Malet Isabelle, Draa Inès, Leducq Valentin, Vuong Fanny, Bonnafous Pascale, Marcelin Anne-Geneviève, Calvez Vincent, Jary Aude
Sorbonne Université, INSERM, Institut Pierre Louis d'Epidémiologie et de Santé Publique, Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpitaux Universitaires Pitié-Salpêtrière Charles-Foix, Laboratoire de Virologie, Paris, Île-de-France, France.
Microbiol Spectr. 2025 Jul;13(7):e0307324. doi: 10.1128/spectrum.03073-24. Epub 2025 Jun 9.
Among the various genotypes of the human papillomavirus (HPV), the HPV16 genotype is the most oncogenic by far, and some (sub)lineages may be associated with an increased risk of cancer. Thus, characterizing the full genome is important to understand the link between the HPV16 genome variability and its transforming role. We set up a multiplex PCR approach combined with Oxford Nanopore Technologies for the HPV16 full-genome characterization. The primers were designed with PrimalScheme, and the optimization of the monoplex/multiplex PCR was performed on SiHa cells. After library preparation, the sequencing was performed on a GridION sequencing device to determine the sensitivity, the specificity, and the performance of the method. Fourteen primer pairs were selected to span the full genome. With the monoplex PCR, 12 primer pairs showed good amplification, while a double concentration of two primers was required to improve the amplification. We then performed a multiplex PCR approach by generating two pools comprising non-overlapping primer pairs. The multiplex PCR showed good sensitivity, allowing the amplification of HPV16 with a Ct value below 27 and providing a coverage greater than 99.9% and a sequencing depth greater than 100×. In addition, the specificity of the method was validated by the absence of amplification of other high-risk HPV genotypes compared with HPV16. We set up an amplicon-based approach to characterize the full genome and diversity of the HPV16 (sub)lineages. This approach shows good sensibility and specificity with limited cost, opening new perspectives in the field of whole-genome HPV sequencing.IMPORTANCEHPV16 is by far the most oncogenic genotype, so characterizing the genetic variability of its genome is important to better understand the link with its transforming role. We developed an amplicon-based approach combined with Oxford Nanopore Technologies next-generation sequencing to overlap the HPV16 genome, which is easy to implement in the laboratory and inexpensive in the field.
在人乳头瘤病毒（HPV）的各种基因型中，HPV16基因型是目前致癌性最强的，某些（亚）谱系可能与癌症风险增加有关。因此，对全基因组进行特征分析对于理解HPV16基因组变异性与其转化作用之间的联系至关重要。我们建立了一种结合牛津纳米孔技术的多重PCR方法来对HPV16全基因组进行特征分析。引物使用PrimalScheme设计，并在SiHa细胞上进行单重/多重PCR的优化。文库制备后，在GridION测序设备上进行测序，以确定该方法的灵敏度、特异性和性能。选择了14对引物覆盖全基因组。对于单重PCR，12对引物显示出良好的扩增效果，而另外两对引物需要双倍浓度才能改善扩增。然后，我们通过生成两个包含不重叠引物对的池进行多重PCR方法。多重PCR显示出良好的灵敏度，能够扩增出Ct值低于27的HPV16，覆盖率大于99.9%，测序深度大于100×。此外，与HPV16相比，其他高危HPV基因型未出现扩增，从而验证了该方法的特异性。我们建立了一种基于扩增子的方法来对HPV16（亚）谱系的全基因组和多样性进行特征分析。这种方法在成本有限的情况下显示出良好的敏感性和特异性，为全基因组HPV测序领域开辟了新的前景。重要性HPV16是目前致癌性最强的基因型，因此对其基因组的遗传变异性进行特征分析对于更好地理解其与转化作用之间的联系至关重要。我们开发了一种结合牛津纳米孔技术下一代测序的基于扩增子的方法来覆盖HPV16基因组，该方法在实验室易于实施，且成本低廉。
