Closing Gaps in MERS-CoV Genome Sequencing

To fill the gaps that remained after mapping Ion reads, we designed 11 primer sets (Table 4) against the NCBI MERS-CoV reference genome assembly (NC_019843.3). Primers were designed and checked using Primer Express software (v3.0) (Applied Biosystems, Foster City, CA, USA) and NetPrimer (Premier Biosoft, Palo Alto, CA, USA), with gap flanking regions incorporated into the primers. Sequences for all the major gaps were amplified by PCR using Platinum^TM Hot Start PCR Master Mix 2X (Thermo Fisher Scientific, Waltham, MA, USA). PCR products were sequenced using the BigDye^® Terminator Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on an Applied Biosystems 3730xl DNA Analyzer. Most minor gaps were sample-specific; to ensure complete genome coverage for phylogenetic analysis, these gaps were closed manually after performing multiple sequence alignment (MSA) on the fully-sequenced genomes. MSA was carried out using Geneious software [34 (link)].