title: Nanopore sequencing of QLD SARS-CoV-2 Isolates
design: Briefly, the nCoV-2019 Nanopore sequencing protocol v3 (Josh Quick, University of Birmingham) was used with minor modifications. RNA was isolated from cell culture supernatant, and cDNA generated using Protoscript II first-strand cDNA synthesis kit as per manufacturers protocol (New England Biolabs, USA). SARS-CoV-2 cDNA was subsequently amplified using ARTIC network v2 primers using two-step PCR amplification with Q5 High-Fidelity DNA Polymerase (New England Biolabs, USA). PCR fragments were purified using AMPure XP beads (Beckman Coulter, USA) and subjected to End Repair/dA-Tailing using the NEBNext Ultra II Module (New England Biolabs, USA). Both P3 QLD02 and QLD935 samples were multiplexed using the Native Barcoding Expansion kit (EXP-NBD104, Oxford Nanopore, UK) and Ligation Sequencing Kit (SQK-LSK109, Oxford Nanopore, UK). Prepared libraries were then quantified and loaded into equimolar concentrations totalling 20 fmol into a Flongle flow cell (FLOFLGOP1, Oxford Nanopore, UK).