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

Wenjuan Yang,Jialiang Yang,Jingjing Li,Weiwei Wang,Bo Meng,Geng Tian,Ying Liu,Jia Liu,Ruyi Dong,Jidong Lang

doi:10.3389/fgene.2020.00660

Abstract

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.

Highlights

Human papilloma virus (HPV) is the major cause of cervical cancer, and HPV16 and 18 are the two most prevalent highrisk HPV types worldwide
nextgeneration sequencing (NGS) is powerful for discovering HPV integration sites, the quality of the results relies on many factors including the probe capture efficiency, sequencing depth, and analysis method
The summary of sequencing results from the two platforms are listed in Table 1 and the sequence coverage of HPV16 genome by Illumina and nanopore sequencer was shown in Supplementary Figure S1

Summary

Introduction

Human papilloma virus (HPV) is the major cause of cervical cancer, and HPV16 and 18 are the two most prevalent highrisk HPV types worldwide. HPV DNA could integrate into human DNA, which is an early and important event during carcinogenesis (Nicolas et al, 2004; Pett and Coleman, 2010; Schmitz et al, 2012; Christiansen et al, 2015; Bodelon et al, 2016; Walline et al, 2017), and involves epigenetic mechanisms that affect the expression of key genes in the tumor transformation process. Maria et al (2015) proposed a scheme of modifications and alterations generated by HPV integration into the host genome that can lead to carcinogenesis. This recent evidence shows that HPV integration often preferably affects those genes that are continuously expressed during DNA transcription and repair to induce carcinogenesis (OyervidesMuñoz et al, 2018). Understanding viral oncogenesis is critical for the clinical management of HPVpositive cancer (Morgan et al, 2017)

Methods

Results

Conclusion