Genome sequencing assays for infectious disease surveillance

“Viruses evolve — that’s a fact of life”, says Dr Nick Downey (NGS collaborations lead, Integrated DNA Technologies), Dr Nikki Freed (Auckland Genomics, University of Auckland, New Zealand) and Dr Olin Silander (School of Natural and Computational Sciences, Massey University, New Zealand) in this DDW exclusive.

Every time a virus replicates itself, there is a small chance that a change or mutation can occur in the genome. Most of the time, these changes do nothing. Unfortunately, these random changes can sometimes result in improved viral traits, such as the ability to bind and infect human cells better. When many people are infected, the number of opportunities for these rare, beneficial mutations grow, which means that multiple viral variants can emerge.

More than 12,000 mutations have arisen in the SARS-CoV-2 virus since it was discovered in late 2019. Remarkably, this is a relatively slow mutation rate for a virus, and many of these mutations are neutral or don’t seem to change the way the virus functions. However, there are, to date, 10 variants of concern or interest being tracked by the World Health Organization (WHO), with many more being monitored in specific regions, such as in Europe and the US. Rapid identification of new mutations that increase the transmissibility, immunity, and/or infection severity of the virus is vital for Covid-19 control, both now and in the future. Although we have monitored mutations in other viruses for decades, many challenges remain.

The ARTIC route

For example, during an outbreak of hemorrhagic fever spread by the Ebola virus in West Africa in 2015, Dr Joshua Quick from the University of Birmingham flew from the UK to Guinea to try to track virus evolution in real-time. Resources for NGS were thin on the ground at the outbreak sites, so Dr Quick deployed his Lab-in-a-Suitcase, which includes portable isolation cabinets built from repurposed hydroponics tents to recreate sterile lab conditions that minimized sample contamination. Additionally, the team used a rapid, inexpensive, portable DNA sequencer from Oxford Nanopore, called a MinION, that enabled real-time DNA sequencing. The set-up allowed for a rapid sequencing protocol that delivered results within 24 hours of sample collection. The kit’s utility was validated when the team moved several times and discovered that there were actually two distinct lineages of the Ebola virus circulating in Guinea. This work indicated that there were frequent transmission events occurring between Sierra Leone and Guinea.

It was during this same outbreak that the ARTIC Network was formed. The ARTIC Network is comprised of genomic epidemiologists looking to develop next-generation sequencing (NGS) tools for viral sequencing during outbreaks to generate actionable epidemiological data in real-time. They work with governments and WHO to facilitate fast and accurate diagnosis and evidence-based containment measures. Since then, the ARTIC Network has spawned multiple NGS protocols in response to outbreaks such as the Zika virus in Brazil and Covid-19. The fast, low-cost, and high-throughput ARTIC Covid-19 protocol has become the most ubiquitous method for sequencing SARS-CoV-2.

The Potential of Faster NGS Methods

However, there is always a desire to make these methods faster, less expensive, and easier to perform. Additionally, in some countries such as New Zealand, real-time genome sequencing (patient swab to uploaded genome in under 10 hours) is routinely used in instances of Covid-19 community transmission. Real-time genome sequencing has enabled health authorities to identify the source of outbreaks in real-time and monitor the spread. In instances like this, an ultrafast NGS method to sequence SARS-CoV-2 provides a major benefit. More specifically, research assays such as the Midnight Panel that sequence the viral genome in bigger sections (i.e., in 1200-base-pair sections with the Midnight protocol rather than 400-base-pair sections with the ARTIC protocol), coupled with rapid enzymatic preparation of the DNA for sequencing, allow researchers to conduct faster, cheaper, and more even sequencing across the viral genome, requiring fewer reads to accurately capture the entire sequence. This process, now known as the Midnight protocol because it sequences in 1200-base pair sections, was described by Freed, et al., in a 2020 study published in Biological Methods & Protocols.

The ability to deliver a viral genome sequence in under 10 hours provides health authorities real-time data on the same day the sample is taken. The consequent identification of index cases and their contacts allows for timely, evidence-driven implementation of quarantines. Additionally, these same ultra-fast methods that use inexpensive portable sequencers (such as those produced by Oxford Nanopore) enable labs or health care facilities with limited resources and skills to sequence high-quality viral genomes themselves.

Less expensive, rapid, and easier methods to sequence viral and bacterial pathogens may become increasingly relevant and critical to general infectious disease surveillance strategies. As we continue combating the Covid-19 pandemic, we must also ensure we are better prepared for a new pandemic. Developing multiple NGS tools for deployment at multiple stages of an outbreak is critical to facilitate rapid and effective outbreak control based on actionable real-time epidemiological data aimed at maximizing public safety.

About the authors

Nick Downey is currently IDT’s NGS Collaborations Lead. Prior to this, Nick served in Applications Support and as Senior Product Manager. He has a PhD in molecular biology, along with postdoctoral experience and time as an assistant professor. Nick has been at IDT since 2012.

Nikki Freed is the Lead Technologist at Auckland Genomics at the University of Auckland, New Zealand. For six years, she was a senior lecturer at the School of Natural and Computational Sciences at Massey University, Auckland. Nikki completed her PhD at ETH Zurich in Switzerland and worked for three years at Novartis Pharmaceuticals in Basel, Switzerland. She originally hails from California.

Olin Silander has been senior lecturer in the School of Natural and Computational Sciences at Massey University, Auckland since 2015. His work focuses on bacterial genetics and evolution. Olin received his PhD in Evolutionary Biology from the University of California, San Diego before a postdoctoral fellowship at ETH Zurich and a research fellowship at the University of Basel.

Reference

Freed NE, Vlkova M, Faisal MB, et al. Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding. Biol Methods Protoc. 2020;5(1):bpaa014.

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