Evaluating the Threat of Antimicrobial Resistance in Agricultural Manures and Slurries

Samples were collected from the slurry tank of a 200-cow dairy farm in the East Midlands once per month between June and October 2017 (n=5). Triplicate extractions were performed on each sample using two extraction kits: PowerFecal Kit (Qiagen) and Isolate II Fecal DNA kit (BioLine) (30 extractions in total). DNA was quantified using a Qubit fluorometer (Invitrogen) while quality was assessed via Nanodrop (ThermoFisher). Extracted DNA was stored at 4˚ C pending sequencing. Metagenomic shotgun sequencing of extracted slurry DNA was performed and demultiplexed by Edinburgh Genomics using the Illumina NovaSeq platform (150bp paired end library).

Viral metagenomes were prepared firstly by homogenising cattle slurry in PBS. The homogenate was ultracentrifuged to pellet unwanted solids and bacteria. To further remove bacteria, the supernatant was passed sequentially through 0.45um and 0.22um filters. The filtrate was concentrated on an Amicon column and DNA was extracted using a standard phenol-chloroform extraction. Sequencing was conducted using Illumina Novaseq with a 2x150bp library.

This data is NERC-funded but not held by the EIDC. This data is archived in the European Nucletotide Archive.