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Related Experiment Video

Updated: Feb 15, 2026

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens
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Identifying Corneal Infections in Formalin-Fixed Specimens Using Next Generation Sequencing.

Zhigang Li1,2, Florian P Breitwieser3, Jennifer Lu3,4

  • 1Department of Ophthalmology, First Affiliated Hospital, Zhengzhou University, Zhengzhou, China.

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Summary

Next-generation sequencing and computational analysis effectively identify diverse pathogens in infectious keratitis cases. This advanced molecular technique shows promise for diagnosing corneal infections in clinical settings.

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Area of Science:

  • Ophthalmology
  • Microbiology
  • Genomics

Background:

  • Infectious keratitis poses a significant threat to vision.
  • Accurate and rapid pathogen identification is crucial for effective treatment.
  • Traditional diagnostic methods may have limitations in sensitivity and specificity.

Purpose of the Study:

  • To evaluate the efficacy of next-generation sequencing (NGS) coupled with computational analysis for identifying a broad spectrum of causative agents in infectious keratitis.
  • To assess the utility of this approach in formalin-fixed corneal specimens.

Main Methods:

  • Retrospective analysis of 16 infectious keratitis cases and 4 control corneas from formalin-fixed tissues.
  • Comparison of NGS data with traditional microbiological analyses (culture, PCR, direct staining).
  • Utilized Kraken and Centrifuge metagenomic classifiers for sequence read analysis, visualized with Pavian software.

Main Results:

  • NGS generated millions of reads per sample, with a small percentage identified as microbial (1.7%).
  • Computational analysis successfully identified fungal, bacterial, and amoebal pathogens in most cases.
  • The method detected all bacterial and mycobacterial, most fungal and Acanthamoeba, and some herpetic keratitis pathogens.

Conclusions:

  • Next-generation sequencing with computational analysis is a powerful tool for pathogen detection in formalin-fixed corneal samples.
  • This approach holds significant potential for improving clinical diagnostics and advancing research in infectious keratitis.