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

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.

Human Papillomavirus Detection by Whole-Genome Next-Generation Sequencing: Importance of Validation and Quality Assurance Procedures

“…However, it requires large amounts of sequencing data, and thus is not applicable in clinical usage, which requires fast and accurate results. To date, the development of new NGS methods for HPV integration detection with high accuracy and prompt reporting capacity is ongoing [ 52 ]. However, the best way currently to detect integration sites is reached by using probe-captured sequencing methods (see section “Enrichment protocols” below).…”

Molecular Tumor Subtypes of HPV-Positive Head and Neck Cancers: Biological Characteristics and Implications for Clinical Outcomes

“…Each DNA assay has its own limitations, for instance, DNA FISH is designed specifically for paraffin-embedded tissue, whereas quantitative PCR measures the relative E2 gene loss compared to E6, which may miss integration events that occur outside the E2 gene region. Nowadays, more studies focus on distinct DNA-seq techniques to capture HPV integration events, having shown to be more accurate and comprehensive [ 51 , 52 ]. However, whereas RNAseq methods may miss integration events that are transcribed but do not result in any viral-host fusion transcripts, the DNA approaches by their very nature cannot determine which integration events are transcribed.…”

Analysis of HPV Integrations in Mexican Pre-Tumoral Cervical Lesions Reveal Centromere-Enriched Breakpoints and Abundant Unspecific HPV Regions

“…HPV is frequently integrated into the human genome [ 17 ]. The integration of high-risk human papillomavirus (HR-HPV) into the host genome is seen in ~85% of cervical squamous cell carcinomas.…”

“…These facts suggest that recurrent loci provide clonal selection advantages that contribute to carcinogenesis. The target host genes observed in these “hot-spots” are enriched in genes that are continuously expressed during transcription and DNA repair [ 14 , 17 , 18 ]. Several integrations sites are located inside the introns of tumor suppressor genes (like SCAI and NR3C2 ), likely altering the original expression patterns and contributing to the complete loss of gene function [ 15 ].…”

“…Moreover, the level of HPVint is positively correlated with cervical intraepithelial neoplasia grades and has been proposed as a marker for CC progression [ 10 , 24 , 25 ]. Therefore, HPVint status may be a biomarker for diagnosis, progression, and CC screening [ 17 ].…”