De Novo Assembly of Human Herpes Virus Type 1 (HHV-1) Genome, Mining of Non-Canonical Structures and Detection of Novel Drug-Resistance Mutations Using Short- and Long-Read Next Generation Sequencing Technologies.
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.
Department of Zoology, University of Oxford, Oxford, United Kingdom.
Timokratis
T
Karamitros
Virus Reference Department, National Infection Services, Public Health England, London, United Kingdom.
Ian
http://orcid.org/0000-0003-4117-961X
I
Harrison
Virus Reference Department, National Infection Services, Public Health England, London, United Kingdom.
Renata
R
Piorkowska
Department of Zoology, University of Oxford, Oxford, United Kingdom.
Aris
A
Katzourakis
Department of Zoology, University of Oxford, Oxford, United Kingdom.|Virus Reference Department, National Infection Services, Public Health England, London, United Kingdom.
Gkikas
G
Magiorkinis
Virus Reference Department, National Infection Services, Public Health England, London, United Kingdom.
Jean Lutamyo
JL
Mbisa
D019943:Amino Acid Substitution; D000818:Animals; D000998:Antiviral Agents / Q000627:therapeutic use; D001482:Base Composition; D002522:Chlorocebus aethiops; D020451:Contig Mapping; D004279:DNA, Viral / Q000235:genetics*; D024882:Drug Resistance, Viral / Q000235:genetics*; D059646:Genome Size; D016679:Genome, Viral; D006561:Herpes Simplex / Q000188:drug therapy / Q000276:immunology* / Q000821:virology; D018259:Herpesvirus 1, Human / Q000145:classification / Q000187:drug effects / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016867:Immunocompromised Host; D009154:Mutation; D014709:Vero Cells
D000998:Antiviral Agents; D004279:DNA, Viral