1D Genome Sequencing on the Oxford Nanopore MinION

Sara Goodwin1, Robert Wappel1, W. Richard McCombie1

1 Genome Center, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 18.11
DOI:  10.1002/cphg.39
Online Posting Date:  July, 2017
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Today's short‐read sequencing instruments can generate read lengths between 50 bp and 700 bp depending on the specific instrument. These high‐throughput sequencing approaches have revolutionized genomic science, allowing hundreds of thousands of full genomes to be sequenced, and have become indispensable tools for many researchers. With greater insight has come the revelation that many genomes are much more complicated than originally thought and include many rearrangements and copy‐number variations. Unfortunately, short‐read sequencing technologies are not well suited for identifying many of these types of events. Long‐read sequencing technologies can read contiguous fragments of DNA in excess of 10 kb and are much better suited for detecting large structural events. The newest long‐read sequencing instrument is the MinION device from Oxford Nanopore. The rapid sequencing speed and low upfront instrument cost are features drawing interest in this device from the genomics community. This unit provides a representative protocol for carrying out human genome sequencing on the Oxford Nanopore MinION. © 2017 by John Wiley & Sons, Inc.

Keywords: DNA sequencng; MinION; long‐read sequencing; Nanopore; WGS

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Table of Contents

  • Introduction
  • Basic Protocol 1: Construction of Long‐Read Libraries for Human Genome Sequencing
  • Support Protocol 1: AMPure XP Bead Purification
  • Support Protocol 2: Measuring DNA Concentration with the Qubit Fluorometer
  • Support Protocol 3: Analyzing Size of DNA Fragments with Agilent Bioanalyzer
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Construction of Long‐Read Libraries for Human Genome Sequencing

  • DNA source
  • Gentra Puregene Kit (Qiagen)
  • Agencourt AMPure XP beads (Beckman Coulter; see protocol 2)
  • Nuclease‐free water
  • FFPE DNA repair kit (NEB)
  • Ligation Sequencing 1D Kit (Oxford Nanopore) containing:
    • DNA CS
    • Adapter mix
    • Adapter bead binding buffer
    • Elution buffer
    • Running buffer (RBF)
    • Library loading beads (LLB)
  • NEBNext Ultra End Repair/dA‐Tailing Module (NEB, cat. no. E7442S)
  • Blunt TA ligase master mix (NEB)
  • Pulsed‐field gel apparatus (e.g., Pippin Pulse, Sage)
  • Qubit (Thermo Fisher) (see protocol 3)
  • Nanodrop (Thermo Fisher)
  • 1.5‐ml protein LoBind microcentrifuge tubes (Eppendorf)
  • Covaris g‐TUBE (Covaris)
  • Eppendorf 5424 centrifuge (or equivalent) or Eppendorf MiniSpin Plus
  • Bioanalyzer (Agilent) (see protocol 4)
  • Thermal cycler
  • 0.5‐ml DNA LoBind PCR tubes (Eppendorf)
  • HulaMixer (ThermoFisher)
  • Magnetic rack
  • Vortex mixer
  • MinION (Oxford Nanopore Technologies)
  • Computer

Support Protocol 1: AMPure XP Bead Purification

  • Agencourt AMPure XP beads (Beckman Coulter)
  • Sample
  • 80% ethanol, prepared fresh
  • Nuclease‐free water
  • 1.5‐ml LoBind microcentrifuge tubes
  • Microcentrifuge
  • End‐over‐end mixer
  • Magnetic bead rack

Support Protocol 2: Measuring DNA Concentration with the Qubit Fluorometer

  • Qubit dsDNA HS assay kit (with standards; ThermoFisher)
  • DNA samples to quantify
  • Qubit assay tubes (ThermoFisher, cat. no. Q32856)
  • Vortexer
  • Qubit 2.0 fluorometer (ThermoFisher)

Support Protocol 3: Analyzing Size of DNA Fragments with Agilent Bioanalyzer

  • DNA 12000 Kit (Agilent, cat. no. 5067‐1509)
  • DNA samples between 0.5 and 50.0 ng/μl
  • Chip Priming Station (Agilent)
  • Bioanalyzer (Agilent)
  • Vortexer
  • 0.5‐ml tubes for sample preparation
  • Microcentrifuge
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Literature Cited

Literature Cited
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