Author: John-Sebastian Eden; Rebecca Rockett; Ian Carter; Hossinur Rahman; Joep de Ligt; James Hadfield; Matthew Storey; Xiaoyun Ren; Rachel Tulloch; Kerri Basile; Jessica Wells; Roy Byun; Nicky Gilroy; Matthew V O’Sullivan; Vitali Sintchenko; Sharon C Chen; Susan Maddocks; Tania C Sorrell; Edward C Holmes; Dominic E Dwyer; Jen Kok
Title: An emergent clade of SARS-CoV-2 linked to returned travellers from Iran Document date: 2020_3_17
ID: izkz1hz4_7
Snippet: In New South Wales (NSW), Australia, WGS for SARS-CoV-2 was developed based on an existing amplicon-based Illumina sequencing approach [6] . Viral extracts were prepared from respiratory tract samples where SARS-CoV-2 was detected by RT-PCR using World Health Organization recommended primers and probes targeting the E and RdRp genes, and then reverse transcribed using SSIV VILO cDNA master mix. The viral cDNA was used as input for multiple overla.....
Document: In New South Wales (NSW), Australia, WGS for SARS-CoV-2 was developed based on an existing amplicon-based Illumina sequencing approach [6] . Viral extracts were prepared from respiratory tract samples where SARS-CoV-2 was detected by RT-PCR using World Health Organization recommended primers and probes targeting the E and RdRp genes, and then reverse transcribed using SSIV VILO cDNA master mix. The viral cDNA was used as input for multiple overlapping PCR reactions (~2.5kb 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 [7] . In short, 400bp tiling . CC-BY-NC 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.15.992818 doi: bioRxiv preprint 4 amplicons designed with Primal Scheme [8] 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 [9] using the ARTIC network nCoV-2019 novel coronavirus bioinformatics protocol [10] . 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) .
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