An emergent clade of SARS-CoV-2 linked to returned travellers from Iran

Eden, John-Sebastian; Rockett, Rebecca; Carter, Ian; Rahman, Hossinur; de Ligt, Joep; Hadfield, James; Storey, Matthew; Ren, Xiaoyun; Tulloch, Rachel; Basile, Kerri; Wells, Jessica; Byun, Roy; Gilroy, Nicky; O’Sullivan, Matthew V; Sintchenko, Vitali; Chen, Sharon C; Maddocks, Susan; Sorrell, Tania C; Holmes, Edward C; Dwyer, Dominic E; Kok, Jen

Viral extracts were prepared from respiratory tract samples where SARS-CoV-2 was detected by reverse-transcription polymerase chain reaction (RT-PCR) using World Health Organisation (2020b) recommended primers and probes targeting the E and RdRp genes. In New South Wales (NSW), Australia, WGS for SARS-CoV-2 was developed based on an existing amplicon-based Illumina sequencing approach (Di Giallonardo et al. 2018 ). Viral extracts were reverse transcribed with SSIV VILO cDNA master mix and then used as input for multiple overlapping PCR reactions (2.5 kb each) spanning the viral genome using Platinum SuperFi master mix (primers provided in Supplementary Table S1 ). Amplicons were pooled equally, purified, and quantified. Nextera XT libraries were prepared and sequencing was performed with multiplexing on an Illumina iSeq (300 cycle flow cell). In New Zealand, the ARTIC network protocol was used for WGS (Quick 2020) . In short, 400-bp tiling amplicons designed with Primal Scheme (Grubaugh et al. 2019a ) were used to amplify viral cDNA prepared with SuperScript III. A sequence library was then constructed using the Oxford NanoPore ligation sequencing kit and sequenced on a R9.4.1 MinION flow cell. Near-complete viral genomes were then assembled de novo in Geneious Prime 2020.0.5 or through reference mapping with RAMPART V1.0.6 (Hadfield 2019) using the ARTIC network nCoV-2019 novel coronavirus bioinformatics protocol (Loman and Rambaut 2020) . In total, 13 SARS-CoV-2 genomes were sequenced from cases in NSW diagnosed between 24 January and 3 March 2020, as well as a single genome from the first patient in Auckland, New Zealand sampled on 27 February 2020 (Table 1) . Australian and New Zealand sequences were aligned to global reference strains sourced from GISAID with MAFFT (Katoh 2002) and then compared phylogenetically using a maximumlikelihood approach-PhyML v2.2.4 (Guindon and Gascuel 2003) .