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Aaron W. Kolb, Curtis R. Brandt, Eric L. Cabot, Marie Adams; Multiplex Genome Sequencing of Seven Clinical Ocular Herpes Simplex Virus Type 1 Isolates and One Syncytial Mutant. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5802.
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© ARVO (1962-2015); The Authors (2016-present)
In HSV-1, the virulence phenotype of a given strain is partly due to a constellation of genes. To better understand this, next generation sequencing techniques were used to sequence the genomes of multiple HSV-1 ocular isolates and begin to characterize them using phylogenetic, sequence similarity and SNP analysis.
The genomes of eight HSV-1 viruses were prepared for multiplexing and then sequenced using the Illumina Genome Analyzer IIx. The sequenced genomes were assembled by mapping the short sequence reads to the HSV-1 strain 17 reference genome. Phylogenetic, sequence similarity and SNP analysis were used to compare the eight genomes
Next generation multiplex sequencing and reference assembly resulted in a minimum of 224x average genome coverage. Low sequence coverage in the VNTR, terminal and internal repeat regions was largely due to high G/C content. Phylogenetic analysis of the genomes from eight sequenced HSV-1 viruses along with previously published strains F and H129 revealed that the viruses form one species and group into three clades. Genome comparison showed that the strains are >99% similar, with the terminal and internal repeats of the genomes displaying the highest sequence variability. SNP analysis demonstrated that each of the strains has approximately 200 coding SNP’s that are evenly distributed across the genome. Analysis of the protein sequences revealed that the UL35, UL47, gK and UL55 protein sequences were conserved across the eight strains. Additionally, the UL1, UL11, UL43, UL49A, gG and gI proteins were significantly more variable than other viral proteins. Phylogenetic analysis of individual genes suggests recombination has occurred. Examination of the genome from a hyper-syncytial variant of the HSV-1 OD4 strain did not clearly identify a mutation responsible for the hyper-syncytial phenotype.
The results demonstrate that multiplexing multiple HSV-1 genomes is a cost effective method of rapidly sequencing HSV-1 genomes. The sequences described in this work are useful for future structure-function studies and lay the groundwork for future virulence studies.
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