Human alphaherpesvirus 1 Genome sequencing and assembly

Using synthetic genomics approaches, we have developed an assembly method for large dsDNA virus genomes utilizing herpes simplex virus type 1 (HSV-1) as a model. Transformation-associated recombination (TAR) cloning in Saccharomyces cerevisiae was used to clone 11 overlapping fragments of the HSV-1 strain KOS genome. The template genomes used to clone the fragments were HSV-1 KOS-37 BAC DNA isolated from Escherichia coli and infected cell DNA from HSV-1 K?25/26, a mutant of strain KOS with a deletion in the UL25/UL26 region. Plasmid DNA containing the cloned HSV-1 fragments was digested to release the HSV-1 genomic fragment and all 11 overlapping HSV-1 genomic fragments were co-transformed into yeast and assembled. The assembled HSV-1 genome is designated KOSYA (KOS yeast-assembled). KOSYA contains yeast centromeric plasmid and bacterial artificial chromosome (BAC) sequences in the intergenic region between UL37/38, the same site as the BAC sequence of KOS-37 BAC. We sequenced the genome of the template strains, KOS-37 BAC and K?25/26, and of KOSYA isolated from E. coli and from infected cell DNA of reconstituted virus.