A versatile method to profile hepatitis B virus DNA integration

Author:

Fukano Kento12,Wakae Kousho1,Nao Naganori345,Saito Masumichi16,Tsubota Akihito7,Toyoshima Takae1,Aizaki Hideki1,Iijima Hiroko8,Matsudaira Takahiro9,Kimura Moto2,Watashi Koichi110,Sugiura Wataru2,Muramatsu Masamichi111

Affiliation:

1. Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan

2. Center for Clinical Sciences, National Center for Global Health and Medicine, Tokyo, Japan

3. Division of International Research Promotion, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan

4. One Health Research Center, Hokkaido University, Sapporo, Japan

5. Institute for Vaccine Research and Development, HU-IVReD, Hokkaido University, Sapporo, Japan

6. Center for Emergency Preparedness and Response, National Institute of Infectious Diseases, Tokyo, Japan

7. Research Center for Medical Science, The Jikei University School of Medicine, Tokyo, Japan

8. Department of Internal Medicine, Division of Hepatobiliary and Pancreatic Disease, Hyogo Medical University, Hyogo, Japan

9. Biotechnological Research Support Division, FASMAC Co., Ltd., Kanagawa, Japan

10. Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan

11. Department of Infectious Disease Research, Foundation for Biomedical Research and Innovation at Kobe, Kobe, Japan

Abstract

Background: 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. 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.

Publisher

Ovid Technologies (Wolters Kluwer Health)

Subject

Hepatology

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3