MinION sequencing; Ecoli MinION sequencing, R7.3 chemistry, SQK-MAP-003, Metrichor 1.9
DNA extraction was performed using the ‘Invisorb Spin Cell Mini Kit’ (Invitek, Birkenfeld, Germany) using the protocol for extraction from serum or plasma according to the manufacturer's instructions.             DNA was quantified using a Qubit fluorometer (Life Technologies, Paisley, UK) and diluted to 23.5 ng/microlitre. 85microlitre was loaded into a G-tube (Covaris, Brighton, UK) and centrifuged at 5000 rpm for 1 minute before inverting the tube then centrifuging again for 1 minute. The fragmented DNA was end-repaired in a total volume of 100microlitre using the NEBNext End-Repair module (NEB, Hitchin, UK) according to the manufacturers instructions with the exception that the incubation time was reduced to 15 minutes.             The resultant blunt-ended DNA was cleaned-up using 1.0x by volume AMPure XP beads (Beckman Coulter, High Wycombe, UK) according to the manufacturers instructions with the exception that 80% ethanol was used instead of 70%, and eluted in 22 microlitre molecular grade water.             A-tailing was performed using the NEBNext dA-tailing module (NEB) in a total volume of 30microlitre according to the manufacturers instructions with the exception that the incubation time was reduced to 15 minutes.             In the following steps, the ‘gDNA library preparation kit’ (Oxford Nanopore Technologies, Oxford, UK) provided as part of the MinION access program (MAP) along with user-supplied reagents were used to generate a sequence-ready library for the MinION.          To the 30 ul dA-tailed DNA, 50 ul Blunt/TA ligase master mix (NEB) was added in addition to 10 ul each of Adapter Mix and HP adapter, the reaction was left to proceed at room temperature for 10 minutes.  The sample was cleaned-up using 0.4x by volume AMPure XP beads according to the manufacturer's instructions with the exceptions that the kit-supplied wash and elution buffers were used, and only a single wash was used. The sample was eluted in 25 ul of elution buffer, leaving to incubate for 10 minutes before pelleting and removal of the library.            A new R7.3 flowcell was removed from storage at 4 degrees celsius and the protective packaging removed. The flowcell was fitted to the minION device and held in place with plastic screws. 150microlitre ‘EP buffer’ was loaded into the sample loading port using a Gilson P1000 pipette and incubated for 10 minutes to prime the flowcell. This was repeated a further time.            The library was quantified using the Qubit fluorometer. 100 ng was diluted in 146 microlitre ‘EP buffer’ and 4 microlitre ‘Fuel mix’. The diluted library was loaded into the sample loading port on the minION using a Gilson P1000 pipette.            A 72-hour sequencing protocol was initiated using the MinION control software, MinKNOW (version 0.56.1.9). Read event data were base-called by the software Metrichor using workflow 1.9.
SQK-MAP_003
UB
Ecoli_R7.3_MAP-003_flowcell20.exp
University of Birmingham, UK
Ecoli_R7.3_MAP-003_flowcell20.exp