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. 2024 Feb 20;25(1):198.
doi: 10.1186/s12864-024-10101-y.

Long-read sequencing reveals the structural complexity of genomic integration of HPV DNA in cervical cancer cell lines

Zhijie Wang #  1   2 , Chen Liu #  1   2 , Wanxin Liu  1   2 , Xinyi Lv  1   2 , Ting Hu  1   2 , Fan Yang  3 , Wenhui Yang  3 , Liang He  4   5 , Xiaoyuan Huang  6   7
Affiliations

Long-read sequencing reveals the structural complexity of genomic integration of HPV DNA in cervical cancer cell lines

Zhijie Wang et al. BMC Genomics. .

Abstract

Background: Cervical cancer (CC) causes more than 311,000 deaths annually worldwide. The integration of human papillomavirus (HPV) is a crucial genetic event that contributes to cervical carcinogenesis. Despite HPV DNA integration is known to disrupt the genomic architecture of both the host and viral genomes in CC, the complexity of this process remains largely unexplored.

Results: In this study, we conducted whole-genome sequencing (WGS) at 55-65X coverage utilizing the PacBio long-read sequencing platform in SiHa and HeLa cells, followed by comprehensive analyses of the sequence data to elucidate the complexity of HPV integration. Firstly, our results demonstrated that PacBio long-read sequencing effectively identifies HPV integration breakpoints with comparable accuracy to targeted-capture Next-generation sequencing (NGS) methods. Secondly, we constructed detailed models of complex integrated genome structures that included both the HPV genome and nearby regions of the human genome by utilizing PacBio long-read WGS. Thirdly, our sequencing results revealed the occurrence of a wide variety of genome-wide structural variations (SVs) in SiHa and HeLa cells. Additionally, our analysis further revealed a potential correlation between changes in gene expression levels and SVs on chromosome 13 in the genome of SiHa cells.

Conclusions: Using PacBio long-read sequencing, we have successfully constructed complex models illustrating HPV integrated genome structures in SiHa and HeLa cells. This accomplishment serves as a compelling demonstration of the valuable capabilities of long-read sequencing in detecting and characterizing HPV genomic integration structures within human cells. Furthermore, these findings offer critical insights into the complex process of HPV16 and HPV18 integration and their potential contribution to the development of cervical cancer.

Keywords: Cervical cancer; HPV integration; HPV16; HPV18.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
HPV16 complex integrated genome structure in SiHa cells. In the top panel, the colored regions in the outer circle represent the HPV16 sequences contained within the integrated structure, while the white regions represent the sequences that were replaced or lost as a result of integration. The inner circle shows the complete HPV16 genome for reference. The bottom panel illustrates the HPV and human reference genomes, which are connected by dotted lines to a contig that covers the integration, demonstrating how it specifically mapped to each genome(1, 2, 3, and 4 represent breakpoints on the HPV16 genome; 1a and 2a represent breakpoints on human chromosome 13; F1, F2, F3, F4, F5 represent consecutive segments on human chromosome 13). Schematic annotations of the integration event were made using data from all PacBio long reads that covered the integration event
Fig. 2
Fig. 2
HPV18 complex integrated genome structure in HeLa cells. A, B Two proposed models of HPV18 integration genome structure in HeLa cells. In the top panel, two proposed HPV18 integration genome structures in HeLa cells are depicted. The colored regions in the outer circle represent the HPV18 sequences contained within the integrated structure, while the white regions represent the sequences that were replaced or lost as a result of integration. The inner circle shows the complete HPV18 genome for reference. The bottom panel illustrates the HPV and human reference genomes, which are connected by dotted lines to a contig that covers the integration, demonstrating how it specifically mapped to each genome. Schematic annotations of the integration event were made using data from all PacBio long reads that covered the integration event
Fig. 3
Fig. 3
Distribution of SVs among Differentially Expressed Genes on Chromosome 13 in SiHa Cells. The x-axis represents the different regions of the genome, including exonic, splicing, ncRNA, UTR5, UTR3, intronic, upstream, downstream, and intergenic regions. The y-axis shows the differentially expressed genes. Different types of SVs are represented by different colors: TRAs (rose red), DELs (dark green), INSs (dark cyan), and INVs (pale purple). The upregulated genes are denoted by triangles, while the downregulated genes are represented by circles
Fig. 4
Fig. 4
HPV integration mechanisms in SiHa and HeLa cells. A, B The figure shows the alignment of the sequence around the integration site between the human genome (rose red) and the HPV16/HPV18 genome in SiHa cells or HeLa cells (dark green), respectively. The black box represents the aligned nucleotides in the microhomology (MH) region of the two reference sequences at the HPV integration site
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