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Related Experiment Video

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A Novel NGS-Based Algorithm for Precise HPV Genotyping and Co-infection Detection in QCMD Samples and FFPE Tissues.

Natalia Kuriata1,2, Katarzyna Zofia Mazur1,2, Magnús Máni Sæmundarson1,2

  • 1Department of Microbiology, University Hospital of Iceland, Reykjavík, Iceland.

Current Protocols
|October 10, 2025
PubMed
Summary

This study introduces a new next-generation sequencing (NGS) pipeline for accurate Human papillomavirus (HPV) genotyping. The method enhances detection of mixed HPV infections, especially in challenging formalin-fixed paraffin-embedded (FFPE) tissues.

Keywords:
HPVNGSQCMD and FFPEalgorithmco‐infection

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Area of Science:

  • Molecular Biology
  • Genetics
  • Virology

Background:

  • Accurate Human papillomavirus (HPV) genotyping is crucial for cervical cancer screening and surveillance.
  • Conventional methods struggle with detecting mixed HPV infections, particularly in formalin-fixed paraffin-embedded (FFPE) tissues.

Purpose of the Study:

  • To develop and validate an optimized next-generation sequencing (NGS) pipeline for comprehensive HPV genotyping.
  • To improve HPV detection sensitivity and accuracy, especially from archival FFPE samples.

Main Methods:

  • Utilized a curated HPV reference database (229 genotypes) and a proportional read-mapping algorithm.
  • Validated the pipeline using QCMD proficiency testing panels and clinical FFPE samples.
  • Included protocols for DNA isolation, PCR amplification, NGS, and data analysis.

Main Results:

  • The NGS pipeline accurately replicated QCMD panel outcomes, including mixed HPV16/18 infections.
  • Identified 44 HPV-positive samples from 50 clinical FFPE specimens.
  • Detected a co-infection scenario missed by traditional diagnostic assays.

Conclusions:

  • The developed low-cost, open-source NGS pipeline offers improved sensitivity and accuracy for HPV detection.
  • This method effectively bridges the gap between research applications and clinical HPV diagnostics.
  • The pipeline is suitable for analyzing FFPE tissues, cervical screening specimens, and fresh tissues.