Project description:Wastewater-based surveillance (WBS) is a proven tool for monitoring population-level infection events. Wastewater contains high concentrations of inhibitors, which contaminate total nucleic acids (TNA) extracted from these samples. We found that TNA extracts from raw influent of Berlin wastewater treatment plants contained highly variable amounts of inhibitors that impaired molecular analyses like dPCR and next-generation sequencing (NGS). By using dilutions, we were able to detect inhibitory effects. To enhance WBS sensitivity and stability, we applied a combination of PCR inhibitor removal and TNA dilution (PIR+D). This approach led to a 26-fold increase in measured SARS-CoV-2 concentrations, practically reducing the detection limit. Additionally, we observed a substantial increase in stability of the time series. We define suitable stability as a mean absolute error (MAE) below 0.1 log10 copies/l and a geometric mean relative absolute error (GMRAE) below 26%. Using PIR+D, the MAE could be reduced from 0.219 to 0.097 and the GMRAE from 65.5% to 26.0% and even further in real-world WBS. Furthermore, PIR+D improved SARS-CoV-2 genome alignment and coverage in amplicon-based NGS for low to medium concentrations. In conclusion, we strongly recommend both the monitoring and removal of inhibitors from samples for WBS.
Project description:Nucleic acids in wastewater provide a rich source of data for detection and surveillance of microbes. We have longitudinally collected 116 RNA samples from a wastewater treatment plant in Berlin/Germany, from March 2021 to July 2022, and 24 DNA samples from May to July 2022. We tracked human astroviruses, enteroviruses, noroviruses and adenoviruses over time to the level of strains or even individual nucleotide variations, showing how detailed human pathogens can be observed using wastewater. For respiratory pathogens, a broad enrichment panel enabled us to detect waves of RSV, influenza, or common cold coronaviruses in high agreement with clinical data. By applying a profile Hidden Markov Model-based search for novel viruses, we identified more than 100 thousand novel transcript assemblies likely not belonging to known virus species, thus substantially expanding our knowledge of virus diversity. Phylogenetic analysis is shown for bunyaviruses and parvoviruses. Finally, we identify Hundreds of novel protein sequences for CRISPR-associated proteins such as Transposase B, a class of small RNA-guided DNA editing enzymes. Taken together, we present a longitudinal and deep investigation into wastewater-derived genomic sequencing data that underlines the value of sewage surveillance for public health, planetary virome research, and biotechnological potential.
