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Using nanopore in COVID-19

If you're getting started with nanopore technology for COVID-19 research, including sequencing of the SARS-CoV-2 virus, get in touch to let us know how we can support. You'll also find some useful resources below.

What do I need to get started?

Starting with nanopore technology

Buy a MinION starter pack

Visit our get started page to understand all you need to get started with nanopore technology — you'll find information about IT requirements, consumables, site preparation requirements, and more.

Starting with COVID-19: whole genome sequencing

Performing genomic epidemiological analysis of the transmission and evolution of the SARS-CoV-2 virus has become a key part of the global public health response to the COVID-19 pandemic. To support this effort we have worked closely with the ARTIC network, who have issued protocols that enable rapid, scalable, and cost-effective whole genome sequencing of SARS-CoV-2 using nanopore technology.

This assay can scale from 1-96 samples per run, and across our range of devices to enable 1-4,500 samples per run. Below you'll find more information about what you need to get started along with the protocol and analysis pipelines.

View the protocol*
*You'll need to login to see the protocol but if you don't already have access you'll be able to create a guest login.

For more information, you can watch Phill James, Applications Manager at Oxford Nanopore, talk through the PCR tiling of COVID-19 virus protocol:

Materials

Flow cell

Input RNA

Native Barcoding Expansion 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114) if multiplexing up to 24 samples, or Native Barcoding Expansion 96 (EXP-NBD196) if multiplexing up to 96 samples

Ligation Sequencing Kit (SQK-LSK109)

Flow Cell Priming Kit (EXP-FLP002)

SFB Expansion (EXP-SFB001)

Consumables

Q5® Hot Start High-Fidelity 2X Master Mix (NEB, M0494)

Random Primer Mix (NEB, S1330S)

10 mM dNTP solution (e.g. NEB N0447)

SuperScript IV reverse transcriptase, 5X RT buffer and 100 mM DTT (ThermoFisher Scientific, 18090010)

RNaseOUT™, 40 U/μl (Life Technologies, 10777019)

COVID-19 primers (lab-ready at 100 µM, IDT)

Nuclease-free water (e.g. ThermoFisher, cat # AM9937)

Agencourt AMPure XP beads

Freshly prepared 80% ethanol in nuclease-free water

Qubit dsDNA HS Assay Kit (ThermoFisher Q32851)

NEBNext Ultra II End repair / dA-tailing Module (E7546)

NEBNext Ultra II Ligation Module (E7595)

NEBNext Quick Ligation Module (E6056)

DNA 12000 Kit & Reagents - optional (Agilent Technologies)

0.2 ml thin-walled PCR tubes

1.5 ml Eppendorf DNA LoBind tubes

Primer sequences Primer sequences

Data analysis

ARTIC Network

Oxford Nanopore recommends the analysis software developed by the ARTIC Network for the exploration of SARS-CoV-2 sequence collections. The ARTIC network has produced a best practices document for the base-calling and aggregation of nanopore sequence reads and for the subsequent steps of alignment against a reference genome and variant calling.

The complete source code and additional instructions for the ARTIC software may be found at their github pages.

RAMPART software

The RAMPART software can be used to assess the performance of a sequencing run. This real-time toolkit provides an intuitive and graphical view of sequence depth-of-coverage across the target genome. It breaks down read distributions by barcode and enables a clean dissection of on-target reads across the sequenced collection. This tool is great for ensuring that sufficient reads have been obtained across all barcoded samples and for identifying when sufficient sequence reads have been produced for the next analytical steps.

Docker images have been prepared and are available, along with supporting documentation, here.

ARTIC Field Bioinformatics software

Described here, this software provides a simplified interface for the aggregation of sequence reads called by the MinKNOW software. The software then enables the mapping of sequence reads to the reference genome (using either BWA or Minimap2). The mapped reads are assessed within the context of the specified primer sets and are judiciously clipped and processed to ensure that primers and synthetic adapters are not propagated through the workflow. A genome SARS-CoV-2 consensus sequence and lists of variants relative to the reference genome are prepared using the Medaka software.

Docker images have been prepared and are available, along with supporting documentation, here.

Bioinformatics workflows for SARS-CoV-2: from raw Nanopore reads to consensus genomes using the ARCTIC protocol

Metagenomic sequencing

Multiple groups are investigating approaches that characterise not only the SARS-CoV-2 virus, but other pathogens or microorganisms present in the sample. These aim to understand co-morbidity patterns of the disease, and also have the potential to be useful in broader surveillance of outbreaks in a population.

Download our Metagenomics Get Started Guide

If you have any questions please contact our Technical Support team.

Download the workflow

Getting started with nanopore sequencing: training materials

If you're new to nanopore sequencing we've got lots of training resources available to help you get started. You'll find a selection below -- visit the Community for the full library 

Flow cell check

Priming and loading a MinION flow cell

Priming and loading a Flongle flow cell

Coming soon...

Nanopore sequencing the SARS-CoV-2 genome: introduction to protocol

Bioinformatics workflows for SARS-CoV-2: from raw Nanopore reads to consensus genomes using the ARCTIC protocol

Troubleshooting

For further support please contact our Technical Support team or join the COVID-19/SARS-CoV-2 group in The Community.

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