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Next-Generation Sequencing for SARS-CoV-2 Surveillance

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SARS-CoV-2 can mutate and evolve over time, leading to the emergence of variants such as B.1.1.7, B.1.351 and P.1. In many cases, mutations are inconsequential, but some could have the potential to increase transmissibility and disease severity, as well as negatively impact the performance of diagnostics, therapeutics and vaccines. Surveillance by the World Health Organization SARS-CoV-2 Virus Evolution Working Group and several national authorities is helping to rapidly identify mutations and understand their significance.

To gain some insights into the role of next-generation sequencing (NGS) in global SARS-CoV-2 surveillance,
Technology Networks spoke to Michelle Fraser, general manager, NGS for PerkinElmer. Michelle also explains how labs working in this area can increase their throughput, decrease associated costs and simplify the analysis of NGS data collected.

Anna MacDonald (AM): Can you tell us about some of the latest advances in NGS technology?

Michelle Fraser (MF):
NGS has been in regular use in research since its introduction in 2005. More recently, NGS has been developed for clinical diagnosis of genetic disease and screening for the risk to develop late onset diseases. This shift from investigational research to reimbursed diagnostics brings with it a maturation of the instruments, kits, and analysis and reporting software to create a complete sample to answer workflow that can be approved by regulatory bodies. 

The continual evolution and advances in NGS have also increased the power of NGS applications. Metagenomics, multiomics and single cell analyses are all examples of where NGS is advancing rapidly.

AM: How is NGS being applied for SARS-CoV-2 surveillance?

When a patient or environmental sample comes up SARS-CoV-2 positive by PCR, the very next question is “where did it come from?” When the virus is transmitted from one person to the next, the viral genome will be identical, or very close to it. The SARS-CoV-2 genome mutates at approximately two point mutations per month, so tracing the virus back to the source and identifying other people that have been in close contact and might not have been screened yet can help to control the spread.

The CDC has designated three classes of variants; Variants of Interest that, based on their genomic sequence, may not be as successfully neutralized by the current vaccines or treatments and are being monitored, Variants of Concern that have been linked with increased transmission, disease severity, reduced neutralization or diagnostic detection failures (false negative tests) and Variants of High Consequence that will require new diagnostics, treatments and are expected to lead to more severe clinical disease and increased hospitalization. For example, the B.1.1.7 variant of concern has been associated with a significantly increased risk of transmission.

AM: What should laboratory staff know today regarding the latest SARS-CoV-2 variants?

SARS-CoV-2 mutations are opportunistic. New variants arise through survival of the fittest. The current rate of mutation is half that of influenza, but it is still a continually changing genome. This can impact the accuracy of PCR tests, so ongoing vigilance is essential. Initially, labs were completely focused on monitoring infection rate through positive or negative detection of SARS-CoV-2. It was a new virus that we knew very little about, other than it spread rapidly and it put immense pressure on global health services. As the pandemic moves towards an endemic, there will be less focus on COVID diagnosis, and more focus on understanding the virus. This is expected to increase the interest in genomics and identifying new variants as they appear.

AM: The rate of sequencing SARS-CoV-2 samples varies greatly across and within countries; how can the global scientific community work against these challenges to keep up with viral mutations?

The SARS-CoV-2 global pandemic has only been able to be brought under a small degree of control because the global scientific community worked together. The first genomic construct was made publicly available, which allowed diagnostic test developers to create testing solutions and pharmaceutical developers to create vaccines in record time. Sequencing will continue to improve our understanding of SARS-CoV-2. The more widespread the sequencing program is, the more variants we will uncover and the better we will understand the infection. If there are regions where there is no sequence information, it is going to be difficult to track variants and understand how the viral mutations are affecting transmission, severity of infection and efficacy of treatments.

AM: How can labs increase their throughput and/or decrease the costs associated with genomic surveillance of SARS-Cov-2 mutations using NGS?

Elimination of steps, such as a secondary nucleic acid isolation and normalization, reduces costs and allow labs to increase their throughput. Automation can free up human resources to focus on more complicated tasks, allow for increased sample numbers to be batched and therefore reduce the cost per sample for sequencing reagents. Additionally, increased multiplexing capabilities allow labs to reduce their sequencing costs, increase throughput, and deliver results faster.

AM: How can labs simplify the analysis of NGS data collected?

The global collaboration to understand SARS-CoV-2 also included NGS data analysis software developers and genomic sequence repositories working closely with academic and industrial research groups to streamline and unify data analysis. This has been an important aspect because it meant that all labs were creating very similar data that could be easily compared between labs across the globe. The analysis pipeline should be
compliant with the CDC requirements for tracking variants and easily submitted to the NCBI and GISAID databases.

What are the advantages and disadvantages of using RT-PCR for identification of SARS-CoV-2 mutations?

RT-PCR is still the gold standard primary diagnostic test for detecting SARS-CoV-2 due to its rapid turn-around time, cost and simple positive/negative results interpretation. However, the RT-PCR tests do not provide a whole genome to analyze. Variants are identified by using the whole genome, so whilst RT-PCR tests can be modified and adapted to detect new variants, the variants first need to be identified by sequencing. We need to sequence a proportion of SARS-CoV-2 RT-PCR positive samples to check for variants, especially if there is an increase in rate of infection, severity of symptoms or another indicator that suggests that there is a new variant that needs to be better understood and more closely tracked.

Michelle Fraser was speaking to Anna MacDonald, Science Writer for Technology Networks.