Abstract
HPV genomic variability and chromosomal integration are important in the HPV-induced carcinogenic process. To uncover these genomic events in an HPV infection, we have developed an innovative and cost-effective sequencing approach named TaME-seq (tagmentation-assisted multiplex PCR enrichment sequencing). TaME-seq combines tagmentation and multiplex PCR enrichment for simultaneous analysis of HPV variation and chromosomal integration, and it can also be adapted to other viruses. For method validation, cell lines (n = 4), plasmids (n = 3), and HPV16, 18, 31, 33 and 45 positive clinical samples (n = 21) were analysed. Our results showed deep HPV genome-wide sequencing coverage. Chromosomal integration breakpoints and large deletions were identified in HPV positive cell lines and in one clinical sample. HPV genomic variability was observed in all samples allowing identification of low frequency variants. In contrast to other approaches, TaME-seq proved to be highly efficient in HPV target enrichment, leading to reduced sequencing costs. Comprehensive studies on HPV intra-host variability generated during a persistent infection will improve our understanding of viral carcinogenesis. Efficient identification of both HPV variability and integration sites will be important for the study of HPV evolution and adaptability and may be an important tool for use in cervical cancer diagnostics.
Highlights
Human papillomavirus (HPV) is the main cause of cervical cancer[1], one of the most common cancers in women worldwide, causing more than 200,000 deaths each year[2,3]
Several important questions remain for HPV integration and more comprehensive analyses of integration sites are needed in order to expand our understanding of HPV pathogenesis
In order to contribute to the understanding of the role of intra-host HPV genomic variability and chromosomal integration in carcinogenesis, we have developed an innovative library preparation strategy followed by an in-house bioinformatics pipeline named TaME-seq
Summary
Human papillomavirus (HPV) is the main cause of cervical cancer[1], one of the most common cancers in women worldwide, causing more than 200,000 deaths each year[2,3]. The most common approaches used in HPV genomic analyses are based on target enrichment using highly multiplexed degenerate primers[29], enrichment by multiplex PCR using HPV16 forward primers[30], bead-based target capture[31,32,33], and rolling circle amplification[34] followed by NGS. These methods are designed to detect either HPV integration or HPV variability. Target capture methods poorly enrich HPV and remain expensive due to high probe cost and off-target sequencing
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