WGS of SARS-CoV-2 from Wastewater
SARS-CoV-2 RNA libraries were produced using the ATOPlex platform, which is a multiplexed amplicon-based sequencing workflow that is tailor-made for detecting SARS-CoV-2 fragments (~500 bp). Following reverse transcription of 10 L RNA, a two-step PCR with 13 and 27 cycles for target enrichment and sequence adapter with dual-barcode primer (Cat No: 1000021626) addition, respectively, was carried out in the first and second PCRs. The amplified PCR products were purified by magnetic bead-based techniques followed by quantity and quality checks using the Qubit dsDNA high sensitivity assay kit (Life Technologies, USA) and gel-based electrophoreses, respectively. Libraries were equally pooled, and single strand circularized DNA (ssDNA) was synthesized using the MGIEasy Dual Barcode Circularization kit (Cat No:1000020570). DNA nanoballs (DNBs) were constructed for both the ssDNA sample library and a dual barcoded balance library (Cat No: 1000022270) in parallel, which was then mixed 3:1 prior to sequencing on the DNBSEQ-G50RS instrument at SAMRCs Genomics Centre. Data analysis was conducted using a sweet of bioinformatics tools