Illumina MiSeq paired end sequencing; Diagnostic Microbiology & Infectious Disease Rapid and sensitive detection of Sars-CoV-2 variants in nasopharyngeal swabs and wastewaters
Diagnostic Microbiology & Infectious Disease Rapid and sensitive detection of Sars-CoV-2 variants in nasopharyngeal swabs and wastewaters
Raw sewage samples (wastewater) were collected between December 2020 and April 2021 from  different WWTPs (Waste Water Treatment Plant) located in the provinces of Naples and Salerno  (Campania Region). Composite samples, representing 24-hour period were collected and stored at  4C until use. On the arrival, samples underwent a 30 min treatment at 56 C to increase the safety of the protocol  and then concentrated with polyethylene glycol (PEG) precipitation. 45 ml of each sample were  centrifuged at 4C for 30 min at 4500g to pellet wastewater solids; then clarified wastewater was  mixed with 10% PEG and 2.25% NaCl and centrifuged at 4C for 2 h at 12000g . The pellet was  finally resuspended in the appropriate volume of PBS and entirely used for RNA extraction with the 147 Total RNA purification kit (Norgen, biotel corp.), following manufacturer instructions. Indexed libraries were prepared from 12 l of viral RNA using a targeted amplicon-based NGS panel  protocol by Paragon Genomics according to manufacturer's instruction. Libraries were pooled in  equimolar amounts.