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Viral surveillance using wastewater

Monitoring the outbreak of viral diseases using amplicon sequencing approaches on wastewater samples has allowed researchers to identify emerging SARS-CoV-2 variants faster and with less bias than clinical surveillance approaches, saving communities precious time and resources during the global COVID-19 health crisis.

What are infectious diseases?

Infectious diseases are illnesses that are spread through external exposure to pathogenic organisms like bacteria, fungi, parasites, or viruses. Sources of infectious diseases include contaminated food, water, animals, or other means such as person-to-person contact. When an infectious disease has ‘jumped’ or become transmissible between animals and humans, it is called a zoonosis or zoonotic infectious disease [1]. Viral zoonotic diseases have recently been of increasing concern with the global SARS-CoV-2 pandemic and outbreaks of Monkeypox [2]. Human immunodeficiency virus (HIV) also started out as a zoonotic disease but has since mutated to only being transmissible via person-to-person contact [3]. Other examples of viral zoonotic diseases that have had global impacts include (but are not limited to) Ebola, Zika, and Middle East Respiratory syndrome (MERS). Since viral zoonotic diseases can be spread between animals and humans, it can be difficult to monitor their rates and patterns of transmission [4]. Moreover, as the frequency of human-animal interactions increases, so does the frequency of the appearance of novel zoonotic diseases [5,6], meaning that being able to monitor and rapidly respond to outbreaks of emerging infectious diseases at a community scale has become more important than ever.

Wastewater-based epidemiology for viral surveillance

Viral surveillance -monitoring the spread of viral infectious diseases at the community level has proven to be instrumental in understanding and responding to the spread of COVID-19, as well as in tracking emerging variants [7,8]. One extremely efficient way researchers have been able to do this throughout the SARS-CoV-2 pandemic has been to use wastewater-based epidemiology, or waste-water surveillance methods [7-9]. This approach to viral surveillance has a number of benefits. One important example is that individuals infected with SARS-CoV-2 shed viral particles in their feces even if they are asymptomatic, meaning that researchers can capture outbreak events even if at the community level people aren’t having symptoms yet [7]. Two main approaches that have been used to monitor wastewater for SARS-CoV-2 are qPCR and amplicon sequencing. qPCR is used to gather information concerning the viral loads present in samples [9], while amplicon sequencing is used to provide data about SARS-CoV-2 variants [7-9]. Amplicon sequencing is a type of targeted next generation sequencing that allows researchers to sequence genetic information from only organisms of interest. When researchers use amplicon sequencing panels, they target multiple DNA regions and amplify or enrich those regions in their samples. Then by adding adapters , researchers can multiplex or pool their samples and sequence them together. These sequences can then be separated bioinformatically and analyzed. Figure 1 describes a standard amplicon sequencing workflow.

xGen Custom Amplicon Panel workflow
Figure 1. xGen Custom Amplicon Panels have a single tube workflow that is done in as little as 2 hours. Creating an NGS library starts with a multiplex PCR reaction using either DNA or, for RNA viruses like SARS-CoV-2, cDNA. Your custom panel is combined with DNA or cDNA to amplify the targets of interest. The samples are then amplified with indexing primers to create a functional dual indexed library. As an optional step, the xGen Normalase reagent can be used after pooling multiple libraries to ensure that each library is equally represented in the final sample on the flow cell.

However, obtaining high-quality sequencing information from wastewater samples can be challenging as these types of samples are complex, tend to have degraded genetic material, low concentrations of viral particles, and high concentrations of PCR inhibitors [7]. Despite these challenges, researchers using the xGen SARS-CoV-2 Amplicon Panel were able to sequence and identify novel variants of SARS-CoV-2 from wastewater samples [7-9]. The xGen SARS-CoV-2 Amplicon Panel was designed to create 345 overlapping amplicons that cover the entire genome of SARS-CoV-2. This high rate of coverage helped researchers identify emerging variants for SARS-CoV-2 from challenging wastewater samples [7-9], in one case, weeks before they had been identified in clinical surveillance networks [7].


The future of viral surveillance for infectious disease outbreaks

Wastewater surveillance of viral outbreaks using next generation sequencing approaches can be challenging, resulting in poor quality sequencing data that makes it difficult to identify novel variants. This is why approaches such as qPCR have often been used in lieu of targeted sequencing because qPCR provides key information about the presence of viruses in wastewater samples as well as their concentration without needing to generate sequencing data. However, as seen in recent studies [6-8], advances in amplicon sequencing panel design have enabled researchers to overcome these sequencing challenges, resulting in the ability to monitor emerging variants at the population level using wastewater samples.

Being able to alert communities about an outbreak of an infectious disease before it has been identified in clinical surveillance studies saves precious time, resources, and overcomes important research biases. Researchers have also suggested that in the future wastewater surveillance could be used to track multiple diseases simultaneously either by using untargeted NGS approaches [9] or expanding targeted NGS technologies to monitor multiple diseases in parallel.


  1. Zoonoses. 2020; Accessed August 19, 2022.
  2. Leon-Figueroa DA, Bonilla-Aldana DK, Pachar M, et al. The never-ending global emergence of viral zoonoses after COVID-19? The rising concern of monkeypox in Europe, North America and beyond. Travel Med Infect Dis. 2022;49:102362.
  3. Sharp PM, Hahn BH. Origins of HIV and the AIDS pandemic. Cold Spring Harb Perspect Med. 2011;1(1):a006841.
  4. Reed KD. Viral Zoonoses. Elsevier. 2018 (Reference Module in Biomedical Sciences).
  5. Jones KE, Patel NG, Levy MA, et al. Global trends in emerging infectious diseases. Nature. 2008;451(7181):990-993.
  6. Holmes EC. COVID-19-lessons for zoonotic disease. Science. 2022;375(6585):1114-1115.
  7. Karthikeyan S, Levy JI, De Hoff P, et al. Wastewater sequencing reveals early cryptic SARS-CoV-2 variant transmission. Nature. 2022.
  8. Fontenele RS, Kraberger S, Hadfield J, et al. High-throughput sequencing of SARS-CoV-2 in wastewater provides insights into circulating variants. Water Res. 2021;205:117710.
  9. Spurbeck RR, Minard-Smith A, Catlin L. Feasibility of neighborhood and building scale wastewater-based genomic epidemiology for pathogen surveillance. Sci Total Environ. 2021;789:147829.

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Published Nov 7, 2022