Development of a long-read high-throughput chromatin conformation capture method for evaluating both cis and trans interactions between integrated Hepatitis B Virus and host genomes.

High-throughput chromatin conformation capture (Hi-C) has been utilized for characterizing the 3-dimensional (3D) interactome of the genome. However, short-read sequencing approaches have limited the information it provides, especially for integrated proviral DNAs. Herein we describe the development of a pipeline to investigate the 3D interactome associated with integrated Hepatitis B Virus (HBV) DNAs (iDNAs) to further understand the role of HBV iDNA in liver carcinogenesis. We employed long-read DNA sequencing combined with a novel target-pull-down library construction approach that protects against DNA shearage resulting in greatly improved sensitivity and specificity for the targeted HBV iDNA. Most importantly, we successfully increased the length of captured Hi-C library to the multi-kilobase-level and report Hi-C contacts based on a custom-developed informatics pipeline. We anticipate this combined laboratory-informatics pipeline will provide a more comprehensive view of HBV iDNA-host genomic interactions associated with their role in liver carcinogenesis, but even more importantly that it should be generalizable for the study of the geno-topological effects associated with any type of integrated DNA.