Shotgun sequencing to determine corneal infection.



Parekh, Mohit ORCID: 0000-0002-5186-068X, Romano, Vito ORCID: 0000-0002-5148-7643, Franch, Antonella, Leon, Pia, Birattari, Federica, Borroni, Davide ORCID: 0000-0001-6952-5647, Kaye, Stephen B ORCID: 0000-0003-0390-0592, Ponzin, Diego, Ahmad, Sajjad ORCID: 0000-0002-8336-4314 and Ferrari, Stefano ORCID: 0000-0003-3983-3328
(2020) Shotgun sequencing to determine corneal infection. American journal of ophthalmology case reports, 19. 100737-.

Access the full-text of this item by clicking on the Open Access link.
[img] Text
Shotgun sequencing to determine corneal infection.pdf - Published version

Download (411kB) | Preview

Abstract

<h4>Purpose</h4>To investigate if shotgun-sequencing method could be useful in detailed diagnosis of herpes simplex virus (HSV) infection and compare it with the conventional diagnostic method.<h4>Observations</h4>Using a sterile scraper, the infectious part of the ocular surface was scraped gently and placed on a glass slide for conventional diagnosis using PCR and histology and in RNA stabilizing reagent for shotgun sequencing respectively. Concentration of the DNA was determined using a sensitive fluorescence dye-based Qubit dsDNA HS Assay Kit. Shotgun-sequencing libraries were generated using the NEBNext DNA ultra II protocol. The samples were sequenced on the Illumina NextSeq 500 in high output mode with 2X150 bp paired-end sequencing. Taxonomic and functional profiles were generated.Conventional diagnostic method suspected herpetic keratitis. The results indicated presence of an amplified product of 92 bp positive HSV-DNA. Conventional diagnostic method detected the presence of Herpes Simplex Virus DNA (type 1). Shotgun sequencing confirmed the diagnosis of HSV along with the taxonomical profiling of the virus. These results were achieved using 1.9 ng/μL of DNA concentration (114 ng in 60 μL) of the total sample volume.<h4>Conclusions and importance</h4>Shotgun sequencing is a hypothesis-free approach that identifies full taxonomic and functional profile of an organism. This technology is advantageous as it requires smaller sample size compared to conventional diagnostic methods.

Item Type: Article
Uncontrolled Keywords: Cornea, Diagnosis, Infection, Sequencing, Shotgun, Virus
Depositing User: Symplectic Admin
Date Deposited: 11 Aug 2020 13:20
Last Modified: 18 Jan 2023 23:37
DOI: 10.1016/j.ajoc.2020.100737
Open Access URL: https://doi.org/10.1016/j.ajoc.2020.100737
Related URLs:
URI: https://livrepository.liverpool.ac.uk/id/eprint/3097161