Human herpesvirus type 1 (HHV-1) has a large double-stranded DNA genome of approximately152 kbp that is structurally complex and GC-rich. This makes the assembly of HHV-1 whole genomes from short-read sequencing data technically challenging. To improve the assembly of HHV-1 genomes we have employed a hybrid genome assembly protocol using data from two sequencing technologies: the short-read Roche 454 and the long-read Oxford Nanopore MinION sequencers. We sequenced 18 HHV-1 cell culture-isolated clinical specimens collected from immunocompromised patients undergoing antiviral therapy. The susceptibility of the samples to several antivirals was determined by plaque reduction assay. Hybrid genome assembly resulted in a decrease in the number of contigs in 6 out of 7 samples and an increase in N(G)50 and N(G)75 of all 7 samples sequenced by both technologies. The approach also enhanced the detection of non-canonical contigs including a rearrangement between the unique (UL) and repeat (T/IRL) sequence regions of one sample that was not detectable by assembly of 454 reads alone. We detected several known and novel resistance-associated mutations in UL23 and UL30 genes. Genome-wide genetic variability ranged from <1% to 53% of amino acids in each gene exhibiting at least one substitution within the pool of samples. The UL23 gene had one of the highest genetic variabilities at 35.2% in keeping with its role in development of drug resistance. The assembly of accurate, full-length HHV-1 genomes will be useful in determining genetic determinants of drug resistance, virulence, pathogenesis and viral evolution. The numerous, complex repeat regions of the HHV-1 genome currently remain a barrier towards this goal.
Bibliographical noteFunding Information:
This work was partly supported by a Public Health England (PHE) Pump-Priming and Small Initiatives Fund to JLM and by a Medical Research Council (MRC) grant [MR/K010565/1] to GM. The views expressed are those of the authors and not necessarily those of PHE, the Department of Health or MRC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
© 2016 Karamitros et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.