Acute respiratory infections cause morethan 4 million deaths a year globally. Most of them are caused by viral pathogens, such as influenza viruses, respiratory syncytial viruses, adenoviruses, human rhinoviruses, and coronaviruses. Infections from these viruses peak in the fall and winter months, when low temperatures and low humidity increase pathogen stability, impair immune response, and alter human behavior.
Health agencies, scientists, and organizations are typically prepared for these recurrent respiratory viruses. However, as viruses mutate and new ones emerge, technology that rapidly detects novel variants and enables scrutiny of the entire viral genome is crucial for monitoring and controlling the spread of disease.
NGS is a powerful, cost-effective tool
Nucleic acid amplification testing (NAAT), a PCR-based method, is powerful for detecting known viruses but is limited in its ability to support surveillance of variants and novel viruses, as it detects only a few target sequences of the viral genome. The recent COVID-19 pandemic demonstrated the power of next-generation sequencing (NGS) for monitoring the clustering, spread, and evolution of viruses. With it becoming affordable, NGS is an ideal tool for generating comprehensive, whole genome viral data for public health initiatives. Target enrichment methods are the most cost-effective sequencing techniques for this purpose.
Techniques for target enrichment
Target enrichment involves enriching or amplifying the viral genome to separate it from the host nucleic acid and other genomes present in complex samples, such as wastewater. Focusing on only the genome-of-interest makes it easier and less costly to sequence and increases the sensitivity to detect low viral loads.
The two common methods of target enrichment are hybridization capture and amplicon sequencing. For hybridization capture, nucleotide probes that hybridize across the viral genome are used to enrich the viral sequences from the host and other contaminating nucleic acids. Although the method is effective for identifying variants and novel strains, the workflow is long and can be cumbersome to adapt for high-throughput processing.
In amplicon sequencing, amplicons are produced from highly multiplexed primer pairs that target regions spanning the length of the viral genome. An advantage of amplicon sequencing over hybridization capture is that amplicon library preparation is fast and simple to perform. Being PCR-based, amplicon sequencing can detect complete viral genomes even from samples with a low viral load.
An improvement on typical amplicon sequencing is to obtain contiguous genome coverage using primers that generate overlapping amplicons in a single tube—a powerful method that allows amplicon formation, even when a mismatch between primer and target prevents binding —enabling full genome coverage and detection of new variants as they emerge, using a single panel design. This method also speeds up virus identification by reducing, or eliminating, the need for panel versioning.
NGS is the method of choice for respiratory virus surveillance
NGS emerged a hero during the COVID-19 pandemic. Using NGS, the SARS-CoV-2 genome was sequenced within a month of the first patient being identified. Having the sequence information in the public domain was instrumental to the developing vaccines and identifying treatment options quickly.
The ability of viruses to continuously mutate results in a constant battle to ensure the timely availability of effective vaccines and treatments. A sensitive and specific tool, such as NGS, that provides a complete picture of the viral genome is key for respiratory virus season preparedness.
With technological improvements driving down the cost of sequencing and more sophisticated amplicon sequencing panels becoming available, health agencies are now better equipped to deploy appropriate testing and response strategies to any viral threat.