One size fits all: development of a simple workflow to characterise any viral pathogen

Effective outbreak control depends on timely information about how the causative pathogen spreads and evolves. Genomic sequencing can provide this information, but many conventional sequencing methods require specialised equipment only available in centralised laboratories with continuous power and trained personnel. Furthermore, transporting samples to sequencing facilities can delay results, particularly for samples from remote and resource-limited settings.

Researchers are overcoming the challenges of monitoring pathogen outbreaks by utilising portable, real-time Oxford Nanopore sequencing and adaptable tiled-amplicon workflows. By bringing sequencing to the outbreak source, teams can generate data rapidly, demonstrating the future potential to inform treatment and control measures in real time.

Sequencing viral genomes in remote areas

Nanopore technology has played a pivotal role in global pathogen surveillance, from the Ebola outbreak in West Africa in 2014–2016^1,2 to the more recent COVID-19 global pandemic in 2020–2023^3,4. To date, more than three million SARS-CoV-2 genomes have been sequenced using nanopore technology globally⁵.

Many known RNA viruses, such as coronaviruses, influenza, and HIV, are fast evolving, which means they continually accumulate changes in their genomes. As viruses evolve, some variants acquire mutations that affect characteristics such as transmission, immune escape, or disease severity. Viral genome sequencing can track these changes, providing data that can guide important control measures, but only if the results are generated quickly enough to inform interventions. This is challenging to achieve with conventional sequencing methods.

With this in mind, Josh Quick and his team based at the University of Birmingham, UK, took advantage of the accessibility, affordability, and portability of nanopore sequencing to successfully perform scalable, field-based genomic surveillance of the Ebola virus in West Africa⁶. Using a laptop, a MinION, and basic lab equipment, the team used nanopore sequencing in the field and generated results within 24 hours of receiving each sample, 'with the sequencing process taking as little as 15–60 minutes'⁶.

Building on this proof-of-concept work, a collaboration of scientists worked together to develop a reproducible pathogen surveillance workflow. Researchers from multiple scientific institutions established the ARTIC network to expand international viral surveillance and support worldwide collaboration in sequencing and analysis of pathogen outbreaks⁷. The network develops and provides open-access resources, including primer schemes and lab protocols, to support real-time genomic surveillance of pathogens.

Delivering an adaptable workflow for pathogen surveillance

The ARTIC network developed a simple, cost-efficient tiled-amplicon workflow, a PCR enrichment approach suitable for samples containing as few as 50 genome copies per reaction⁸. The workflow uses overlapping amplicons to cover the viral genome for complete coverage, with a multiplex PCR step to combine multiple primer pairs into a streamlined step. Additionally, the primer scheme for the PCR step can be designed for specific viruses, making the approach adaptable.

Researchers have since adapted the approach to a range of viruses that vary substantially in genome size and structure using primer designs specific to the target virus (Table 1).

Capturing variants of concern with wastewater surveillance

Researchers have also extended tiled-amplicon sequencing to monitor viruses at the population level. Wastewater surveillance captures viral material shed across a community, offering researchers a way to track circulating variants and mutations of potential clinical relevance.

During the COVID-19 pandemic, researchers used genomic surveillance to identify and monitor SARS-CoV-2 variants of concern (VOC). To apply genomic surveillance to the population level, Barbé et al. adapted the tiled-amplicon workflow for wastewater samples. They achieved near-complete genome coverage, demonstrating how wastewater surveillance could support population-level monitoring for early detection of emerging VOC²².

More recently, John et al. extended amplicon-based wastewater surveillance to influenza A virus. Over the course of two winter seasons, the team collected wastewater samples across four treatment sites in Switzerland. The researchers used an optimised tiled-amplicon workflow to target the HA, NA, and M segments of the virus. By targeting these segments, the team characterised circulating lineages and monitored mutations at the drug-target and vaccine-relevant sites²³.

This study shows how researchers have adapted the tiled-amplicon method to address seasonal and emerging respiratory viruses present in populations. This approach could also provide timely data to support public health responses and inform vaccine strain selection.

Extending the approach to animal-virus surveillance

Researchers have also adapted tiled-amplicon workflows for viruses that affect animal health, including rabies, avian influenza, African swine fever virus (ASFV), and classical swine fever virus (CSFV). ASFV presents a particularly difficult sequencing challenge because of its large, complex genome, so many previous studies only characterised part of it¹⁵.

Using the MinION, Warr et al. developed a tiled-amplicon approach to sequence ASFV in the field, reducing the need to transport samples to centralised facilities¹⁵. This method could enable near-live monitoring of outbreak situations with rapid turnaround times and minimal transportation of samples. More recently, McDowell et al.²⁴ similarly generated near-full-length genomes of both ASFV and CSFV enabled by nanopore sequencing generating reads of unrestricted length, ‘providing more complete genetic information for phylogenetic analysis and viral evolution studies of these pathogens’. Both viruses can cause highly contagious disease and substantial mortality in domestic pigs, with outbreaks disrupting pork production and local economies, making effective genomic surveillance essential²⁴.

Collaboration with the research community

From portable Ebola sequencing to wastewater monitoring of SARS-CoV-2 and influenza A, researchers have repeatedly adapted tiled-amplicon sequencing with Oxford Nanopore technology to meet different surveillance needs. By combining virus-specific primer schemes with portable, scalable nanopore sequencing, teams can generate targeted genomic data closer to the source of an outbreak and rapidly inform public health measures.

Collaboration between the ARTIC network and the wider research community has expanded this viral pathogen surveillance approach. By sharing open-access primer schemes, protocols, and analysis resources, the network provides a flexible framework that researchers can adapt as viral threats and public health priorities change.

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