Whole Genome Sequencing of Vibrio aestuarianus isolate S/27/15 A
DNA was extracted from the isolates using the MasterPure Gram positive DNA extraction kit (Cat. No. MGP04100; Epicentre). In summary, a 1 l loopful of bacteria (previously sub-cultured onto seawater agar (SWA)) was placed into a 1.5 ml Eppendorf tube containing 1 ml 0.9% saline. The solution was centrifuged at 1500 rpm, supernatant removed and 150 l TE buffer was added and samples vortexed to re-suspend the pellet. 150 l of a premade dilution of proteinase K in Gram-positive lysis solution was added to each sample, at a concentration of 1 l Proteinase K per 150 l of Gram-positive lysis solution. The samples were vortexed and subsequently incubated at 65-70C for 15 min which included vortexing every 5 min. Samples were cooled to 37C then put on ice for 3-5 minutes, following which 175 l of MPC Protein Precipitation Reagent was added to each sample. Samples were vortexed and centrifuged at 1500 rpm at 4C for 10 min. The supernatant was collected (pellets discarded) and 500 l of isopropanol was added, samples inverted 30-40 times and centrifuged again at 1500 rpm at 4C for 10 min. The supernatant was removed, 70% ethanol was added and samples centrifuged for a final time at 1500 rpm at 4C for 5 min. Finally, the supernatant was removed and samples were re-suspended in 100 l of molecular grade water and stored at -80C until future use.  The extracted DNA was quantified using a Quantus fluorometer (Promega), and quality assessed using NanoDrop ND-1000 Spectrophotometer (Thermo). DNA was normalised, following which a standard tagmentation programme was run on the libraries using a thermocycler. Subsequently, the genomic libraries where amplified (thermocycler) using the appropriate Index 1 (i7) and Index 2 (i5) primers. The libraries were then cleaned up and quality assessed with an Agilent Technology 2100 Bioanalyzer using a High sensitivity DNA chip. Finally, libraries were pooled and combined with the PhiX control before loading into the reagent cartridge and run on the Miseq.