MinION sequencing; Ebola virus surveillance with portable nanopore sequencing
11 or 19 overlapping RT-PCR products designed to cover Ebola virus genome
RNA was extracted from 50 µl whole blood or 140 µl of resuspended swab using the QIAamp Viral RNA Mini Kit (Qiagen, Manchester, UK), following manufacturer’s instructions.          Sample inactivation was done in a glovebox by adding 560 µl of Buffer AVL (Qiagen) and 560 µl of 100% ethanol.         Eleven individual RT-PCR reactions were set-up using primers designed to generate 1500-2000 base pair overlapping amplicons covering the Ebola virus genome.         RT-PCR was performed using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Life Technologies Ltd., Paisley, UK) on a 25-sample Eppendorf MasterCycler Personal (Eppendorf UK, Stevenage, UK)          Following amplification each product was quantified on a Qubit 3.0 fluorimeter using the dsDNA HS assay.          90 ng of each product (normalised by amplicon length) was pooled into a single tube and cleaned-up using an equal volume of MAGBIO HighPrep PCR beads (AutoQ Biosciences, Reading, UK).          Pooled amplicons were diluted to 85 µl, and end-repaired in 100 µl, using the NEBNext End Repair Module (New England Biolabs, Hitchin, UK) before being cleaned-up using an equal volume of HighPrep PCR beads.          3’ dA-tailing was performed using the NEBNext dA-Tailing Module (New England Biolabs) in a volume of 30 µl, before being cleaned-up using an equal volume of HighPrep PCR beads.          10 µl of ‘Adapter mix’ and 10 µl ‘HP adapter’ supplied in the SQK-MAP005 library preparation kit (Oxford Nanopore Technologies, Oxford, UK) were added to the dA-tailed amplicons along with 50 µl, Blunt/TA Ligase Master Mix (New England Biolabs) in a Protein LoBind tube (Eppendorf UK) and incubated for 10 minutes.          The resulting sequencing library was purified using Dynabeads His-Tag Isolation and Pulldown beads (Life Technologies, Stevenage, UK) according the SQK-MAP005 protocol supplied by Oxford Nanopore Technologies as part of the MinION Access Program.          The final library was quantified using the Qubit to confirm the process had been successful.          6 µl, of library was diluted using 75 µl  ‘2x Running Buffer’, 66 µl Nuclease-free water (Promega UK, Chilworth, UK) and 3 µl, ‘Fuel Mix’.