MinION sequencing; Ecoli MinION sequencing, R7.3 chemistry, SQK-MAP-004, 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 4200 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 0.4x 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 25 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 SQK-MAP-004 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, 10 ul of Adapter Mix, 2 ul HP adapter and 50 ul Blunt/TA ligase master mix (NEB) was added and was left at room temperature for 10 minutes.           Molecules with an 'HP motor' were isolated using His-Tag pulldown beads (Life Technologies, Carlsbad, USA) by adding 100 ul washed beads to the ligated DNA, incubating for 5 minutes, then eluting in 25 ul '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. 150 microlitre ‘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.            141 microlitre ‘EP buffer’, 3 microlitre ‘Fuel mix’ and 6 ul library were mixed in a tube. The diluted library was loaded into the sample loading port on the minION using a Gilson P1000 pipette.            A 48-hour sequencing protocol was initiated using the MinION control software, MinKNOW (version 0.47.3.7). Read event data were base-called by the software Metrichor using workflow 1.9.
SQK-MAP-004
UB
Ecoli_R7.3_MAP-004_flowcell32.exp
University of Birmingham, UK
Ecoli_R7.3_MAP-004_flowcell32.exp