Data_Sheet_1_Accurate Detection of HPV Integration Sites in Cervical Cancer Samples Using the Nanopore MinION Sequencer Without Error Correction.xlsx

During the carcinogenesis of cervical cancer, the DNA of human papillomavirus (HPV) is frequently integrated into the human genome, which might be a biomarker for the early diagnosis of cervical cancer. Although the detection sensitivity of virus infection status increased significantly through the Illumina sequencing platform, there were still disadvantages remain for further improvement, including the detection accuracy and the complex integrated genome structure identification, etc. Nanopore sequencing has been proven to be a fast yet accurate technique of detecting pathogens in clinical samples with significant longer sequencing length. However, the identification of virus integration sites, especially HPV integration sites was seldom carried out by using nanopore platform. In this study, we evaluated the feasibility of identifying HPV integration sites by nanopore sequencer. Specifically, we re-sequenced the integration sites of a previously published sample by both nanopore and Illumina sequencing. After analyzing the results, three points of conclusions were drawn: first, 13 out of 19 previously published integration sites were found from all three datasets (i.e., nanopore, Illumina, and the published data), indicating a high overlap rate and comparability among the three platforms; second, our pipeline of nanopore and Illumina data identified 66 unique integration sites compared with previous published paper with 13 of them being verified by Sanger sequencing, indicating the higher integration sites detection sensitivity of our results compared with published data; third, we established a pipeline which could be used in HPV integration site detection by nanopore sequencing data without doing error correction analysis. In summary, a new nanopore data analysis method was tested and proved to be reliable in integration sites detection compared with methods of existing Illumina data analysis pipeline with less sequencing data required. It provides a solid evidence and tool to support the potential application of nanopore in virus status identification.

在子宫颈癌的致癌过程中，人乳头瘤病毒（HPV）的DNA频繁整合至人类基因组中，这或许成为子宫颈癌早期诊断的生物标志物。尽管通过Illumina测序平台，病毒感染状态的检测灵敏度显著提高，但仍存在进一步优化的不足，包括检测的准确性以及复杂整合基因组结构的识别等问题。纳米孔测序已被证实为一种快速且准确的临床样本病原体检测技术，其测序长度显著较长。然而，利用纳米孔平台进行病毒整合位点的识别，尤其是HPV整合位点的识别，却鲜有实施。在本研究中，我们评估了通过纳米孔测序仪识别HPV整合位点的可行性。具体而言，我们通过纳米孔和Illumina测序重新测序了一个先前发表的样本的整合位点。经过分析，得出三点结论：首先，在全部三个数据集中（即纳米孔、Illumina和发表的数据），从19个先前发表的整合位点中发现了13个，显示出三者之间高度的重叠率和可比性；其次，与我们之前发表的论文相比，我们的纳米孔和Illumina数据流程识别出了66个独特的整合位点，其中13个通过Sanger测序得到了验证，这表明我们的结果相较于发表数据具有更高的整合位点检测灵敏度；第三，我们建立了一个无需进行错误校正分析的流程，可用于纳米孔测序数据的HPV整合位点检测。总之，一种新的纳米孔数据分析方法被测试并证明在整合位点检测方面相较于现有的Illumina数据分析流程具有可靠性，且所需的测序数据更少。这为纳米孔在病毒状态识别中的潜在应用提供了坚实的证据和工具。