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• The latest version of Nanopore improves precision in wastewater viral surveillance. • Efficiency in fragmented viral genome detection is maximized with shorter amplicons. • As little as 0.001 Gb Nanopore data provides accurate real-time variant information. • Nanopore demonstrates utility in revealing dynamic SARS-CoV-2 patterns in Hong Kong. • Optimal cutoffs enable uncovering novel mutations and linking them with variants. Wastewater genomic sequencing stands as a pivotal complementary tool for viral surveillance in populations. While long-read Nanopore sequencing is a promising platform to provide real-time genomic data, concerns over the sequencing accuracy of the earlier Nanopore versions have somewhat restrained its widespread application in wastewater analysis. Here, we evaluate the latest improved version of Nanopore sequencing (R10.4.1), using SARS-CoV-2 as the model infectious virus, to demonstrate its effectiveness in wastewater viral monitoring. By comparing amplicon lengths of 400 bp and 1200 bp, we revealed that shorter PCR amplification is more suitable for wastewater samples due to viral genome fragmentation. Utilizing mock wastewater samples, we validated the reliability of Nanopore sequencing for variant identification by comparing it with Illumina sequencing results. The strength of Nanopore sequencing in generating real-time genomic data for providing early warning signals was also showcased, indicating that as little as 0.001 Gb of data can provide accurate results for variant prevalence. Our evaluation also identified optimal alteration frequency cutoffs (>50 %) for precise mutation profiling, achieving >99 % precision in detecting single nucleotide variants (SNVs) and insertions/deletions (indels). Monitoring two major wastewater treatment plants in Hong Kong from September 2022 to April 2023, covering over 4.5 million population, we observed a transition in dominant variants from BA.5 to XBB lineages, with XBB.1.5 being the most prevalent variants. Mutation detection also highlighted the potential of wastewater Nanopore sequencing in uncovering novel mutations and revealed links between signature mutations and specific variants. This study not only reveals the environmental implications of Nanopore sequencing in SARS-CoV-2 surveillance but also underscores its potential in broader applications including environmental health monitoring of other epidemic viruses, which could significantly enhance the field of wastewater-based epidemiology. [Display omitted] [ABSTRACT FROM AUTHOR]

• Achieving reliable results with sequencing throughput above 0.5 Gb. • Validating performance of variant deconvolution pipeline. • Deciphering the variant prevalence through wastewater genomic sequencing. • Tracking variant trends 16 days earlier than clinical data using wastewater data. • Uncovering novel SARS-CoV-2 mutations and alleles in wastewater. Wastewater-based epidemiology (WBE) has been widely used as a complementary approach to SARS-CoV-2 clinical surveillance. Wastewater genomic sequencing could provide valuable information on the genomic diversity of SARS-CoV-2 in the surveyed population. However, reliable detection and quantification of variants or mutations remain challenging. In this study, we used mock wastewater samples created by spiking SARS-CoV-2 variant standard RNA into wastewater RNA to evaluate the impacts of sequencing throughput on various aspects such as genome coverage, mutation detection, and SARS-CoV-2 variant deconvolution. We found that wastewater datasets with sequencing throughput greater than 0.5 Gb yielded reliable results in genomic analysis. In addition, using in silico mock datasets, we evaluated the performance of the adopted pipeline for variant deconvolution. By sequencing 86 wastewater samples covering more than 6 million people over 7 months, we presented two use cases of wastewater genomic sequencing for surveying COVID-19 in Hong Kong in WBE applications, including the replacement of Delta variants by Omicron variants, and the prevalence and development trends of three Omicron sublineages. Importantly, the wastewater genomic sequencing data were able to reveal the variant trends 16 days before the clinical data did. By investigating mutations of the spike (S) gene of the SARS-CoV-2 virus, we also showed the potential of wastewater genomic sequencing in identifying novel mutations and unique alleles. Overall, our study demonstrated the crucial role of wastewater genomic surveillance in providing valuable insights into the emergence and monitoring of new SARS-CoV-2 variants and laid a solid foundation for the development of genomic analysis methodologies for WBE of other novel emerging viruses in the future. [Display omitted] [ABSTRACT FROM AUTHOR]