title: Pooled sample of 29 untreated sewage samples from treatment works in the East of England amplified for GI and GII norovirus using optimised PCR conditions
design: For each sample 1L of untreated sewage was collected and 150 mL processed using ammonium sulphate precipitation. Nucleic acid extraction using a Kingfisher Flex (Thermo Scientific, UK) and NucliSENS (BioMrieux, France) reagents was performed. Multiple samples from the same region of England (UK) were collected in October 2021 and pooled together. Nucleic acid extracts were cleaned with Mag-Bind TotalPure NGS beads using 25L of nucleic acids. LunaScript RT SuperMix was used for reverse transcription, with a nucleic acid volume of 10 L and a final sample volume of 20 L.   Separate semi-nested PCR assays for norovirus GI and GII were performed with either 5 L of cDNA or 5 L first-round PCR product as template. Platinum Taq polymerase was used with 25 L reaction volumes. Cycling conditions and mastermix recipe were as described by the manufacturer. Annealing temperatures of 47.4C and 57.2C for GI first-round and semi-nested PCRs and 55.7C for both GII PCRs were used. Primers are as outlined by Scott et al., 2024. PCR products (10 L) were cleaned using ExoSAP-IT and quantified using the Qubit fluorometer HS dsDNA kit. GI and GII PCR amplicons were pooled prior to library prep (85.3 fmol of GI and 114.7 fmol GII) to account for non-specific amplification in the GII assay. Library preparation was performed using the Native Barcoding Kit 96 V14 with sequencing using R10.4.1 flow cells, a GridION (MinKNOW software release 22.10.7) and 260 bps super accurate basecalling.