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 packVisit 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. You'll need to log in to see the protocol, but if you don't already have access, you'll be able to create a guest login.
| Materials |
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 |
LunaScript™ RT SuperMix Kit Q5® Hot Start High-Fidelity 2X Master Mix (NEB, M0494) SARS-CoV-2 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) NEB Blunt/TA Ligase Master Mix (M0367) NEBNext Ultra II End repair / dA-tailing Module (E7546) NEBNext Quick Ligation Module (E6056) DNA 12000 Kit & Reagents - optional (Agilent Technologies) 1.5 ml Eppendorf DNA LoBind tubes Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Cat # 0030129504) with heat seals 96-well deepwell plates |
| 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 command line interface for the aggregation and analysis of sequence reads called by the MinKNOW software. The pipeline curates sequence reads by barcode and maps the reads to the reference SARS-CoV-2 genome using 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 SARS-CoV-2 consensus genome sequence and lists of variants relative to the reference genome are prepared using the Medaka consensus polishing software and the LongShot variant calling software.
A workflow that introduces the usage of the Field Bioinformatics workflow for SARS-CoV-2 sequence analysis is provided in the EPI2ME Labs software.
Bioinformatics workflows for SARS-CoV-2: from raw Nanopore reads to consensus genomes using the ARTIC 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. This infographic describes some of the approaches available for metagenomic & metatranscriptomic sequencing of SARS-CoV-2 samples.
Preparation of clinical research samples via SISPA (Sequence-independent, single primer amplification), followed by nanopore sequencing, is an effective method of rapidly identifying unknown and novel infectious agents and generating consensus sequences. View the workflow here.
Download our Metagenomics Getting Started Guide for sample-to-answer guidance on nanopore metagenomic sequencing.
If you have any questions please contact our Technical Support team.
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.
