Global patterns in coronavirus diversity

Anthony, Simon J.; Johnson, Christine K.; Greig, Denise J.; Kramer, Sarah; Che, Xiaoyu; Wells, Heather; Hicks, Allison L.; Joly, Damien O.; Wolfe, Nathan D.; Daszak, Peter; Karesh, William; Lipkin, W. I.; Morse, Stephen S.; Mazet, Jonna A. K.; Goldstein, Tracey

RNA was extracted from all samples, and cDNA prepared using superscript III (Invitrogen). Two broadly reactive consensus PCR assays targeting non-overlapping fragments of the orf1ab were used to detect both known and novel CoVs (Quan et al. 2010; Watanabe et al. 2010 ). The first [the 'Watanabe' assay (Watanabe et al. 2010) ] amplified a $434 bp fragment of the RNA-dependent RNA polymerase (RdRp) corresponding to nucleotides (NTs) 15,156-15,589 in the human CoV OC43 genome (NC_005147), while the second [the 'Quan' assay (Quan et al. 2010) ] amplified a $332 bp fragment of a different peptide downstream of the RdRp, corresponding to NTs 18,323-18,654. Amplified products of the expected size were cloned and sequenced (traditional Sanger dideoxy sequencing) according to standard protocols, and sequences edited manually in Geneious Pro (ver 9.1.3, Biomatters, Auckland, NZ). We note that this approach was adopted to facilitate viral discovery in resourcepoor settings, which are the target of pandemic preparedness initiatives such as USAID PREDICT, and where techniques such as high throughput sequencing are largely unavailable.