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Single-cell whole-genome sequencing identifies human papillomavirus integration in cervical tumour cells prior to and following radiotherapy.

ABSTRACT: Single-cell sequencing technology is a promising systematic and comprehensive approach to delineate clonal associations between cells. The present study collected 13 and 12 cervical cells from fresh tumour tissue prior to and following radiotherapy, respectively, from a 46-year-old female patient with exogenous-type cervical carcinoma. Next, single-cell whole-genome sequencing analysis was performed on each cell. Examination revealed that normal cells could be clearly distinguished from tumour cells among the 25 cells. Tumour cells prior to and following radiotherapy almost represented two independent clones, with the main subpopulation prior to radiotherapy being killed and the minor subpopulation prior to radiotherapy becoming the main subpopulation following radiotherapy. A human papillomavirus (HPV) integration site was detected in POU class 5 homeobox 1B (POU5F1B) in tumour cells following radiotherapy, which has been reported to be a frequent HPV integration site in cervical carcinoma. These results indicate that tumour cells with HPV integration in POU5F1B survive radiotherapy, and that tumour cells prior to and following radiotherapy exhibit distinct characteristics.

SUBMITTER: Yang D

PROVIDER: S-EPMC6004640 | biostudies-other | 2018 Jun

REPOSITORIES: biostudies-other

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Single-cell whole-genome sequencing identifies human papillomavirus integration in cervical tumour cells prior to and following radiotherapy.

Yang Dong D Zhang Weiyuan W Liu Yang Y Liang Junqing J Zhang Tongqing T Bai Yunbo Y Hao Wenjing W Ma Kexin K Lu Danni D Chen Jing J

Oncology letters 20180425 6

Single-cell sequencing technology is a promising systematic and comprehensive approach to delineate clonal associations between cells. The present study collected 13 and 12 cervical cells from fresh tumour tissue prior to and following radiotherapy, respectively, from a 46-year-old female patient with exogenous-type cervical carcinoma. Next, single-cell whole-genome sequencing analysis was performed on each cell. Examination revealed that normal cells could be clearly distinguished from tumour c ...[more]

PMID: 29928338

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Project description:Cervical cancer is caused by high-risk human papillomaviruses (HPV), in more than half of the worldwide cases by HPV16. Viral DNA integration into the host genome is a frequent mutation in cervical carcinogenesis. Because integration occurs into different genomic locations, it creates unique viral-cellular DNA junctions in every single case. This singularity complicates the precise identification of HPV integration sites enormously. We report here the development of a novel multiplex strategy for sequence determination of HPV16 DNA integration sites. It includes DNA fragmentation and adapter tagging, PCR enrichment of the HPV16 early region, Illumina next-generation sequencing, data processing, and validation of candidate integration sites by junction-PCR. This strategy was performed with 51 cervical cancer samples (47 primary tumors and 4 cell lines). Altogether 75 HPV16 integration sites (3'-junctions) were identified and assigned to the individual samples. By comparing the DNA junctions with the presence of viral oncogene fusion transcripts, 44 tumors could be classified into four groups: Tumors with one transcriptionally active HPV16 integrate (n = 12), tumors with transcribed and silent DNA junctions (n = 8), tumors carrying episomal HPV16 DNA (n = 10), and tumors with one to six DNA junctions, but without fusion transcripts (n = 14). The 3'-breakpoints of integrated HPV16 DNA show a statistically significant (p<0.05) preferential distribution within the early region segment upstream of the major splice acceptor underscoring the importance of deregulated viral oncogene expression for carcinogenesis. Half of the mapped HPV16 integration sites target cellular genes pointing to a direct influence of HPV integration on host genes (insertional mutagenesis). In summary, the multiplex strategy for HPV16 integration site determination worked very efficiently. It will open new avenues for comprehensive mapping of HPV integration sites and for the possible use of HPV integration sites as individualized biomarkers after cancer treatment of patients for the early diagnosis of residual and recurrent disease.

Project description:BackgroundWe recently described a method for unbiased detection of all known human papillomaviruses (HPV) types with the potential for the determination of their variant and integration from the resulting whole genome sequence data. Considering the complex workflow for target-enriched next generation sequencing (NGS), we focused on the reproducibility and limit of detection (LOD) of this new universal HPV typing assay in this study.ResultsWe evaluated the reproducibility and LOD for HPV genotyping based on our recently published method that used RNA-baits targeting whole genomes of 191 HPV types, Agilent SureSelect protocol for target enrichment and Illumina HiSeq 2500 for sequencing (eWGS, enriched whole genome sequencing). Two libraries, prepared from pooled plasmids representing 9 vaccine HPV types at varying input (1-625 copies/reaction), were sequenced twice giving four replicates for evaluating reproducibility and LOD. eWGS showed high correlation in the number of reads mapped to HPV reference genomes between the two flow-cell lanes within (R2 = 1) and between experiments (R2 = 0.99). The number of mapped reads was positively correlated to copy number (β = 13.9, p < 0.0001). The limit of blank (LOB) could be calculated based on mapped reads to HPV types not included in each sample. HPV genotyping was reproducible for all 9 types at 625 copies using multiple cut-off criteria but LOD was 25 copies based on number of reads above LOB even when multiple types were present. eWGS showed no bias for HPV genotyping under single or multiple infection (p = 0.16-0.99).ConclusionsThe universal eWGS method for HPV genotyping has sensitivity, competitive with widely used consensus PCR methods with reduced type competition, and with the potential for determination of variant and integration status. The protocol used in this study, using defined samples varying in complexity and copy number, analyzed in replicate and duplicate assays, is applicable to most WGS methods.

Dataset Information

Single-cell whole-genome sequencing identifies human papillomavirus integration in cervical tumour cells prior to and following radiotherapy.

Publications

Single-cell whole-genome sequencing identifies human papillomavirus integration in cervical tumour cells prior to and following radiotherapy.

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