permit #NY0271420). Furthermore, the river
 runs through areas of rural and exurban habitation and agricultural land
 use. To control for this we sampled both upstream (Above) and downstream
 (Below) of the Bard WWTP outflow (Outflow). <strong>Sample
 collection</strong> Samples
 were collected on ten different occasions over a period of five months
 ranging from June 6, 2015, and October 20, 2015 (Table 1). For each
 collection date, we collected 2 L of treated wastewater directly from the
 WWTP outflow, 2 L of surface water from two sites below the outflow, and
 from two sites above the outflow and two ~500 mg sediment cores at each
 collection site. Sampling began with the most downstream site and worked
 upstream, to eliminate influence of water content or sediment disturbance
 on subsequent samples. In total, we thus collected 130 samples, including
 40 surface water samples, 80 sediment samples, and 10 wastewater samples
 from the WWTP outflow (Table 1). For each site, we first collected the water sample
 in the mid-channel using heat and acid-sterilized Nalgene bottles
 submerged ~0.5 m below the surface of the stream. To avoid possible
 contamination that could be present on the surface of the bottles, all
 sample bottles were rinsed three times with surface waters from the site
 immediately before collection. After collecting the water samples,
 sediment samples were collected using a stainless-steel corer, which was
 cleaned with a wipe, sterilized with 70% ethanol, and air-dried in between
 each sampling. At each site, duplicate cores about 7 cm deep were
 collected from undisturbed sediment and placed in a sterile 50 ml falcon
 tube using a sterilized metal spatula. Finally, for each collection date,
 a single 2 L samples from the WWTP were taken from the end of the outflow
 pipe using a sterilized 1,000-ml scoop and stored in heat and
 acid-sterilized Nalgene bottles. Following collection, all samples were
 placed in a cooler for transport to the lab and processed within 2 hours
 of collection.  <strong>Nucleic acid
 extraction</strong> We
 extracted total DNA from water samples by filtering 750 mL of water onto a
 0.22 µm Sterivex filter. We then extracted DNA from the filter using the
 PowerWater DNA Isolation kit (MoBio Laboratores Carlsbad, CA, USA), now
 available as the DNeasy PowerWater DNA Isolation Kit (QIAGEN, Hilden,
 Germany), per manufacturer’s instructions. As for sediment samples, we
 weighed 250 mg of sediment samples and used the PowerSoil DNA Isolation
 Kit (MoBio Laboratories, Carlsbad, CA, USA), now available as the DNeasy
 PowerSoil DNA Isolation kit (QIAGEN, Hilden, Germany) as per
 manufacturer’s instructions. <strong>Physico-chemical water quality
 indicators</strong> At each
 sampling site, before collecting water and sediment samples, we measured
 water temperature (°C), conductivity (µmhos/cm), dissolved oxygen (ppm),
 and salinity (ppm) using a handheld YSI field probes (YSI, TN, USA)
 suspended at &lt; 0.5 m depth mid-channel. Turbidity was measured
 using 15 mL aliquots from shaken 2L sample bottles with a Hach 2100P
 Turbidimeter (Loveland, CO). Precipitation and air temperature data
 collected by a local public weather station. <strong>Concentrations of microbial water
 quality indicators</strong> In each sample, we measured the abundance of three
 fecal indicators: <em>Enterococcus</em>,
 <em>Escherichia</em> <em>coli</em>, and Total
 Coliforms. All three indicators were measured using the IDEXX MPN method
 (IDEXX Laboratories, ME, USA), as per manufacturer instruction and within
 2 hours of sample collection in the field. To run the IDEXX Colilert
 assay, which estimates both E. coli and coliform concentrations, on
 mid-channel water samples, a 100 mL undiluted sample and one 100 mL sample
 diluted 1:10 with sterile DI water were assayed. For the WWTP outflow
 samples, a 1:10 and 1:100 dilution with sterile DI water were assayed. For
 sediments, slurries were prepared by adding 250 mg of centrifuged sediment
 to 50 mL of sterile DI water and mixing gently. A 1:10 and 1:100 dilution
 of the sediment slurry was assayed. For each sample assayed, Colilert
 reagents were dissolved in the sample in a sterile 100 mL vial. Once
 dissolved, the mixture was poured into a 49-well sterile Quanti-Tray
 (IDEXX) and sealed. The trays were then incubated for 24 hours at 35°C.
 Following incubation, the Quanti-Tray were enumerated for positive counts
 where all cells that have turned yellow are considered positive for
 coliform and all yellow cells that fluoresce under UV excitation are
 considered positive for <em>E</em>. <em>coli</em>.
 The concentrations of <em>E</em>. <em>coli</em>
 and Coliforms indicators were then calculated as CFU/mL by applying the
 Most Probable Number (MPN) method to the number of positive
 cells. To estimate the
 concentration of <em>Enterococcus</em> sp. in each sample, we
 used the IDEXX Enterolert assay (IDEXX, ME, USA). For surface water and
 outflow samples, we assayed 100 mL of undiluted sample while we used
 undiluted slurry (see above for details) for sediment samples. Enterolert
 reagents were dissolved in the 100 mL of sample in a sterile 100 mL vial.
 Once dissolved, the mixture was poured into a 49-well sterie Quanti-Tray
 (IDEXX) and sealed. The trays were then incubated for 24 hours at 41°C.
 After incubation, we used the fluorescence under UV light to estimate
 CFU/mL using the standard MPN method. <strong>Concentration of
 endotoxins</strong> Endotoxins were measured in water samples within 4
 hours of sampling using the Charles River Endosafe system (Charles River,
 Cambridge, MA, USA) with cartridges supporting a 10-0.1 EU/ml measurement
 range. Prior to measurement, using endotoxin-free pipette tips, 20 uL of
 water sample was diluted with 1980 uL of sterile, endotoxin-free Hyclone
 water in a sterile and endotoxin-free glass test tube to create a 1:100
 dilution. As per Charles River’s Endosafe protocol, once a new sterile
 cartridge was validated by the Endosafe system, 25 uL of samples were then
 pipetted into each of the 4 cartridge wells without introducing bubbles.
 These were treated as duplicate raw and duplicate spike readings. The
 assay was run for 5-15 minutes of testing time before displaying data.
 Readings that were fully validated by the instrument (those whose spike
 returns were between 50 and 200%, and whose replicate variations (both
 sample and spike) had a coefficient of variation &lt; 25%)  were
 recorded. Invalid tests prompted a second assay, using a 1:1000 dilution
 of the sample to dilute contaminants and/or bring the sample into
 measurement range.  <strong>Relative abundance of Integron
 1</strong> Using the primers
 (Int1F2: TCGTGCGTCGCCATCACA, Int1R2: GCTTGTTCTACGGCACGTTTGA), (Gaze, et
 al. 2011). We processed each sample in triplicate using the PowerUp SYBR
 Green Master Mix (Applied Biosystems, Foster City, CA, USA) and using the
 Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories,
 Hercules, CA, USA).  For each run, we built an internal standard curve
 using at least three dilutions of the strain
 <em>Escherichia</em> <em>coli</em> SK4903 with
 IncPβ R751, which was constructed to contain seven
 <em>16S</em> <em>rRNA</em> copies and six
 <em>intI1</em> copies. Finally, we adjusted the total number
 of <em>16S</em> <em>rRNA</em> copies found in each
 sample by dividing that number by 4.2, which is the estimated average
 number of <em>16S</em> <em>rRNA</em> copies each
 bacteria cell harbors (Větrovský, et al. PLOS ONE, 2013). <strong>Amplification of
 <em>16S</em> <em>rRNA</em> sequences and
 analysis</strong> A
 <em>16S</em> <em>rRNA</em> gene amplicon
 sequencing library targeting the V4 region was amplified using primers
 515F and 806R. Samples were shipped to Wright Labs (Huntingdon, PA, USA)
 for sequencing with the Illumina Miseq platform using 250-bp paired
 ends. Sequences were filtered and
 trimmed with Trimmomatic, ver. 0.39 (Bolger, et al. Bioinformatics, 2014)
 using the following parameters: ILLUMINACLIP:TruSeq3-PE.fa:2:30:10
 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:100. All subsequent
 analysis was performed using QIIME2, ver 2019.2 (Bolyen et al. Nat
 Biotechnol 2019). Reads were resolved, denoised, and clustered into
 amplicon sequence variants (ASVs) using DADA2 (denoisepaired,
 --p-trim-left-f 13, --p-trim-left-r 13, --p-trunc-len-f 150,
 --p-trunc-len-r 130). Taxonomic assignment was performed using QIIME2’s
 naive Bayes scikit-learn classifier trained using the 16S rRNA gene
 sequences in SILVA database (Silva SSU 132), (McDonald, et al. ISME J
 2012). <strong>Works
 cited</strong> Bolger, A. M., Lohse, M. & Usadel, B.
 Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics
 30, 2114–2120 (2014). Bolyen, E.,
 Rideout, J. R., Dillon, M. R., Bokulich, N. A., Abnet, C. C., Al-Ghalith,
 G. A., Alexander, H., Alm, E. J., Arumugam, M., Asnicar, F., Bai, Y.,
 Bisanz, J. E., Bittinger, K., Brejnrod, A., Brislawn, C. J., Brown, C. T.,
 Callahan, B. J., Caraballo-Rodríguez, A. M., Chase, J., Cope, E. K., Da
 Silva, R., Diener, C., Dorrestein, P. C., Douglas, G. M., Durall, D. M.,
 Duvallet, C., Edwardson, C. F., Ernst, M., Estaki, M., Fouquier, J.,
 Gauglitz, J. M., Gibbons, S. M., Gibson, D. L., Gonzalez, A., Gorlick, K.,
 Guo, J., Hillmann, B., Holmes, S., Holste, H., Huttenhower, C., Huttley,
 G. A., Janssen, S., Jarmusch, A. K., Jiang, L., Kaehler, B. D., Kang, K.
 B., Keefe, C. R., Keim, P., Kelley, S. T., Knights, D., Koester, I.,
 Kosciolek, T., Kreps, J., Langille, M. G. I., Lee, J., Ley, R., Liu,
 Y.-X., Loftfield, E., Lozupone, C., Maher, M., Marotz, C., Martin, B. D.,
 McDonald, D., McIver, L. J., Melnik, A. V., Metcalf, J. L., Morgan, S. C.,
 Morton, J. T., Naimey, A. T., Navas-Molina, J. A., Nothias, L. F.,
 Orchanian, S. B., Pearson, T., Peoples, S. L., Petras, D., Preuss, M. L.,
 Pruesse, E., Rasmussen, L. B., Rivers, A., Robeson, M. S., Rosenthal, P.,
 Segata, N., Shaffer, M., Shiffer, A., Sinha, R., Song, S. J., Spear, J.
 R., Swafford, A. D., Thompson, L. R., Torres, P. J., Trinh, P., Tripathi,
 A., Turnbaugh, P. J., Ul-Hasan, S., van der Hooft, J. J. J., Vargas, F.,
 Vázquez-Baeza, Y., Vogtmann, E., von Hippel, M., Walters, W., Wan, Y.,
 Wang, M., Warren, J., Weber, K. C., Williamson, C. H. D., Willis, A. D.,
 Xu, Z. Z., Zaneveld, J. R., Zhang, Y., Zhu, Q., Knight, R. &
 Caporaso, J. G. Reproducible, interactive, scalable and extensible
 microbiome data science using QIIME 2. Nat Biotechnol 37, 852–857
 (2019). Gaze, W., Zhang, L.,
 Abdouslam, N. et al. Impacts of anthropogenic activity on the ecology of
 class 1 integrons and integron-associated genes in the environment. ISME J
 5, 1253–1261 (2011). https://doi.org/10.1038/ismej.2011.15 McDonald, D., Price, M. N., Goodrich, J., Nawrocki, E. P., DeSantis, T. Z., Probst, A., Andersen, G. L., Knight, R. & Hugenholtz, P. An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J 6, 610–618 (2012). Větrovský, T. & Baldrian, P. The Variability of the 16S rRNA Gene in Bacterial Genomes and Its Consequences for Bacterial Community Analyses. PLOS ONE 8, e57923 (2013). <strong id="docs-internal-guid-af998b87-7fff-9346-fbf8-79b068999de8" style="font-weight:normal;"></strong>
description: Original raw sequences are available in NCBI SRA (Accession:
 PRJNA565393)<br>Sample sites and sample names are in
 "Sawkill_mapping_and_env_var.csv"<br>All scripts used are
 located in "Combined_Scripts.rmd".<br><br>This
 dataset includes the following files: 
 Sampling_site_metadata_table.csv - Sample ID, Field ID, Date,
 Cumulative Rain Fall (mm), and Air Temperature (°C)
 Physicochemical_characteristics.csv - Sample ID, Water
 Temperature (ºC), Turbidity (TU), Conductivity (µmhos/cm), Dissolved
 Oxygen (mgL), and Salinity (ppt)
 Escherichia_coli_concentration.csv - Sample ID, E. coli
 (MPN·100mL-1) Total_coliforms_concentration.csv -
 Sample ID, Coliform (MPN·100mL-1)
 Enterococcus_sp_concentration.csv - Sample ID, Enterococcus sp.
 (MPN·100mL-1) Endotoxins_concentation.csv - Sample
 ID, Endotoxin (EU/mL)
 Integron_1_relative_abundance.csv - Sample ID, intI1 rel.
 abun. ASV_and_taxa_assignment.tsv - Tab separated
 file with each ASV, assigned taxa, and percent confidence (aka,
 ASV_to_taxa_confidece.tsv)
 Taxa_abundance_by_sample.csv - All taxa at species level
 resolution (or lowest possible) and abundances in each sample (aka,
 Species_resolved_taxa_and_counts.csv)
 Sawkill_mapping_and_env_var.csv - comma-separated file
 containing sample names and sample sites and measured environmental
 variables. detail: SampleID - Unique Identifier:
 SK-&lt;sort_number&gt;    sort - Unique
 Identifier index number FieldID - Combination field:
 &lt;site&gt;_&lt;date&gt; combination of site column and
 date column Date - Calendar date
 Day - Sample index starting at one (06/22/2015) and incrementing
 by one for each additional sample date Site -
 Location of Sample site and Replicate number (Counting from 0):
 OW = Outflow Water AS# = Above Outflow
 Sediment (Far) (NB: AS = replicate 0, AS1 = replicate 1)
 A1S# = Above Outflow Sediment (Near) (NB: A1S = replicate 0,
 A1S1 = replicate 1) BS# = Below Outflow Sediment
 (Near) (NB: BS = replicate 0, BS1 = replicate 1) B1S#
 = Below Outflow Sediment (Far) (NB: B1S = replicate 0, B1S1 = replicate
 1) AW = Above Outflow Sediment (Far)
 A1W = Above Outflow Sediment (Near) BW =
 Below Outflow Sediment (Near) B1W = Below Outflow
 Sediment (Far) GeoLocation - Spatial location of
 sample site O = Outflow A =
 Above Outflow (Far) A1 = Above Outflow
 (Near) B = Below Outflow (Near)
 B1 = Below Outflow (Far) Type - Physical
 matrix sampled (Outflow, Sediment, Water) Desc -
 Brief text description of Sample Pool - Brief text
 description of Sample Group Rain12 - Cumulative
 Measure of Rain (mm) collected over the previous 12 hours.   
 Rain24 - Cumulative Measure of Rain (mm) collected over the
 previous 24 hours.    Rain48 - Cumulative Measure of
 Rain (mm) collected over the previous 48 hours.   
 Rain72 - Cumulative Measure of Rain (mm) collected over the
 previous 72 hours. AirTemp - Ambient air temperture
 (Celsius) WaterTemp - Temperature of water at
 collection site (Celsius) Turbidity - Water Turbidity
 (TU) Conductivity - Water Conductivity
 (µmhos/cm) DO_mgl - Water Dissolved Oxygen
 (mgL) salinity_ppt - Water Salinity (ppt)
 intI1 - Relative abundance of intI1 using qPCR
 Ecoli - IDEXX Colilert assay (MPN·100mL-1)
 Coliform - IDEXX Colilert assay (MPN·100mL-1)
 Entero - IDEXX Enterolert (MPN·100mL-1)
 Endotoxins - Charles River Endosafe system (EU/mL)
 Denoising_qc_stats.tsv - tab separated
 file with the following read abundances per sample 'input',
 filtered', 'denoised', 'merged', and
 'non-chimeric'. Sample_frequency_detail.csv
 - Reads used per sample Sample_site_GPS.txt - Map
 refined estimated GPS coordinates (decimal format) of all sample
 sites. Sample_type_date_site_season_name.csv - Comma
 separated file containing Sample, type, date, site and season.
 For ease of use, we have also included the original
 visualization files generated by QIIME2 (using the code in
 'combined_scripts.rmd'). These are viewable using the QIIME2
 viewer or manually by decompressing them. 
 Combined_Scripts.rmd - Rmarkdown containing code for scripts
 used for data processing and presentation
 demux.qzv denoising_stats.qzv
 rep-seqs.qzv table-dada2.qzv
 taxa-bar-plots.qzv