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.
Oxford Nanopore Technologies provides the fastest SARS-CoV-2 Sequencing, at low cost, and high quality.
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 in use by scientists around the world. Their foundational work, and that of Nikki Freed and Olin Silander, form the core of the workflows available for the nanopore sequencing of SARS-CoV-2.
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 set up SARS-CoV-2 sequencing quickly and easily in your own lab. Starter pack and bundle options are available, providing everything you need to start sequencing, whether you’re new to nanopore sequencing or getting started with SARS-CoV-2 analysis, at low cost per sample.
Visit the Resource Centre to view the latest publications, presentations, and more from scientists using nanopore sequencing to research COVID-19.
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 South Korea have performed direct RNA sequencing of the virus, providing insights into the transcriptome of the virus and epigenetic modifications present.
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.
Sequencing of SARS-CoV-2: how does it work?
There are two methods available for whole-genome nanopore sequencing of SARS-CoV-2: Midnight and ARTIC Classic. Both methods employ a PCR tiling approach in which the viral genome is amplified in overlapping sections, maximising coverage across the full genome.
Midnight is a simple, rapid method of sequencing SARS-CoV-2 genomes at low cost per sample. The approach is highly flexible, allowing the on-demand sequencing of small numbers of samples or scaling up to high-throughput sequencing needs. Hands-on time is also minimal, facilitating automation. In the Midnight protocol, the SARS-CoV-2 genome is amplified in ~1,200 bp overlapping segments, making it more resilient to drop-out caused by mutations in the viral genome.
ARTIC Classic was the first SARS-CoV-2 nanopore sequencing protocol to be utilised, and has been used by scientists around the world. In this method, the SARS-CoV-2 genome is amplified in ~400 bp fragments. This shorter length may help improve coverage for RNA samples that are likely to be degraded - for example, due to freeze-thaw cycles or storage at temperatures above -80°C.
Update: January 2022
From 1st February 2022, the SARS-CoV-2 sequencing Midnight Kits shipped will feature an upgraded primer mix. This update, based on the collaborative efforts of Nikki Freed and the ARTIC Community, will ensure complete coverage of amplicon 28 when sequencing the Omicron variant. The new primer mix will not require any modifications of the library preparation protocol.
As new mutations are identified in the Omicron sub-lineages, we are continuing to monitor for any potential impacts on the sequencing of complete SARS-CoV-2 genomes and rapidly respond. If you encounter any amplicon dropouts in your SARS-CoV-2 genomes, please let us know by emailing firstname.lastname@example.org.
The Midnight workflow for preparation of SARS-CoV-2 whole-genome sequencing. This method is similar to the ARTIC amplicon sequencing protocol for MinION for SARS-CoV-2 v3 (LoCost) by Josh Quick and the method used in Freed et al., 2020. Timings shown are for preparation of 24 samples.
Sequencing SARS-CoV-2: which approach should I choose?
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 provides two key analysis workflows: FieldBioinformatics and RAMPART.
The FieldBioinformatics software provides a command line interface for the aggregation and analysis of sequence reads called by the Oxford Nanopore MinKNOW software. The pipeline curates sequence reads by sample barcode and maps to a 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.
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.
The ARTIC bioinformatics pipeline is implemented in the EPI2ME Labs Workflow, wf-artic. Our recommended solution for SARS-CoV-2 analysis, the approach enables high-throughput analysis of both Midnight and ARTIC Classic sequencing data in an hour or less, providing detailed results in an HTML report. An easy-to-follow, step-by-step EPI2ME Labs Tutorial is available, with an example dataset, for those getting started with the pipeline. For beginners to bioinformatics, the cloud-based EPI2ME Workflow provides a simple, interactive method of real-time analysis, with no prior bioinformatics experience required.
Real-time assessment of a SARS-CoV-2 sequencing run with RAMPART
Midnight SARS-CoV-2 whole-genome sequencing: 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.