The bacteriophage population used for sequencing was obtained from the same M. oxyfera bioreactor enrichment culture. See Gambelli et al. (2016) (link) for a full description. Bioreactor material was collected over a period of about three months, stored at 4 °C and viral particles were obtained as described before (Gambelli et al., 2016 (link)). Briefly, the aggregated microbial biomass was disrupted to free the bacteriophages and viral particles were precipitated using PEG8000 (Guo et al., 2012 (link)). Free bacteriophages present in the bioreactor supernatant medium and not within the bacterial aggregates were precipitated by iron chloride flocculation (Cunningham et al., 2015 (link)).
The two samples obtained by iron chloride flocculation and PEG 8000 precipitation were pooled together and bacteriophages were concentrated by ultracentrifugation (Optima XE90; Beckman-Coulter, High Wycombe, UK; Rotor: Type 90 Ti; Beckman-Coulter, High Wycombe, UK) at 77,000× g at 4 °C for 1 h. The pellet was resuspended in 1 ml of supernatant and the total DNA was extracted according to the protocol published by Thurber et al. (2009) (link). Using the Qubit dsDNA HS assay kit (Thermo Scientific, Waltham, MA, USA), the extracted DNA was quantified at 0.2 ng DNA.
The two samples obtained by iron chloride flocculation and PEG 8000 precipitation were pooled together and bacteriophages were concentrated by ultracentrifugation (Optima XE90; Beckman-Coulter, High Wycombe, UK; Rotor: Type 90 Ti; Beckman-Coulter, High Wycombe, UK) at 77,000× g at 4 °C for 1 h. The pellet was resuspended in 1 ml of supernatant and the total DNA was extracted according to the protocol published by Thurber et al. (2009) (link). Using the Qubit dsDNA HS assay kit (Thermo Scientific, Waltham, MA, USA), the extracted DNA was quantified at 0.2 ng DNA.
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