The analysis of HPV integration sites based on nanopore sequencing and the profiling changes along the course of photodynamic therapy.

OBJECTIVE: To detect the HPV genotype and integration sites in patients with high-risk HPV infection at different stages of photodynamic therapy using nanopore technology and to evaluate the treatment effect. METHODS: Four patients with HPV infection were selected and subjected to photodynamic therapy, and cervical exfoliated cell was sampled at before treatment, after three courses of treatment and six courses of treatment, their viral abundance and insertion sites were analyzed by nanopore technology, and pathological examinations were performed before and after treatment. In this study, we developed a novel assay that combined viral sequence enrichment and Nanopore sequencing for identification of HPV genotype and integration sites at once. The assay has obvious advantages over qPCR or NGS-based methods, as it has better sensitivity after viral sequences enrichment and can generate long-reads (kb to Mb) for better detection rate of structure variations, moreover, fast turn-around time for real-time viral sequencing and analysis. RESULTS: The pathological grade was reduced in all four patients after photodynamic therapy. Virus has been cleared in two cases after treatment, the virus amount reduced after treatment but not completely cleared in one case, and two type viruses were cleared and one type virus persisted after treatment in the last patient with multiple infection. Viral abundance and the number of integration sites were positively correlated. Gene enrichment analysis showed complete viral clearance in 1 patient and 3 patients required follow-up. CONCLUSION: Nanopore sequencing can effectively monitor the abundance of HPV viruses and integration sites to show the presence status of viruses, and combined with the results of gene enrichment analysis, the treatment effect can be dynamically assessed.

Exosomal RNA Sequencing from Body Fluid Samples.

Exosomes are microcapsules released by different types of cells. It contains DNA, RNA, proteins, and other substances. Exosomes spread in body fluids, and their contents can be phagocytosed by other cells and become an important mediator of intercellular communication. Previous studies show that exosomes secreted by cancer cells are involved in tumorigenesis, growth, invasion, and metastasis. With the improvement of technologies for exosome isolation and deep sequencing, we can identify exosomal RNA profiling for further detection of cancer-related biomarkers. In this chapter, we introduce the experiment procedure and bioinformatic pipeline on exosomal RNA sequencing.