Dataset Information

Analysis of HBV Genomes Integrated into the Genomes of Human Hepatoma PLC/PRF/5 Cells by HBV Sequence Capture-Based Next-Generation Sequencing.

ABSTRACT: Hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma (HCC) worldwide. The integration of HBV genomic DNA into the host genome occurs randomly, early after infection, and is associated with hepatocarcinogenesis in HBV-infected patients. Therefore, it is important to analyze HBV genome integration. We analyzed HBV genome integration in human hepatoma PLC/PRF/5 cells by HBV sequence capture-based next-generation sequencing (NGS) methods. We confirmed the results by using Sanger sequencing methods. We observed that HBV genotype A is integrated into the genome of PLC/PRF/5 cells. HBV sequence capture-based NGS is useful for the analysis of HBV genome integrants and their locations in the human genome. Among the HBV genome integrants, we performed functional analysis and demonstrated the automatic expression of some HBV proteins encoded by HBV integrants from chromosomes 3 and 11 in Huh7 cells transfected with these DNA sequences. HBV sequence capture-based NGS may be a useful tool for the assessment of HBV genome integration into the human genome in clinical samples and suggests new strategies for hepatocarcinogenesis in HBV infection.

SUBMITTER: Ishii T

PROVIDER: S-EPMC7348787 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Analysis of HBV Genomes Integrated into the Genomes of Human Hepatoma PLC/PRF/5 Cells by HBV Sequence Capture-Based Next-Generation Sequencing.

Ishii Tomotaka T Tamura Akinori A Shibata Toshikatsu T Kuroda Kazumichi K Kanda Tatsuo T Kanda Tatsuo T Sugiyama Masaya M Mizokami Masashi M Moriyama Mitsuhiko M

Genes 20200618 6

Hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma (HCC) worldwide. The integration of HBV genomic DNA into the host genome occurs randomly, early after infection, and is associated with hepatocarcinogenesis in HBV-infected patients. Therefore, it is important to analyze HBV genome integration. We analyzed HBV genome integration in human hepatoma PLC/PRF/5 cells by HBV sequence capture-based next-generation sequencing (NGS) methods. We confirmed the results by using ...[more]

PMID: 32570699

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Project description:BackgroundMitochondrial DNA (mtDNA) typing can be a useful aid for identifying people from compromised samples when nuclear DNA is too damaged, degraded or below detection thresholds for routine short tandem repeat (STR)-based analysis. Standard mtDNA typing, focused on PCR amplicon sequencing of the control region (HVS I and HVS II), is limited by the resolving power of this short sequence, which misses up to 70% of the variation present in the mtDNA genome.MethodsWe used in-solution hybridisation-based DNA capture (using DNA capture probes prepared from modern human mtDNA) to recover mtDNA from post-mortem human remains in which the majority of DNA is both highly fragmented (<100 base pairs in length) and chemically damaged. The method 'immortalises' the finite quantities of DNA in valuable extracts as DNA libraries, which is followed by the targeted enrichment of endogenous mtDNA sequences and characterisation by next-generation sequencing (NGS).ResultsWe sequenced whole mitochondrial genomes for human identification from samples where standard nuclear STR typing produced only partial profiles or demonstrably failed and/or where standard mtDNA hypervariable region sequences lacked resolving power. Multiple rounds of enrichment can substantially improve coverage and sequencing depth of mtDNA genomes from highly degraded samples. The application of this method has led to the reliable mitochondrial sequencing of human skeletal remains from unidentified World War Two (WWII) casualties approximately 70 years old and from archaeological remains (up to 2,500 years old).ConclusionsThis approach has potential applications in forensic science, historical human identification cases, archived medical samples, kinship analysis and population studies. In particular the methodology can be applied to any case, involving human or non-human species, where whole mitochondrial genome sequences are required to provide the highest level of maternal lineage discrimination. Multiple rounds of in-solution hybridisation-based DNA capture can retrieve whole mitochondrial genome sequences from even the most challenging samples.

Project description:BackgroundAccumulating evidence implicates mitochondrial DNA (mtDNA) alleles, which are independent of the nuclear genome, in disease, especially in human metabolic diseases. However, this area of investigation has lagged behind in researching the nuclear alleles in complex traits, for example, in gout.MethodsNext-generation sequencing was utilized to investigate the relationship between mtDNA alleles and phenotypic variations in 52 male patients with gout and 104 age-matched male non-gout controls from the Taiwan Biobank whole-genome sequencing samples. Differences from a reference sequence (GRCh38) were identified. The sequence kernel association test (SKAT) was applied to identify gout-associated alleles in mitochondrial genes. The tools Polymorphism Phenotyping, Sorting Intolerant From Tolerant (SIFT), Predict the pathology of Mutations (PMUT), Human Mitochondrial Genome Database (mtDB), Multiple Alignment using Fast Fourier Transform (MAFFT), and Mammalian Mitochondrial tRNA Genes (Mamit-tRNA) were used to evaluate pathogenicity of alleles. Validation of selected alleles by quantitative polymerase chain reaction of single nucleotide polymorphisms (qPCR SNPs) was also performed.ResultsWe identified 456 alleles in patients with gout and 640 alleles in non-gout controls with 274 alleles shared by both. Mitochondrial genes were associated with gout, with MT-CO3, MT-TA, MT-TC, and MT-TT containing potentially pathogenic gout-associated alleles and displaying evidence of gene-gene interactions. All heteroplasmy levels of potentially pathogenic alleles exceeded metabolic thresholds for pathogenicity. Validation assays confirmed the next-generation sequencing results of selected alleles. Among them, potentially pathogenic MT-CO3 alleles correlated with high-density lipoprotein (HDL) levels (P?=?0.034).ConclusionThis study provided two scientific insights. First, this was the most extensive mitochondrial genomic profiling associated with gout. Second, our results supported the roles of mitochondria in gout and HDL, and this comprehensive analysis framework can be applied to other diseases in which mitochondrial dysfunction has been implicated.

Dataset Information

Analysis of HBV Genomes Integrated into the Genomes of Human Hepatoma PLC/PRF/5 Cells by HBV Sequence Capture-Based Next-Generation Sequencing.

Publications

Analysis of HBV Genomes Integrated into the Genomes of Human Hepatoma PLC/PRF/5 Cells by HBV Sequence Capture-Based Next-Generation Sequencing.

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