Pipelines to do MinION sequencing of Zika virus

Protocols for Zika sequencing

Allison Black1,2

1Department of Epidemiology, University of Washington, Seattle, WA, USA, 2Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

Multiplex PCR for MinION sequencing of Zika virus

This protocol is a bench-ready version of the protocol presented in the following paper:

Quick J, Grubaugh ND, Pullan ST, Claro IM, Smith AD, Gangavarapu K, Oliveira G, Robles-Sikisaka R, Rogers TF, Beutler NA, Burton DR, Lewis-Ximenez LL, Goes de Jesus J, Giovanetti M, Hill S, Black A, Bedford T, Carroll MW, Nunes M, Alcantara LCJ, Sabino EC, Baylis SA, Faria N, Loose M, Simpson JT, Pybus OG, Andersen KG, Loman NJ. 2017. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. In prep.

Additional thanks to Josh Quick and Nathan Grubaugh for lots of useful advice, which I've tried to work in to the protocol as helpful hints.

Bench protocols

  1. Viral RNA extraction
  2. Two-step RT-PCR for amplicon generation
  3. Post-PCR clean-up and amplicon quantification
  4. MinION library preparation
  5. MinION flowcell priming and library loading

Equipment and Reagent Checklist

Tiling amplicon generation
  • QIAamp Viral RNA Mini Kit (Qiagen, cat. no. 52906)
  • Random Hexamers (50 ┬ÁM) (Thermo Fisher, cat. no. N8080127)
  • Protoscript II First Strand cDNA Synthesis Kit (NEB, cat. no. E6560)
  • Deoxynucleotide (dNTP) Solution Mix (NEB, cat. no. N0447)
  • Q5 Hot Start High-Fidelity DNA Polymerase (NEB, cat. no. M0493)
  • Custom PCR primers click here for primer sequences
  • Agencourt AMPure XP beads (Beckman Coulter, cat. no. A63881)
  • Qubit dsDNA HS Assay Kit (Thermo Fisher, cat. no. Q32854)
  • HyClone Molecular Biology Grade Water (GE Life Sciences, cat. no. SH30221.10)
  • 100% Ethanol
MinION sequencing
  • SpotON Flow Cell Mk I (R9.4) (Oxford Nanopore Technologies, cat. no. FLO-MIN106)
  • Nanopore Sequencing Kit (R9) (Oxford Nanopore Technologies, cat. no. SQK-NSK007)
  • Native Barcoding Kit (Oxford Nanopore Technologies, cat. no. EXP-NBD002)
  • NEBNext Ultra II End-repair/dA-tailing Module (NEB, cat no. E7546)
  • NEB Blunt/TA Ligase Master Mix (NEB, cat. no. M0367)
  • MyOne C1 Streptavidin beads (Thermo Fisher, cat. no. 65001)