From initial characterisation of the SARS-CoV-2 virus genome to the rapid identification of variants, researchers have been utilising nanopore sequencing to generate data essential to combating the spread of COVID-19.
Here, you’ll find information on the use of nanopore sequencing for genomic epidemiology, including what to expect from the workflows available, how to choose a sequencing approach to suit your requirements, and what you’ll need to get started.
This page focuses on the use of nanopore sequencing for research into the SARS-CoV-2 virus and COVID-19. For information on LamPORE, the COVID-19 diagnostic test developed by Oxford Nanopore, visit the Oxford Nanopore Diagnostics website.
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What is genomic epidemiology?
Genomic epidemiology is the study of how variations in the genomes of pathogens, or their hosts, influence health and disease, including how common specific variations are, how they interact with environmental factors, and how they contribute to disease risk.
Scientists around the world are using nanopore sequencing to rapidly sequence and analyse SARS-CoV-2 virus genomes. In combination with rapid data sharing across the scientific community, this enables genomic epidemiological analysis, which has become a key part of the global public health response to the COVID-19 pandemic.
By rapidly sequencing and sharing SARS-CoV-2 genomic data, it is possible to:
- Quickly identify variants and track their prevalence and distribution. These may impact the nature of the disease caused by the virus, or inform future treatment strategies and vaccine design.
- Determine how strains of the virus are related. This can help indicate, or rule out, routes of transmission, enable identification and investigation of clusters, and help inform strategies to control the spread of the virus.
Sequencing to fight COVID-19
Why nanopore for SARS-CoV-2 whole-genome sequencing?
The unique features of nanopore technology have enabled its use by the scientific community for the rapid sequencing of pathogens in multiple outbreak situations, including Zika, Ebola, Yellow Fever, and Swine flu. This experience has supported the rapid deployment of nanopore sequencing in the COVID-19 pandemic.
Oxford Nanopore have worked closely with the ARTIC Network, who have developed workflows for the rapid preparation and sequencing of SARS-CoV-2 whole genomes. These protocols are now available in the Nanopore Community, optimised for nanopore sequencing technology. Making use of fast, streamlined library preparation methods, real-time nanopore sequencing and data streaming, and highly scalable nanopore sequencing technology, these workflows enable you to quickly and easily set up SARS-CoV-2 sequencing in your own lab.
In addition to investigating the virus itself, nanopore sequencing can be employed to investigate the interactions between SARS-CoV-2 and the infected host. Whole human genome sequencing can enable research into what might cause different responses to the virus in different individuals. Furthermore, the sequencing of full-length immune cell receptor genes via long nanopore reads allows research into the response of the immune system to SARS-CoV-2 infection.
SISPA is an effective method of analysing both the genome of a pathogen of interest (in the above example, a novel pathogen is identified) plus that of co-infecting pathogens in the same sample.
Extending COVID-19 research: metagenomics, epigenetics, and immune response
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 and 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.
Nanopore sequencing can also be used to directly sequence RNA, without conversion to cDNA. This enables base modifications to be preserved and detected in nanopore sequencing data. Researchers in Australia and Korea have performed direct RNA sequencing of the virus, providing insights into the transcriptome of the virus and epigenetic modifications present.
Sequencing of SARS-CoV-2: how does it work?
The ARTIC SARS-CoV-2 library preparation method employs a PCR tiling approach to ensure even depth of coverage across the full RNA virus genome, even where RNA degradation has occurred. Starting from extracted RNA which has tested positive for SARS-CoV-2 via a clinically approved test, samples are first reverse transcribed, then amplified in overlapping ‘tiled’ sections covering the whole genome. The latest primer designs for this method can be found here. The amplified samples are then barcoded for sequencing in multiplex and pooled, before the library is prepared for nanopore sequencing.
Take a look at our overview of the ARTIC Classic protocol for an introduction the approach. Below, you’ll find more information on how to choose a protocol to suit your needs.
Knowledge Exchange: Nanopore sequencing the SARS-CoV-2 genome
During this presentation, Hazel and Anthony from Oxford Nanopore’s Technical Services team provided an overview to sequencing the SARS-CoV-2 virus, using nanopore technology. They walked through the process, from sample preparation, to selection of the right protocol for your experiment, and then provided some insight into the analysis approaches available to you.
Sequencing SARS-CoV-2: which approach should I choose?
The ARTIC Classic protocol was the first SARS-CoV-2 nanopore sequencing protocol to be utilised, and is the most well established. Featuring a normalisation step, the method is ideal for the routine sequencing of smaller batches of samples spanning a wide range of Ct values, and is optimised for maximum coverage across all genomes sequenced. The protocol is best for those with some prior experience of nanopore sequencing.
The recently released Midnight protocol is optimised for high throughput requirements, for highly multiplexed SARS-CoV-2 genome sequencing. Making use of the Rapid Sequencing Kits, the approach has the fastest turnaround time, least hands-on time, and lowest cost-per-sample; it is also a good choice for those wishing to incorporate automation.
Sequencing SARS-CoV-2: how do I analyse my data?
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 basecalling 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 via GitHub.
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. 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.
The Field Bioinformatics 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. 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.
ARTIC Classic: in action
For on-demand, flexible sequencing of 12-480 SARS-CoV-2 genomes in a sequencing run, we recommend:
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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.