Optimised protocol for monitoring SARS-CoV-2 in wastewater using reverse complement PCR-based whole-genome sequencing

Area of Science:

• Environmental microbiology
• Genomic epidemiology
• Public health virology

Background:

• Population-level viral surveillance is vital for epidemic response and understanding transmission dynamics.
• Genomic sequencing of wastewater offers unbiased data, capturing infections missed by clinical sampling.
• Early detection of outbreaks and novel variants is crucial for effective public health interventions.

Purpose of the Study:

• To present an optimized protocol for high-throughput genomic sequencing of SARS-CoV-2 in wastewater.
• To validate the protocol's efficacy and sensitivity for real-world application in England during the COVID-19 pandemic.

Main Methods:

• Developed an optimized protocol for SARS-CoV-2 quantification and sequencing in wastewater.
• Utilized reverse complement PCR for efficient, single-step library preparation and adapter addition.
• Employed high-throughput sequencing and validated with synthetic SARS-CoV-2 RNA.

Main Results:

• The protocol demonstrated efficacy and sensitivity in sequencing SARS-CoV-2 from wastewater samples.
• Successful application for high-throughput genomic surveillance in England during the COVID-19 pandemic.
• Provided guidance on quality control for library preparation and data analysis.

Conclusions:

• The presented method offers an effective approach for high-throughput SARS-CoV-2 wastewater sequencing.
• This protocol can be adapted for surveillance of other human and animal viruses and pathogens.
• Enhanced wastewater surveillance contributes to robust public health monitoring during epidemics.