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Global Translational Medicine The research advances in HPV integration

pipeline with a pair-assembling strategy, integration capacity to reveal more extensive changes, such as changes
sites are identified through data analysis. This approach in gene copy number resulting from the integration of
has proven successful in several studies for pinpointing the HPV genome into the host genome. The development
meaningful integration sites . Another strategy, tagging, of long-read single-molecule sequencing technologies
[65]
enrichment, and NGS of HPV16 (TEN16), relies on targeted by Pacific Biosciences (PacBio) and Oxford Nanopore
next-generation DNA sequencing . The process involves Technologies (ONT) has introduced new possibilities for
[66]
Nextera in vitro transposition for DNA fragmentation and the application of direct nucleic acid enrichment tools.
adapter-tagging, followed by multiplex PCR with HPV16- Long-read sequencing enables the elucidation of complex
specific primers for HPV16 DNA enrichment. This gene rearrangements, and Nanopore analysis requires
approach successfully identified 75 HPV16 integration smaller amounts of sequencing data compared to Illumina
sites in a mixed tumor sample pool. RNA-sequencing methods while maintaining similar accuracy [73,74] .
stands out as another method of choice for detecting One study successfully identified four new candidate
viral-host fusion transcripts. Koneva et al. successfully target genes for CC by integrating multi-omics data
[67]
identified 320 integration breakpoints distributed across from The Cancer Genome Atlas with patient-matched
the human and HPV genomes using RNA-seq data from long-read sequencing of HPV integration sites. The
84 HNSCC tumors. In addition, whole-exome sequencing identified genes are BNC1, RSBN1, USP36, and TAOK3.
and high-throughput chromosome conformation capture The study employed the HPV-targeted PacBio long-
sequencing have been adapted for the identification of read sequencing method and validated the potential
HPV integration sites [56,65,68] .
involvement of these genes in tumorigenesis by assessing
Parsing the NGS data is crucial for the identification of their expression levels. It is essential to note that the study
integration sites, and various novel bioinformatic pipelines had a limited sample size, including samples from only
have been developed to process the data to obtain the eight women, and as such, its findings may not be broadly
necessary information. ViFi, a computational approach in representative .
[75]
2018, utilizes phylogenetic methods combined with reference- Pooled CRISPR inverse PCR sequencing (PCIP-seq)
based read mapping for calling integration sites . ViFi is a method employing a pool of CRISPR guide RNAs
[69]
achieves not only high precision and sensitivity in identifying for selective cleavage of circular DNA fragments carrying
fusion reads but also detects circular extrachromosomal viral DNA. Subsequently, reverse long-fragment PCR and
DNA (ecDNA) human-viral fusion structures. SearcHPV is
another novel pipeline based on targeted capture technology, multiplex sequencing are performed using the MinION
specifically designed for the analysis of targeted capture DNA platform. The DNA is then sheared into approximately 8 kb
sequencing data. Notably, SearcHPV demonstrates higher fragments, followed by intramolecular ligation and removal
sensitivity and specificity for detecting HPV-host integration of remaining linear DNA through digestion. To selectively
sites compared to VirusFinder and VirusSeq, two integration linearize circular DNA containing viral sequences, specific
callers developed earlier. It is important to acknowledge that regions of the HPV18 genome are targeted for selective
a significant limitation of SearcHPV is its validation on only CRISPR-mediated cleavage, and PCR amplification is
three samples . In addition to SearcHPV, deep-learning performed before sequencing. The identified insertion
[70]
models such as DeepHPV have been designed for predicting sites support the hypothesis that rearrangements occur
HPV integration sites . A comprehensive database is through a “looping” mechanism, as the two sets of breaks
[71]
[76]
essential for systematic searches, comparisons, and analyses share a common breakpoint .
of viral integration sites. The Viral Integration Site Database Xdrop technology is a novel microfluidics system
was developed as a user-friendly web tool for browsing, designed for targeted enrichment in low-input and long-
searching, analyzing, visualizing, and downloading data . fragment DNA. The technology relies on the isolation
[72]
The utilization of well-organized, comprehensive, and of long DNA fragments within millions of droplets,
continuously updated databases, along with the availability wherein the droplets containing a fluorescently labeled
of various evolving intelligent analysis pipelines, has target sequence of interest are sorted using fluorescence-
significantly alleviated the burden of analyzing large amounts activated cell sorting. The sorted DNA fragments
of sequencing data in advanced biomedical research. undergo amplification through multiple displacement
amplification (MDA) and are subsequently subjected to
4.3. Third-generation sequencing-based techniques sequencing. This approach successfully resolved three
Short reads are suitable for investigating specific HPV18-chr8 integration sites at base-pair resolution .
[77]
structural variations at insertion sites, such as deletions Table 1 summarizes the studies conducted on the
or chromosomal translocations. However, they lack the identification of HPV integration sites for various types of

Volume 2 Issue 4 (2023) 7 https://doi.org/10.36922/gtm.2034