SRP213791 PRJNA553308 HBV infection accelerates clone evolution in HBV-HCC Chronic Hepatitis B virus (HBV) integration into the human genome is a risk factor for Hepatocellular carcinoma (HCC). HCC is very heterogeneous and frequently contains multifocal tumors. Bulk tissue-based whole genome sequencing or transcriptome sequencing can detect only somatic mutations or HBV integration events of high allele frequency so that heterogeneity at cell level can't be accurately estimated. To interrogate heterogeneity of HBV-HCC, we developed a HBV genome enriched single cell sequencing (HGE-scSeq) procedure and applied the approach to a HBV-HCC cell line MHCC97H. We developed a computational method to identify HBV integration sites and infer DNA copy number variations (CNVs) based on HGE-scSeq data, and validated HGE-scSeq results using MHCC97H's whole genome sequencing data. To understand heterogeneity and potential mechanisms of HBV integrations and tumor clone evolution, we performed HGE-scSeq on 269 cells from 4 tumor sites and 2 tumor thrombi of a HBV-HCC patient. HBV integrations were identified in 142 out of 269 (53%) cells sequenced, and were enriched in two HBV integration hot spots on chr1: 34,397,059 (CSMD2) and chr8: 118,557,327 (MED30/EXT1). The enrichment of cells with HBV integrations at the hot spots was likely due to expansion of clones with loss of CSMD2 expression and enhanced expression of EXT1, which promoted cell growth. There were also 162 rare integration sites. HBV integration sites were enriched in DNA fragile sites and sequences around HBV integration sites were enriched for microhomologous sequences between human and HBV genomes, which suggest that human genome sequence properties determine HBV integration events. Even though only 53% cells contain HBV-integration, all of 269 cells carried chromosome 1q amplification, a hall mark of HCC tumors, consistent with 40-80% sensitivity of identifying HBV integration sites by HGE-scSeq. Cells were grouped into 4 clonal groups based on CNVs. The HBV integration heterogeneity was associated with single cell's CNVs. Less HBV integrations were identified in cells with DNA amplification at genomic regions enriched for genes involved in immune response. The clonal compositions of primary tumor and secondary tumors were different. Non-primary tumors were enriched for clones of high metastasis potentials. In conclusion, our HGE-scSeq data reveals high heterogeneity of HCC tumor cells and potential mechanisms of HBV integrations and tumor clone evolution.