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Development of a simplified and cost-effective sample preparation method for genotyping of human papillomavirus by next-generation sequencing

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Abstract

High-risk human papillomavirus (HPV) infection is the most common cause of cervical cancer, but low-risk HPV strains can sometimes also be involved. Although HPV genotyping techniques used in clinical diagnosis cannot detect low-risk HPV, next-generation sequencing (NGS) can detect both types. However, DNA library preparation is complicated and expensive. The aim of this study was to develop a simplified, cost-effective sample preparation procedure for HPV genotyping based on next-generation sequencing (NGS). After DNA extraction, a first round of PCR was performed using modified MY09/11 primers specific for the L1 region of the HPV genome, followed by a second round of PCR to add the indexes and adaptors. Then, the DNA libraries were purified and quantified, and high-throughput sequencing was performed using an Illumina MiSeq platform. The sequencing reads were compared with reference sequences for HPV genotyping. The limit of detection for HPV amplification was 100 copies/µl. Analysis of the correlation of pathological cytology with the HPV genotype in individual clinical samples showed that HPV66 was the most common genotype found in the normal stage, whereas HPV16 was the main genotype found in low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and cervical cancer. This NGS method can detect and identify several HPV genotypes with 92% accuracy and 100% reproducibility, and it shows potential as a simplified and cost-effective technique for large-scale HPV genotyping in clinical samples.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Funding

This research was funded by the Thailand Science Research and Innovation Fund (TSRI) (grant number CU_FRB65_hea (27)_034_30_15), a scholarship from the Graduate School, Chulalongkorn University to commemorate the 72nd Anniversary of his Majesty King Bhumibol Adulyadej.

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Authors and Affiliations

  1. Division of Biochemistry, Department of Preclinical Science, Faculty of Medicine, Thammasat University, 10120, Pathum Thani, Thailand

    Rungrat Jitvaropas

  2. Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, 10330, Bangkok, Thailand

    Ukrit Thongpoom, Kesmanee Praianantathavorn & Sunchai Payungporn

  3. Center of Excellence in Systems Microbiology, Faculty of Medicine, Chulalongkorn University, 10330, Bangkok, Thailand

    Vorthon Sawaswong, Kritsada Khongnomnan & Sunchai Payungporn

  4. Research Affairs, Faculty of Medicine, Chulalongkorn University, 10330, Bangkok, Thailand

    Witthaya Poomipak

  5. Center of Excellent in Clinical Virology, Faculty of Medicine, Chulalongkorn University, 10330, Bangkok, Thailand

    Pornjarim Nilyanimit & Yong Poovorawan

Authors
  1. Rungrat Jitvaropas
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  2. Ukrit Thongpoom
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  4. Kritsada Khongnomnan
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  9. Sunchai Payungporn
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Contributions

SP contributed to the study’s conception and design. Material preparation and data collection were performed by UT. Sample collection and laboratory screening were supported by PN. The experiments were carried out by UT, KK, WP, and KP. Data analysis and evaluation were performed by RJ, VS, and SP. The first draft of the manuscript was written by RJ and UT. The tables and figures were prepared by RJ and VS. The manuscript was revised by RJ and SP. YP supervised the study and provided samples. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Sunchai Payungporn.

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Conflict of interest

The authors declare no potential conflicts of interest.

Ethical approval

This study was approved by the Institutional Review Board of the Faculty of Medicine Chulalongkorn University (IRB number 603/2558).

Additional information

Communicated by Graciela Andrei

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Jitvaropas, R., Thongpoom, U., Sawaswong, V. et al. Development of a simplified and cost-effective sample preparation method for genotyping of human papillomavirus by next-generation sequencing. Arch Virol 168, 185 (2023). https://doi.org/10.1007/s00705-023-05810-w

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