Background
HBV DNA integration into the host genome is frequently found in HBV-associated HCC tissues and is associated with hepatocarcinogenesis. Multiple detection
Conclusions
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.
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. Conclusions: 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.