Tis’ the Season for Sequencing

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Responsible for more than four million deaths per year, acute respiratory infections are the world’s third leading cause of death from disease. Approximately 80 percent of these infections are caused by viral pathogens; particularly, influenza viruses, respiratory syncytial virus, adenoviruses, human rhinoviruses, and, more recently, coronaviruses (1). As we approach respiratory virus season in the Northern Hemisphere – with the lower temperatures and low humidity of the fall and winter months helping to stabilize viral pathogens (2) — health agencies, epidemiologists, virologists, and scientists developing vaccines and antivirals are striving to ensure maximum protection against disease.

The world is still recovering from the crippling humanitarian and economic effects of the COVID-19 pandemic. Next generation sequencing (NGS) played a major role in bringing the pandemic under control relatively quickly; thanks to the power of NGS, SARS-CoV-2 was sequenced within a month of the first known case being detected. Armed with the virus’ sequence, pharmaceutical companies were able to quickly pivot and adapt to produce and deploy novel vaccines worldwide. Knowledge of the viral sequence also supported the rapid identification of effective therapies from existing antivirals and development of monoclonal antibody therapies – saving thousands of high-risk patients.

Although we know plenty about the viruses that recur in the colder months, we must remain vigilant for the unknowns – variants of existing viruses that may be resistant to preventative and curative measures, as well as emerging viruses that could lead to an outbreak. NGS has proven to be an effective and powerful technology for identifying novel viruses and detecting variants of known viruses. The technology enables scrutiny of the entire genome of viruses, providing comprehensive information that can be used to monitor clustering, spread, and evolution.

A toolbox of options

There are a variety of NGS methods available, and choosing the correct approach has implications for speed, ease of use, and cost-effectiveness. Typical methods are shotgun, hybridization capture, and amplicon-based sequencing. In shotgun sequencing, the viral nucleic acid is fragmented or reverse-transcribed and ligated with adapters for sequencing. Shotgun sequencing is a costly technique because contaminating host nucleic acid increases the sequencing cost; however, the method is useful for identifying novel viruses because it requires no prior knowledge of the viral genome.

Hybridization capture targets viral sequences of interest. In this technique, the viral genome is enriched from the host nucleic acid using nucleotide probes that hybridize to the target sequences of interest – significantly lowering sequencing cost. Hybrid capture is insensitive to mutations within the genome, making it an effective technique for identifying variants and emerging strains; however, the workflow is long and more complicated to adapt for high throughput processing.

Figure 1. Overlapping amplicons create “super amplicons” that provide coverage in situations of primer dropout. In a two-tube amplicon approach, Variant 1 is detected, but because of the failure of primer F2 to bind, Amplicon 2 is not generated, so Variant 2 is not detected. Using a one-tube amplico

Amplicon sequencing is a powerful technique that is fast and simple to perform even with samples that have a low viral load. Highly multiplexed primer pairs designed to target regions along the length of the viral genome are used to produce short amplicons that are easily sequenced. An even more rigorous and reliable approach is to use overlapping amplicons in a single-tube multiplex PCR to enable contiguous genome coverage, even in instances where a mismatch between primer and target prevents binding. In specific single-tube chemistries, super amplicons – which cannot be formed when alternating primer pairs are placed into two separate reactions – are produced that enable full target coverage (see Figure 1). With super amplicon sequencing, a single panel design can be used to detect a variety of mutations and viral strains without experiencing coverage dropouts.

A cost-effective solution for routine clinical use

The common method for virus detection is nucleic acid amplification testing (NAAT), a PCR-based method. Although this method is useful for diagnosing diseases caused by existing viruses and may support surveillance of point mutations to detect variants, it is limited in the number of changes that can be detected and in its ability to ascertain different variants at the same base. Using multiplexed assays, it is possible to analyze multiple loci simultaneously; however, it is a significantly lower throughput method than NGS, and variant strains and novel viruses can evade detection.

The cost of NGS has decreased significantly over the years, making it possible to use the technology to generate comprehensive data in clinical settings. Keeping costs low requires careful consideration of the methods and platforms that will be used – from experimental design to data analysis. It is imperative to start with a robust experimental design and ensure that your method is compatible with available sequencers or is platform-agnostic. In a clinical setting, amplicon-based sequencing would typically be the method of choice because of its speed, ease of use, and relatively low cost. Amplicon-based sequencing is less labor-intensive and requires less sequencing capacity than shotgun and hybrid capture sequencing, but still provides full viral genome coverage and detection of novel mutations. Additionally, the ability to multiplex more than 1,500 reactions supports the investigation of many samples at a low cost per sample.

For any sequencing method, another important consideration is the choice of target capture panel. This is especially true if cost is a primary concern when using amplicon-based sequencing. Comprehensive panels that target many known pathogenic viruses may be useful for exploratory studies where the specific pathogens might be unknown. However, the amount of sequencing required to generate useful data from such large panels when using complex samples, such as wastewater (representing community-wide viral content) would negate the cost benefits of amplicon-based sequencing for routine monitoring of known respiratory viruses and their variants. To keep costs down when targeting known respiratory viruses and possible variants, it would be prudent to identify focused panels that have the sensitivity to identify both known and unknown variants.

What’s next?

The utility of NGS for monitoring virus clustering, evolution, and spread was demonstrated during the recent COVID-19 pandemic – and was key to the quick deployment of vaccines and treatments. At the right cost, NGS can be a powerful tool for studying pathogenic viruses in preparation for and during respiratory virus season. As improvements in sequencing technology continue to drive down the cost and adoption increases, more comprehensive panels can be used to quickly and effectively detect novel viruses and variants. In turn, pharmaceutical companies can design and manufacture vaccines for timely deployment to prevent major outbreaks – preserving life and maintaining economic stability.

References

X Wang et al., “Detection of respiratory viruses directly from clinical samples using next-generation sequencing: A literature review of recent advances and potential for routine clinical use,” Rev Med Virol, 32, e2375 (2022). PMID: 35775736.
M Moriyama et al., “Seasonality of respiratory virus infections,” Annu Rev Virol, 7, 83 (2020). PMID: 32196426.