基于二代测序的口咽癌HPV-16病毒整合位点检测新方法的建立与应用

A novel method in determining HPV-16 integration sites in oropharyngeal cancer based on next generation sequencing

目的:构建一个基于二代测序的口咽癌HPV-16病毒整合位点检测新方法,明确建库条件和流程,评估测序结果和质量,为口咽癌临床诊疗提供技术手段和理论支持。方法:对HPV-16阳性的口咽癌细胞UM-SCC-47以及5对临床标本利用多重PCR方法进行DNA建库和测序,评估测序深度及偏倚性,对测序数据进行分析,找出HPV-16整合位点所在基因。结果:成功构建了测序体系,可在96孔板中批量实现并于12小时内完成。石蜡包埋组织与新鲜组织测序质量并无差异。分析得到HPV-16整合位点所在的19个基因。结论:一个基于二代测序的口咽癌HPV-16整合位点检测新方法已成功构建。该方法建库时间短,成本低,测序深度有保证,方便应用和推广。

Objective:To construct a new method for the detection of HPV-16 virus integration sites in oropharyngeal cancer based on next-generation sequencing,to explore the conditions and procedures of library construction,to evaluate the sequencing results and quality,and provide technical means and theories for clinical diagnosis and treatment of oropharyngeal cancer.Methods:DNA library construction and sequencing of HPV-16-positive oropharyngeal cancer cells UM-SCC-47 and 5 pairs of clinical specimens were performed by multiplex PCR method to evaluate the sequencing depth and bias,and analyze the sequencing data to find out the integrated sites.Results:The library construction and sequencing system was successfully constructed,which could be realized in batches of 96-well plates and completed within 12 hours.There was no difference in sequencing quality between paraffin-embedded tissue and fresh tissue.Nineteen genes were obtained where the HPV-16 integration site was located.Conclusion:A new method for the detection of HPV-16 integration sites in oropharyngeal carcinoma based on next-generation sequencing has been successfully constructed.The method has short library construction time,low cost,with guaranteed sequencing depth,and is convenient for clinical application and promotion.