Efficient wastewater sample filtration improves the detection of SARS-CoV-2 variants: An extensive analysis based on sequencing parameters

Abstract

During the SARS-CoV-2 pandemic, many countries established wastewater (WW) surveillance to objectively monitor the level of infection within the population. As new variants continue to emerge, it has become clear thatWWsurveillance is an essential tool for the early detection of variants. The EU Commission published a recommendation suggesting an approach to establish surveillance of SARS-CoV-2 and its variants in WW, besides specifying the methodology forWWconcentration and RNA extraction. Therefore, different groups have approached the issue with different strategies, mainly focusing onWWconcentration methods, but only a few groups highlighted the importance of prefilteringWWsamples and/ or purification of RNA samples. Aiming to obtain high-quality sequencing data allowing variants detection, we compared four experimental conditions generated from the treatment of: i)WWsamples byWWfiltration and ii) the extracted RNA by DNase treatment, purification and concentration of the extracted RNA. To evaluate the best condition, the results were assessed by focusing on several sequencing parameters, as the outcome of SARS-CoV-2 sequencing fromWWis crucial for variant detection. Overall, the best sequencing result was obtained by filtering theWWsample. Moreover, the present study provides an overview of some sequencing parameters to consider when optimizing a method for monitoring SARS-CoV-2 variants fromWWsamples, which can also be applied to any sample preparation methodology.