A versatile method to profile hepatitis B virus DNA integrationKento Fukano, Kousho Wakae, Naganori Nao, Masumichi Saito, Akihito Tsubota, Takae Toyoshima, Hideki Aizaki, Hiroko Iijima, Takahiro Matsudaira, Moto Kimura, Koichi Watashi, Wataru Sugiura, Masamichi Muramatsu
HBV DNA integration into the host genome is frequently found in HBV-associated HCC tissues and is associated with hepatocarcinogenesis. Multiple detection methods, including hybrid capture-sequencing, have identified integration sites and provided clinical implications; however, each has advantages and disadvantages concerning sensitivity, cost, and throughput. Therefore, methods that can comprehensively and cost-effectively detect integration sites with high sensitivity are required. Here, we investigated the efficiency of RAISING (Rapid Amplification of Integration Site without Interference by Genomic DNA contamination) as a simple and inexpensive method to detect viral integration by amplifying HBV-integrated fragments using virus-specific primers covering the entire HBV genome.
Methods and Results:
Illumina sequencing of RAISING products from HCC-derived cell lines (PLC/PRF/5 and Hep3B cells) identified HBV-human junction sequences as well as their frequencies. The HBV-human junction profiles identified using RAISING were consistent with those determined using hybrid capture-sequencing, and the representative junctions could be validated by junction-specific nested PCR. The comparison of these detection methods revealed that RAISING-sequencing outperforms hybrid capture-sequencing in concentrating junction sequences. RAISING-sequencing was also demonstrated to determine the sites of de novo integration in HBV-infected HepG2-NTCP cells, primary human hepatocytes, liver-humanized mice, and clinical specimens. Furthermore, we made use of xenograft mice subcutaneously engrafted with PLC/PRF/5 or Hep3B cells, and HBV-human junctions determined by RAISING-sequencing were detectable in the plasma cell-free DNA using droplet digital PCR.
RAISING successfully profiles HBV-human junction sequences with smaller amounts of sequencing data and at a lower cost than hybrid capture-sequencing. This method is expected to aid basic HBV integration and clinical diagnosis research.