De novo assembly and characterisation of drug resistance in human herpesvirus type 1 (HHV-1)

Between 60‑90% of the general population are infected with human herpesvirus type 1 (HHV‑1)1. Despite the majority of infections being asymptomatic, up to 45% of the population suffer from recurrent labial lesions2. While a number of drugs are available to treat infection with HHV‑1, they are all susceptible to resistant mutations. HHV‑1 is a large double‑stranded DNA virus (152 kb), with a structurally complex and GC‑rich genome. This makes the assembly of HHV‑1 whole genomes from short‑read sequencing data technically challenging.

To improve the assembly and analysis of HHV‑1 genomes, enabling the characterisation of drug resistance mutations, researchers at the University of Oxford, UK, and Public Health England, UK, combined long‑read nanopore sequencing data with data derived from a short‑read sequencing platform3. The team sequenced 18 HHV‑1 samples cultured from immunocompromised patients undergoing antiviral therapy. Genome assembly was performed using the MIRA48 and LINKS49 tools.

The length of the nanopore reads allowed the merging of existing short‑read derived contigs that had been disrupted by repetitive elements. This allowed the generation of improved genome assemblies as evidenced by fewer contigs and increased N50 values (Table 1).

The team was also able to characterise regions of structural variance, including duplications, deletions and rearrangements. In addition, high levels of nucleotide variation were found in UL23, a gene, within which, the majority of drug resistance mutations occur. The researchers believe that these variations most likely arose during the antiviral therapy that had been administered prior to sample acquisition3.

According to the team, this study proves that nanopore sequencing is capable of improving the reference‑free de novo assembly of the viral genome and can pass through repetitive elements that terminate the contigs generated by the short‑read sequencing platform3.

Table (below): Addition of nanopore long‑read data to existing short‑read data allowed the generation of improved genome assemblies. Data from a single HHV‑1 sample (p1A_454) shown. Table adapted from Karamitros et al.3

Assembly# contigsLargest contig (bp)Total length (bp)N50
Short read2162,373136,93543,352
Short + long nanopore reads18111,746136.978111,746

This case study is taken from the Microbiology white paper.

  1. Chayavichitsilp, P., Buckwalter, J.V., Krakowski. A.C., & Friedlander, S.F. Herpes simplex. Pediatr Rev. 30(4): 119‑29 (2009).
  2. Harmenberg, J., Oberg, B. & Spruance,  S. Prevention of ulcerative lesions by episodic treatment of recurrent herpes labialis: A literature review. Acta dermato-venereologica. 90(2):122–30 (2010).
  3. Karamitros, T. et al. De novo assembly of Human Herpes Virus Type 1 (HHV‑1) genome, mining of noncanonical structures and detection of novel drug‑resistance mutations using short‑ and long‑ read next generation sequencing technologies. PLoS One 11(6) (2016).