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Illumina MiSeq sequenced 16S library of Microbial Community Response to Oil and Corexit in Coastal Water Mesocosms

Authors: Shawn Doyle, Jason Sylvan
Published On: May 04 2017 20:30 UTC
DOI: https://doi.org/10.7266/N72F7KGG
UDI: R4.x263.189:0003
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File Format(s):
fastq

Project Information

Funded By:
Gulf of Mexico Research Initiative

Funding Cycle:
RFP-IV

Research Group:
Aggregation and Degradation of Dispersants and Oil by Microbial Exopolymers (ADDOMEx)

Point of Contact

Shawn Doyle
Texas A&M University / Department of Oceanography
sdoyle2@gmail.com

Theme keywords

16S rRNA V4 hypervariable region sequences, EPS, hydrocarbon degrading microorganisms, mesocosm

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Abstract:

Two independent mesocosm experiments were carried out in August (Mesocosm#1) and October of 2015 (Mesocosm#2) using coastal water collected near Galveston Bay, Texas. Four mesocosm treatments were prepared in triplicate for this study: (1) Control (seawater only); (2) WAF (oil + seawater); (3) Chemically-enhanced WAF (CE-WAF; oil + Corexit + seawater); (4) Diluted CE-WAF (1:10 dilution). Initial mesocosm volumes were 79L. Prior to experiment initiation, phytoplankton were collected from coastal Galveston, TX waters and added to each tank to stimulate interactions between phytoplankton, oil, and Corexit. Starting at time-point zero and every 12 hours thereafter, water was collected from each mesocosm in clean, opaque Nalgene bottles with PTFE-lined diaphragm pumps. DNA was extracted from filtered water samples and sequenced using barcoded primers specific for the V4 hypervariable region of the 16S rRNA gene.

Suggested Citation:

Shawn Doyle, Jason Sylvan. 2017. Illumina MiSeq sequenced 16S library of Microbial Community Response to Oil and Corexit in Coastal Water Mesocosms. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N72F7KGG

Publications:

Doyle, S. M., Whitaker, E. A., De Pascuale, V., Wade, T. L., Knap, A. H., Santschi, P. H., … Sylvan, J. B. (2018). Rapid Formation of Microbe-Oil Aggregates and Changes in Community Composition in Coastal Surface Water Following Exposure to Oil and the Dispersant Corexit. Frontiers in Microbiology, 9. doi:10.3389/fmicb.2018.00689

Purpose:

Characterize the microbial community composition and structure in Mesocosm1 and Mesocosm2.

Data Parameters and Units:

Raw sequence reads in FASTQ format for each replicate of each treatment (i.e. Seawater only, WAF, CE-WAF, Diluted CE-WAF).

Methods:

The hyper-variable V4 region of the 16S rRNA gene was PCR amplified from the DNA extracts with GoTaq Flexi DNA Polymerase (Promega) using a methodology similar to that described by Caporaso et al. 2011. Each sample was amplified in triplicate 25μL reactions with the following cycling parameters: 95°C for 3 min, 30 cycles of 95°C for 45 s, 50°C for 60 s, and 72°C for 90 s, and a final elongation step at 72°C for 10 min. All reactions were performed using the 515F-806R primer pair (10µM each) which was modified to include Golay barcodes and adapters for Illumina MiSeq sequencing (Caporaso et al. 2012). The primer pair was also modified to include recently published revisions that reduce bias against the Crenarchaeota/Thaumarchaeota archaeal lineages and the SAR11 bacterial clade (Parada et al. 2015; Apprill et al. 2015). Following amplification, triplicate reactions were combined together and run on a 1.5% agarose gel to assess amplification and relative band intensity. Amplicons were then quantified with the QuantiFluor dsDNA System (Promega), pooled at equimolar concentrations, and purified with an UltraClean PCR Clean-Up Kit (MoBio Laboratories). The purified library, along with aliquots of the three sequencing primers, were sent to the Georgia Genomics Facility (Athens, GA, USA) for MiSeq sequencing (v2 chemistry, 2 x 250bp).

Instruments:

Illumina MiSeq Sequencing: V2-Chemistry; PE-250