Abstract
Human herpesvirus type 1 (HHV-1) has a large double-stranded DNA genome of approximately 152 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.
Highlights
Human herpesvirus type 1 (HHV-1), known as Herpes simplex virus type 1 (HSV-1), has seroprevalence that ranges from 60 to 90% in the general population [1]
The de novo assembly of HHV-1, and other herpesviruses, is challenging due to the increased length and the unique structure of the genome, which incorporates extended regions that are repeated and omnipresent Variable Number Tandem Repeats (VNTRs) that usually exceed the read length of existing generation sequencing platforms [22, 44, 45]
In this study we investigated the potential of a newly released sequencer, the MinION, to improve the de novo assembly pipeline of HHV-1 by assisting with the length of contigs generated by the Roche 454 GS Junior Sequencer
Summary
Human herpesvirus type 1 (HHV-1), known as Herpes simplex virus type 1 (HSV-1), has seroprevalence that ranges from 60 to 90% in the general population [1]. Despite the majority of the infections being asymptomatic, 15 to 45% of the adult population suffers from recurrent labial lesions [2]. Antiviral drugs used include the nucleoside analogs acyclovir (ACV)–the drug of choice -, and penciclovir (PCV), as well as foscarnet (FOS), a pyrophosphate analog [9]. The mechanism of these drugs is through inhibition of the viral DNA polymerase (Pol) by acting as competitive inhibitors and/or as chain polymerization terminators. The mono-phosphorylated nucleoside analog Cidofovir (CDV) inhibits Pol, but is not approved for the treatment of HHV-1 infections [10]. Mutations that are selected in TK are normally insertions or deletions in homopolymeric regions (runs of Gs or Cs) that result in frameshift mutations and premature stop codons [13]
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