Effects of produced water from oil drilling on mouse microbiome using Nanopore Technologies Sequencing

Produced water is a wastewater byproduct of oil and gas extraction, containing a complex mixture of formation fluid and chemical additives used in production and maintenance. In our area of concern, the National Pollutant Discharge Elimination System permits the release of this water for livestock and wildlife watering, including the irrigation of crops. Little is known about the impacts of this water on humans and surrounding wildlife, therefore this study (as well as others in a larger project) was run to determine the impacts of produced water on mice gut microbiomes. In this project, 30 female nonpregnant mice (strain C578L/6N) were exposed to produced water at varying concentrations (0% (lab water), 6.25%, 12.5%, 25%, 50%, and 100%) through the drinking water (sipper sacks). Over the course of the study no changes in body weight gains, organ weights, or histopath changes occurred. However, we did see a decrease in water consumption at the two highest concentrations. Caecum and feces samples were removed from the mice post mortum. Microbiome DNA was isolated from feces and whole caecum using the Qiagen DNeasy PowerSoil Pro Kit according to the manufacturer's instructions.After isolation DNA concentration and quality was quantified using a Qubit Flex Fluorometer, Nanodrop 2000c Spectrophotometer, and an Agilent Bioanalyzer System. 16S rDNA was amplified using the Oxford Nanopore Technologies 16S Barcoding Kit. The DNA was amplified by PCR using specific 16S primers, 27F adn 1492R. The amplified DNA was sequenced using a R9.4.1 flow cell on the Oxford Nanopore Technologies MinION sequencer.