Reliable Detection of Herpes Simplex Virus Sequence Variation by High-Throughput Resequencing

Abstract

High-throughput sequencing (HTS) has resulted in data for a number of herpes simplex virus (HSV) laboratory strains and clinical isolates. The knowledge of these sequences has been critical for investigating viral pathogenicity. However, the assembly of complete herpesviral genomes, including HSV, is complicated due to the existence of large repeat regions and arrays of smaller reiterated sequences that are commonly found in these genomes. In addition, the inherent genetic variation in populations of isolates for viruses and other microorganisms presents an additional challenge to many existing HTS sequence assembly pipelines. Here, we evaluate two approaches for the identification of genetic variants in HSV1 strains using Illumina short read sequencing data. The first, a reference-based approach, identifies variants from reads aligned to a reference sequence and the second, a de novo assembly approach, identifies variants from reads aligned to de novo assembled consensus sequences. Of critical importance for both approaches is the reduction in the number of low complexity regions through the construction of a non-redundant reference genome. We compared variants identified in the two methods. Our results indicate that approximately 85% of variants are identified regardless of the approach. The reference-based approach to variant discovery captures an additional 15% representing variants divergent from the HSV1 reference possibly due to viral passage. Reference-based approaches are significantly less labor-intensive and identify variants across the genome where de novo assembly-based approaches are limited to regions where contigs have been successfully assembled. In addition, regions of poor quality assembly can lead to false variant identification in de novo consensus sequences. For viruses with a well-assembled reference genome, a reference-based approach is recommended.

The CCCTC Binding Factor, CTRL2, Modulates Heterochromatin Deposition and the Establishment of Herpes Simplex Virus 1 Latency In Vivo

“…The results of this sequence analysis revealed no indels within coding regions of genes, and only one single nucleotide base change in UL36, which was synonymous. There were four additional single nucleotide base substitutions and one two-nucleotide deletion that occurred in noncoding regions (mainly in reiterated regions in the repeats) (36). Considering these sequencing data, it is unlikely that any sequence changes resulted in changes to the fitness of the 17ΔCTRL2 recombinant.…”

“…All experiments were performed using HSV-1 strain 17synϩ or the recombinant ΔCTRL2 (constructed in the 17synϩ background). The ΔCTRL2 virus was constructed by homologous recombination, as previously reported, with a 135-bp deletion spanning nucleotides 120500 to 120635 (GenBank accession number NC_001806) (36) (Fig. 1).…”

Genetic diversity and connectivity of the Ostreid herpesvirus 1 populations in France: a first attempt to phylogeographic inference for a marine mollusc disease

“…To solve this problem, we used the approach proposed by Morse et al . (Morse et al, 2017): contigs generated by de novo assembly were ordered using a reference-based approach to ultimately assemble non-redundant genomes in which only one copy of each repeated element was kept. These non-redundant genomes constituted the keystone of our bioinformatics analyses.…”

Complete Genome Sequence of Herpes Simplex Virus 1 Strain McKrae

“…The genome termini were manually determined by the a sequence (303 bp) bracketed by two DR1 elements, which also had an inverted repeat in the L-S junction (15). The draft sequence was then refined by the PacBio genome resequencing pipeline (SMRT Analysis v6.0), with default settings (16). A total of 4,834,368 subreads (mean length, 2,183; maximum length, 26,927; total bases, 10,553,016,021) were mapped to the final resolved genome sequence.…”