MERS-CoV Genome Sequencing Protocol

Thirty-two pairs of nested PCR assays that span the genome were designed based on alignment of available MERS-CoV genomes.^{39 (link), 40 (link)} MERS-CoV genomes or partial genomes were amplified as needed to fill the gaps missed by Fluidigm Access Array PCR. Positive bands of the expected size that had strong signal and without additional bands were cleaned up using Exonuclease I (New England Biolabs) and Shrimp Alkaline Phosphatase (Roche). Samples were incubated at 37 °C for 15 min followed by 80 °C for 15 min to inactivate the Exonuclease and Shrimp Alkaline Phosphatase. Purified PCR amplicons were sequenced with the PCR primers in both directions on an ABI Prism 3130 Automated Capillary Sequencer (Applied Biosystems, Foster City, CA, USA) using Big Dye 3.1 cycle sequencing kits (Life Technologies, Carlsbad, CA, USA). The Sanger sequence data were analyzed using Sequencher 5.0. Ends and low-quality regions were trimmed manually. Contigs and consensus sequences were generated.