Oligonucleotide Enrichment of HSV-1 Genomic DNA from Clinical Specimens for Use in High-Throughput Sequencing

Mackenzie M. Shipley, Molly M. Rathbun, Moriah L. Szpara

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

To date more than 400 genomes of herpes simplex virus 1 (HSV-1) and the distantly related HSV-2 have been examined using deep sequencing techniques. This powerful approach has been especially useful for revealing the global genetic diversity that exists within and between strains of each virus species. However, most early methods for high-throughput sequencing required the input of abundant viral genomic DNA to enable the successful production of sequencing libraries, and the generation of sufficient short-read sequencing data for de novo genome assembly and similar applications. Therefore, the majority of sequenced HSV strains have been cultured and expanded in vitro prior to genomic analysis, to facilitate isolation of sufficient viral DNA for sequencing-library preparation. Here, we describe an in-solution targeted enrichment procedure for isolating, enriching, and sequencing HSV genomic DNA directly from clinical specimens. When this enrichment technique is combined with traditional sequencing-library preparation procedures, the need for in vitro culturing, expansion, and purification of viral DNA is eliminated. Furthermore, enrichment reduces the large amount of nonviral DNA that is typically present in specimens obtained directly from natural infections, thereby increasing the likelihood of successful viral genome sequencing and assembly. We have used this approach to prepare viral DNA libraries from clinical specimens derived from skin swabs, saliva, blood, and similar sources. We then use these libraries for deep sequencing and successful de novo assembly of the ~152 kb viral genomes, at coverage depths exceeding 100–1000×, for both HSV-1 and HSV-2.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages199-217
Number of pages19
DOIs
StatePublished - Jan 1 2020

Publication series

NameMethods in Molecular Biology
Volume2060
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

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All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

Cite this

Shipley, M. M., Rathbun, M. M., & Szpara, M. L. (2020). Oligonucleotide Enrichment of HSV-1 Genomic DNA from Clinical Specimens for Use in High-Throughput Sequencing. In Methods in Molecular Biology (pp. 199-217). (Methods in Molecular Biology; Vol. 2060). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-9814-2_11