Long-read sequencing reveals the structural complexity of genomic integration of HBV DNA in hepatocellular carcinoma

Zhongling Zhuo,Fei Su,Hongyuan Cui,Ye Liu,Ying Li,Fei Xiao,Hexin Li,Weiqi Rong,Xiaokun Tang,Lili Zhang,Xuanmei Luo

doi:10.1038/s41525-021-00245-1

Abstract

The integration of HBV DNA into the human genome can disrupt its structure in hepatocellular carcinoma (HCC), but the complexity of HBV genomic integration remains elusive. Here we applied long-read sequencing to precisely elucidate the HBV integration pattern in the human hepatocellular genome. The DNA library was sequenced using the long-read sequencing on GridION and PacBio Sequel II, respectively. The DNA and mRNA were sequenced using next-generation sequencing on Illumina NextSeq. BLAST (Basic Local Alignment Search Tool) and local scripts were used to analyze HBV integration patterns. We established an analytical strategy based on the long-read sequences, and analyzed the complexity of HBV DNA integration into the hepatocellular genome. A total of 88 integrated breakpoints were identified. HBV DNA integration into human genomic DNA was mainly fragmented with different orientations, rarely with a complete genome. The same HBV integration breakpoints were identified among the three platforms. Most breakpoints were observed at P, X, and S genes in the HBV genome, and observed at introns, intergenic sequences, and exons in the human genome. Tumor tissue harbored a much higher integrated number than the adjacent tissue, and the distribution of HBV integrated into human chromosomes was more concentrated. HBV integration shows different patterns between cancer cells and adjacent normal cells. We for the first time obtained the entire HBV integration pattern through long-read sequencing and demonstrated the value of long-read sequencing in detecting the genomic integration structures of viruses in host cells.

Highlights

Hepatocellular carcinoma (HCC) ranks fifth in global cancer incidence and represents the third leading cause of cancer deaths[1]
Illumina sequencing of the tumor yielded a total of 475 hepatitis B virus (HBV)-human chimeric reads, of which HBV was integrated into repeat region in 265 reads, and other 210 reads were kept for further analysis
Illumina sequencing of the adjacent tissue demonstrated a total of 36 HBV-human chimeric reads, of which HBV was integrated into repeat region in 21 reads and the other ten reads were kept for further analysis

Summary

INTRODUCTION

Hepatocellular carcinoma (HCC) ranks fifth in global cancer incidence and represents the third leading cause of cancer deaths[1]. Traditional techniques such as polymerase chain reaction (PCR), Southern blot, and Northern blot focus on known genes and typically investigate the integration of HBV in HCC in small samples[10,11,12]. Based on the Nanopore and PacBio platforms, we utilized longread sequences to detect the HBV complex integrated genome structure in HCC and its adjacent tissues. Using this new method, we obtained the entire HBV complex integrated structures for the first time. We found differences in HBV integration patterns and gene expressions between HCC and its adjacent tissues

RESULTS

90 Gb 150 bp 458 21

DISCUSSION

METHODS