You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


1. Introduction {#sec1-genes-11-00661}
===============

Hepatitis B virus (HBV) infection occasionally induces hepatocellular carcinoma (HCC) through direct and indirect mechanisms and is an important cause of morbidity and mortality worldwide. HBV is a partially double-stranded DNA hepatotropic virus of \~3.2 kb in length. The HBV DNA sequence consists of four open reading frames encoding the surface (HBsAg), core (HBcAg), polymerase, and X (HBx) proteins \[[@B1-genes-11-00661],[@B2-genes-11-00661]\].

Soon after HBV infection, HBV DNA is converted into a covalently closed circular DNA molecule (HBV cccDNA) in the nucleus of HBV-infected cells as a stable episomal template \[[@B3-genes-11-00661]\]. HBV cccDNA is responsible for the chronic persistent HBV infection of hepatocytes. On the other hand, the integration of HBV DNA into the host genome occurs randomly, early after infection, and seems to continue \[[@B4-genes-11-00661],[@B5-genes-11-00661],[@B6-genes-11-00661],[@B7-genes-11-00661],[@B8-genes-11-00661]\]. Although the frequent observation of somatic integration of HBV DNA suggests a possible benefit for HBV replication, the mechanism of integration, its functions, and the clinical impact on hepatocarcinogenesis remain largely unknown \[[@B9-genes-11-00661],[@B10-genes-11-00661]\].

Next-generation sequencing (NGS) has been applied in various fields of virology and cancer research \[[@B6-genes-11-00661],[@B7-genes-11-00661]\]. However, the low abundance of viral integration occasionally prevents viral identification. Specific target-sequence capture-based NGS has been developed as a method for detecting low levels of virus particles \[[@B11-genes-11-00661]\]. After fragments with HBV sequence were enriched by a set of HBV probes, high-throughput sequencing could detect the location of HBV integration breakpoints in the HCC genome \[[@B12-genes-11-00661]\]. Capture sequencing methods have higher sensitivity and efficiently detect sequences with low costs, compared to conventional methods \[[@B12-genes-11-00661],[@B13-genes-11-00661]\].

In this report, we analyze HBV genome integration in human hepatoma PLC/PRF/5 cells \[[@B14-genes-11-00661]\] by more sensitive HBV sequence capture-based NGS methods. We constructed an HBV genome sequence and determined the HBV genotype (GT) in human hepatoma PLC/PRF/5 cells. We also focus on the topical subject of HBV DNA integration, which is linked HCC progression. We reveal HBV DNA integrants and their locations in the human genome. HBV sequence capture-based NGS is a useful and powerful tool for the assessment of HBV genome integration.

2. Materials and Methods {#sec2-genes-11-00661}
========================

2.1. Cell Culture {#sec2dot1-genes-11-00661}
-----------------

Human hepatoma PLC/PRF/5 and Huh7 cells were purchased from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan) \[[@B14-genes-11-00661]\]. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum and 100 U/mL penicillin/100 μg/mL streptomycin at 37 °C in a 5% CO~2~ atmosphere. We used PLC/PRF/5 cells within 5 passages in the present study.

2.2. Cellular DNA Extraction and DNA Fragmentation {#sec2dot2-genes-11-00661}
--------------------------------------------------

Total DNA was isolated using a QIAamp DNA Mini Kit (Qiagen, Tokyo, Japan) according to the manufacturer's instructions. A total of 10 μg DNA was fragmented using an E-200 ultrasonicator (Covaris, Unit H. Woburn, MA, USA) \[[@B15-genes-11-00661]\], and the size of the resulting DNA fragments was \~300 bp, which was confirmed by using an Agilent Bioanalyzer 2100 (Agilent Technologies, San Jose, CA, USA).

2.3. Library Preparation and HBV Sequence Capture-Based Next-Generation Sequencing (NGS) {#sec2dot3-genes-11-00661}
----------------------------------------------------------------------------------------

After the generation of blunt-ended fragments, a sequence adapter was added with a TruSeq Nano DNA Sample Prep Kit (Illumina, San Diego, CA, USA). Target-capture sequencing on the Roche SeqCap platform was performed across HBV full genomes: GT-A (AP007263), GT-B (AB287327), and GT-C (AB368296). The DNA libraries from PLC/PRF/5 cells were captured using probes generated from these 3 HBV sequences by using SeqCap EZ Libraries (Roche NimbleGen, Tokyo, Japan).

HBV DNA-specific fragments were selectively collected with Invitrogen Dynabeads (Invitrogen, Carlsbad, CA, USA) and used as genome libraries. NGS of these libraries was performed by using a HiSeq 2000 (Illumina) system with a HiSeq PE Cluster Kit cBot (Invitrogen). Sequence data were processed using a standard pipeline in CLC Genomics Workbench (Qiagen). We focused on the accumulation of HBV-integrated sequences/fused/chimeric sequences and analyzed the sequences of the HBV and human genomes in each read by HBV sequence capture-based NGS. The integrated sequences of the HBV and human genomes with sequence reads \>100 were analyzed.

HBV short genome sequences isolated at NGS were mapped to the reference sequence of HBV full genome GT-C (AB014378), resulting in the full-length HBV sequence (PLC-HBV) derived from PLC/PRF/5 cells being obtained.

All sequence reads have been submitted to the DNA Data Bank of Japan (DDBJ; temporary submission ID: SSUB015333).

2.4. Confirmation of HBV Genome Integration by Sanger Sequencing Methods {#sec2dot4-genes-11-00661}
------------------------------------------------------------------------

PCR primers were designed at the following locations in the human genome (hg19): 131170441--131170444 and 131172081--131172105, 64808026--64808415 and 64808438--64808453, and 80063555--80063577 and 80065509--80065534 for HBV integrants in chromosomes 3, 11, and 17, respectively. PCR primers were also designed at the following locations, the human genome (hg19): 1296892--1296914 on chromosome 5 and 33662450--33662472 for translocation of chromosome (5; 13). A total of 100 ng DNA was amplified by PCR with KOD FX Neo (KFX-201, Toyobo, Osaka, Japan), according to the manufacturer's instructions.

The PCR products were cloned into the pCR-Blunt II-TOPO vector (Invitrogen). Sanger sequencing was performed with M13 forward and reverse primers using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Tokyo, Japan) and an ABI 3730xl DNA Genetic Analyzer (Thermo Fisher Scientific), according to the manufacturer's instructions.

2.5. Phylogenetic Analysis {#sec2dot5-genes-11-00661}
--------------------------

GENETYX version 10 (GENETEX Corp., Shibuya, Tokyo, Japan) was used to analyze the nucleotide sequences and perform phylogenetic tree analysis by neighbor-joining (NJ) methods. The statistical reliability of the phylogenetic trees was assessed using the bootstrap method on 10,000 times and the Kimura two-parameter model \[[@B16-genes-11-00661]\]. The accession numbers of sequences of various HBV GTs are indicated \[[@B17-genes-11-00661],[@B18-genes-11-00661],[@B19-genes-11-00661],[@B20-genes-11-00661],[@B21-genes-11-00661]\].

2.6. Immunofluorescence Study {#sec2dot6-genes-11-00661}
-----------------------------

HBV integrants from chromosomes 3 and 11 were cloned into the pCR-Blunt II-TOPO vector. Plasmids were transfected into Huh7 cells using Lipofectamine 2000 (Thermo Fisher Scientific). After 24 h of transfection, the cells were fixed with 4% paraformaldehyde (Gibco, Palo Alto, CA, USA) and incubated with an HBsAg-specific mouse monoclonal antibody (M3506, Dako, Carpinteria, CA, USA) at a dilution of 1:50, or HBV polymerase-specific mouse monoclonal antibody (2C8, sc-81590, Santa Cruz Biotechnology, Dallas, TX, USA) at a dilution of 1:50 for 1 h. The cells were washed and incubated with anti-mouse Ig conjugated with an Alexa 594 secondary antibody (Invitrogen) at a dilution of 1:500 for 1 h at room temperature. Finally, the cells were washed and mounted for fluorescence microscopy (BIOREVIO BZ-9000, Keyence, Osaka, Japan). PLC/PRF/5 cells were used as control.

3. Results {#sec3-genes-11-00661}
==========

3.1. HBV DNA Sequence Derived from PLC/PRF/5 Cells {#sec3dot1-genes-11-00661}
--------------------------------------------------

Using HBV sequence capture-based NGS technology, we analyzed PLC/PRF/5 cells to reveal the HBV signature. HBV genome sequences were found in PLC/PRF/5 cells (1,784,734 reads out of 6,165,629 reads, including the mitochondrial genome; see [Table 1](#genes-11-00661-t001){ref-type="table"}). We mapped these sequences onto a previously reported HBV sequence to obtain the full-length HBV sequence (PLC-HBV) derived from PLC/PRF/5 cells. PLC-HBV has been deposited in the DNA Data Bank of Japan (<https://www.ddbj.nig.ac.jp/index-e.html>; accessed on 28 March 2020 under accession number LC533934).

Phylogenetic tree analysis by the neighbor-joining (NJ) method demonstrated that PLC-HBV belongs to HBV GT-A ([Figure 1](#genes-11-00661-f001){ref-type="fig"}), in agreement with the production of subtype adw of HBsAg by PLC/PRF/5 cells \[[@B14-genes-11-00661],[@B17-genes-11-00661]\] and the finding that the HBV genome sequences recovered from PLC/PRF/5 cells belong to HBV GT-A \[[@B18-genes-11-00661]\]. PLC-HBV shows nucleotide homology of 94% (3054/3221), 90% (2919/3220), and 91% (2932/3220) to the HBV sequences used for the generation of HBV capture probes in the present study (GT-A, HB-JI444AF (AP007263); GT-B, JPN Bj A53 (AB287327); GT-C C2, HBV-CH48-201w (AB368296), respectively) \[[@B22-genes-11-00661],[@B23-genes-11-00661]\].

PLC/PRF/5 cells do not harbor an intracellular free HBV genome or infectious HBV virions in the conditioned medium \[[@B19-genes-11-00661],[@B24-genes-11-00661],[@B25-genes-11-00661],[@B26-genes-11-00661]\]. Although there is one report showing that PLC/PRF/5 cells harbor full-length HBV genomes \[[@B20-genes-11-00661]\], the PLC-HBV genome is constructed from multiple partial HBV genome sequences in PLC/PRF/5 cells. Here, we demonstrate that HBV GT-A is integrated into the genome of PLC/PRF/5 cells.

3.2. Analysis of HBV Genome Integrants and Their Locations in the Human Genome {#sec3dot2-genes-11-00661}
------------------------------------------------------------------------------

We focused on the accumulation of HBV-integrated sequences/fused/chimeric sequences and analyzed the sequences of the HBV and human genomes in each read by HBV sequence capture-based NGS. HBV-integrated sequences/fused/chimeric sequences consist of one derived from the host and the other from HBV. The results of the analysis for the integrated sequences of the HBV and human genomes with sequence reads \>100 are shown in [Table 2](#genes-11-00661-t002){ref-type="table"}.

[Figure 2](#genes-11-00661-f002){ref-type="fig"} shows the results regarding the integrated sequences of HBV and human genome chromosomes 3, 11, and 17, confirmed by Sanger sequencing methods. These results were different to some extent from those of NGS. [Figure 2](#genes-11-00661-f002){ref-type="fig"}A presents the results for integrated HBV and human chromosome 3 sequences. Human genome chromosome 3 exhibits a 1637-bp deletion with the insertion of a partial 2623-bp HBV DNA sequence. The HBV regions included in this integrant are shown in [Figure 2](#genes-11-00661-f002){ref-type="fig"}D.

[Figure 2](#genes-11-00661-f002){ref-type="fig"}B shows the results regarding integrated HBV and human chromosome 11 sequences. Human genome chromosome 11 exhibits a 17-bp deletion with the insertion of a partial 1591-bp HBV DNA sequence. The HBV regions included in this integrant are shown in [Figure 2](#genes-11-00661-f002){ref-type="fig"}D. The insertion point of human chromosome 11 is the intron of the SAC3 domain-containing 1 (SAC3D1) region. The SAC3D1 mRNA is significantly associated with the overall survival of patients with HCC (B Cox value, +0.540; HR (95% CI), 1.717 (0.179--3.0 0); *p* \< 0.0001). Higher expression of SAC3D1 mRNA in HCC is considered a high-risk factor associated with short survival \[[@B29-genes-11-00661]\]. SAC3D1 is associated with centrosome abnormalities, and SAC3D1 could be a prognostic marker for HCC recurrence after surgical treatment \[[@B30-genes-11-00661]\].

[Figure 2](#genes-11-00661-f002){ref-type="fig"}C provides the results for integrated HBV and human chromosome 17 sequences. Human genome chromosome 17 harbors a 1932-bp deletion with the insertion of a partial 1699-bp HBV DNA sequence. The HBV regions included in this integrant are shown in [Figure 2](#genes-11-00661-f002){ref-type="fig"}D. The insertion point of human chromosome 17 is the coiled-coil domain-containing 57 (CCDC57) coding region. It has been reported that CCDC57 is one of the genes targeted by the integration of human papillomavirus 16 (HPV 16) \[[@B31-genes-11-00661]\].

3.3. Translocation of Chromosomes (5; 13) with HBV Integrants {#sec3dot3-genes-11-00661}
-------------------------------------------------------------

We also observed the translocation of chromosomes (5; 13) with HBV genome integrants ([Figure 3](#genes-11-00661-f003){ref-type="fig"}). The results confirmed by Sanger sequencing methods demonstrate that the insertion point in human chromosome 5 is 2409 bp upstream of the telomerase reverse transcriptase (TERT) gene, which is located near the TERT promoter region. The HBV regions included in this integrant are shown in [Figure 3](#genes-11-00661-f003){ref-type="fig"}. The insertion point in human chromosome 13 is also 15021 bp downstream of the StAR-related lipid transfer domain containing 13 (STARD13) genes ([Figure 3](#genes-11-00661-f003){ref-type="fig"}).

3.4. Automatic Expression of Proteins of HBV Integrants from Chromosomes 3 and 11, from the Vector without Any Promoter Sequences {#sec3dot4-genes-11-00661}
---------------------------------------------------------------------------------------------------------------------------------

To examine the function of HBV integrant DNA from chromosome 3 in Huh7 cells after 24 h of transfection, we examined the expression of each HBV protein by immunofluorescence analysis. Compared to the expression of HBV proteins in PLC/PRF/5 ([Figure 4](#genes-11-00661-f004){ref-type="fig"}A), we observed higher expression of both HBsAg and HBV polymerase protein in HBV integrant DNA-transfected Huh7 cells ([Figure 4](#genes-11-00661-f004){ref-type="fig"}B). We did not observe HBcAg or HBx protein expression in HBV integrant DNA-transfected Huh7 cells (data not shown).

After 24 h of transfection of the HBV integrant from chromosome 11 into Huh7 cells, we observed only HBsAg expression ([Figure 4](#genes-11-00661-f004){ref-type="fig"}C). As the pCR-Blunt II-TOPO vector is a cloning vector and does not contain any promoters for the expression of coding proteins in mammalian cells, these integrants should automatically express the HBV-host fusion, HBsAg or HBV polymerase protein. No previous studies have shown polymerase expression in PLC/PRF/5 cells.

4. Discussion {#sec4-genes-11-00661}
=============

We observed that HBV GT-A is a major GT of HBV integrated into 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 these HBV genome integrants, we performed functional analysis and demonstrated the automatic expression of some HBV proteins of HBV integrants from chromosomes 3 and 11 in Huh7 cells transfected with these DNA sequences.

The integration of a viral genome could lead to the disruption of the function of the human genome \[[@B32-genes-11-00661],[@B33-genes-11-00661],[@B34-genes-11-00661],[@B35-genes-11-00661]\]. The deregulation of key cellular genes by HBV integration, which may present a selective growth advantage to hepatocytes and result in hepatocarcinogenesis, is thought to occur through several distinctive mechanisms.

PLC/PRF/5 cells can produce HBsAg in the cell culture medium \[[@B14-genes-11-00661],[@B19-genes-11-00661]\]. Edman et al. reported that the PLC/PRF/5 cell line contains at least six (four complete and two incomplete) HBV genomes integrated into high-molecular-weight host DNA \[[@B20-genes-11-00661]\]. Northern blot analysis demonstrated the presence of RNA transcripts specific for the surface antigen sequences of HBV DNA and the absence of detectable transcripts corresponding to the hepatitis B core antigen, supporting the results of the immunofluorescence analysis conducted in the present study ([Figure 4](#genes-11-00661-f004){ref-type="fig"}).

A previous in situ hybridization study with an HBV DNA probe for metaphase chromosomes of the PLC/PRF/5 cell line followed by statistical analysis identified 3 integration sites, namely, 11q22, 15q22-q23 and 18q12 \[[@B36-genes-11-00661]\], and we also found partial HBV genomes in chromosomes other than 11, 15, and 18 ([Table 1](#genes-11-00661-t001){ref-type="table"}). It is possible that our methods are more sensitive and less error-prone.

HBV genome integration into exons and introns results in truncated proteins and decreased protein expression levels, respectively. We found HBV integrants in the intron of chromosome 3 ([Figure 2](#genes-11-00661-f002){ref-type="fig"}A) and part of the exon of CCDC57 in chromosome 17 ([Figure 2](#genes-11-00661-f002){ref-type="fig"}C). We also found HBV integrants in the exon of SAC3D1 in chromosome 11 ([Figure 2](#genes-11-00661-f002){ref-type="fig"}B).

Monjardino et al. reported that a defective HBV DNA molecule (approx. 2.8 kilobase pairs) appears to be integrated in a head-to-tail tandem arrangement, and they proposed that such defective molecules may be involved in the induction of hepatocarcinogenesis by HBV \[[@B26-genes-11-00661]\].

HBV genome integration induces aberrant promoter function in the host genome. [Figure 3](#genes-11-00661-f003){ref-type="fig"} shows HBV genome integration in a region close to the TERT promoter. Telomerase activity, which restores the length of telomere repeat arrays, is frequently observed in various malignancies, including HCC. As TERT is a protooncogene, the integration of HBV could result in hepatocarcinogenesis through its amplification and/or overexpression \[[@B37-genes-11-00661]\]. We also measured TERT mRNA by real-time RT-PCR, but we did not see any difference in TERT mRNA among PLC/PRF/5, Huh7, and HepG2 cells.

STARD13/Deleted in Liver Cancer (DLC) proteins belong to the RhoGAP family, and this protein is more abundantly expressed in HCC tissue, in particular, in the association with inflammation background \[[@B38-genes-11-00661]\]. STARD13 is related to HCC growth and hepatocarcinogenesis \[[@B38-genes-11-00661],[@B39-genes-11-00661],[@B40-genes-11-00661]\], although there is a report that HCC-patients with higher STARD13 or Fas expression levels have longer overall survival \[[@B41-genes-11-00661]\]. The consequences of the detected HBV integration sites and the clinical consequences or integration impact on hepatocarcinogenesis are yet unclear.

Interestingly, the automatic expression of some proteins encoded by HBV integrants from chromosomes 3 and 11 was observed in Huh7 cells transfected with these DNA sequences. This phenomenon may be involved in hepatocarcinogenesis in patients infected with HBV. Chromosome 11 HBV integrants did not code HBV full-length polymerase ([Figure 2](#genes-11-00661-f002){ref-type="fig"}D). This may be the reason why we did not observe HBV polymerase protein in Huh7 cells transfected by chromosome 11 HBV integrants ([Figure 4](#genes-11-00661-f004){ref-type="fig"}C). Further studies, including the functional analysis of these mechanisms, are needed.

In the present study, we enhanced NGS using HBV-targeted sequence capture. Although metagenomic shotgun sequencing is an important tool for the characterization of viral populations, metagenomic shotgun sequencing occasionally lacks sensitivity and may yield insufficient data for detailed analysis \[[@B42-genes-11-00661],[@B43-genes-11-00661]\]. A targeted sequence-capture panel enhances metagenomic shotgun sequencing \[[@B42-genes-11-00661],[@B43-genes-11-00661]\]. As genetic libraries must be generated from samples with low concentrations of HBV DNA and a high content of nucleic acids from a host in many cases \[[@B44-genes-11-00661]\], HBV hybridization-based enrichment may be useful for improving the sensitivity of the detection of HBV genome integration in hepatocytes. However, it is difficult to demonstrate how many copies of HBV deletion/rearrangements exist per cell, and single-cell genome sequencing may be helpful in this case \[[@B45-genes-11-00661]\].

Characteristics of human hepatoma PLC/PRF/5 cells have been reported for 40 years \[[@B36-genes-11-00661],[@B46-genes-11-00661],[@B47-genes-11-00661],[@B48-genes-11-00661]\], and several genome sequences in the present study were not reported in detail. Watanabe et al. extensively analyzed the characteristics of human hepatoma PLC/PRF/5 cells using other NGS strategies \[[@B49-genes-11-00661]\]. However, they did not report the translocation of chromosomes (5; 13) with HBV integrants, which the present study has mentioned, suggesting that the HBV sequence capture-based NGS method is the more powerful and sensitive tool for the analysis of HBV integrants.

Characteristics of human hepatoma PLC/PRF/5 cells may not reflect those of human HCC samples. It may be useful to analyze the HBV integrants in human HCC samples by our methods, although there may be false-positives in their analysis and their impact when real clinical samples that contain high levels of circulating virus are used. A similar approach has already been used for the identification of HBV integration in the human genome of clinical samples \[[@B12-genes-11-00661]\]. Compared to the method reported by Li et al. \[[@B12-genes-11-00661]\], our method does not require special computational analysis. The limitation of our study is that we did not use clinical samples. Further study is needed.

5. Conclusions {#sec5-genes-11-00661}
==============

The HBV replication mechanism and the mechanism of integration, its functions, and the clinical impact on hepatocarcinogenesis remain largely unknown. HBV sequence capture-based NGS is a useful and powerful tool for the assessment of HBV genome integration to explore HBV genome integrants and their locations in the human genome. Clinical impact on hepatocarcinogenesis and new treatment strategies can be provided through this method.

The authors thank all staff members at the Nihon University School of Medicine, Itabashi Hospital, for the care provided.

Conceptualization, T.I., A.T., K.K., and M.M. (Mitsuhiko Moriyama); methodology, T.I., A.T., M.S., K.K., and M.M. (Mitsuhiko Moriyama); software, M.S., A.T., T.S., and K.K.; validation, M.S., A.T., T.S., and K.K.; formal analysis, M.S., A.T., T.S., and K.K.; investigation, T.I., M.S., A.T., T.S., and K.K.; resources, M.S., A.T., T.S., and K.K.; data curation, M.S., A.T., T.S., and K.K.; writing---original draft preparation, T.I., A.T., and T.K.; writing---review and editing, T.I. and T.K.; visualization, T.K.; supervision, M.M. (Masashi Mizokami); funding acquisition, M.M. (Mitsuhiko Moriyama); All authors have read and agreed to the published version of the manuscript.

This research was funded by a Nihon University Multidisciplinary Research Grant (15-009 to M.M. (Mitsuhiko Moriyama)).

The authors declare no conflict of interest.

![Phylogenetic tree for the hepatitis B virus (HBV) full-length genome obtained in the present study as determined by neighbor-joining (NJ) methods. **Black circle**, PLC-HBV (LC533934); **blue square**, reference sequences in the present study. GT-Ae, the original European genotype A; GT-Aa, the new African/Asian genotype A. The accession numbers of sequences of various HBV genotypes (GTs) are indicated \[[@B21-genes-11-00661],[@B22-genes-11-00661],[@B23-genes-11-00661],[@B27-genes-11-00661],[@B28-genes-11-00661]\].](genes-11-00661-g001){#genes-11-00661-f001}

![Results for integrated sequences of hepatitis B virus (HBV) and human genome chromosomes (Chrs) 3, 11, and 17 determined by Sanger sequencing. (**A**) Chr 3, (**B**) Chr 11, (**C**) Chr 17, deletion of human genome sequence including part of an exon, (**D**) summary of HBV integrants in PLC/PRF/5 cells confirmed by Sanger methods. SNX15, sorting nexin 15; SAC3D1, SAC3 domain-containing 1; NAALADL1, N-acetylated alpha-linked acidic dipeptidase such as 1; CCDC57, coiled-coil domain-containing 57; S, surface antigen; C, core; P, polymerase; X, HBx.](genes-11-00661-g002){#genes-11-00661-f002}

![Translocation of chromosomes (5; 13) with hepatitis B virus (HBV) integrants. The results were confirmed by Sanger sequencing methods. Chr, chromosome (Hg19); TERT, telomerase reverse transcriptase; STARD13, StAR-related lipid transfer domain containing 13 genes.](genes-11-00661-g003){#genes-11-00661-f003}

![Automatic expression of proteins of HBV integrants from chromosomes 3 and 11. Representative images are shown (40×). Fluorescent immunostaining for HBsAg (**S**) and HBV polymerase (**P**) in PLC/PRF/5 cells (**A**), Huh7 cells transfected with the pCR-Blunt II-TOPO-HBV integrant from chromosome 3 (**B**), or Huh7 cells transfected with the pCR-Blunt II-TOPO-HBV integrant from chromosome 11 (**C**).](genes-11-00661-g004){#genes-11-00661-f004}

genes-11-00661-t001_Table 1

###### 

The mapped reads and average coverage of hepatitis B virus (HBV) sequence capture-based next-generation sequencing in the present study.

  Reference Sequence   Reference Length   Consensus Length   Total Reads   Single Reads   Reads in Pairs   Average Coverage
  -------------------- ------------------ ------------------ ------------- -------------- ---------------- ------------------
  Chr. 1               2.49 × 10^8^       1,545,531          190,160       18,518         171,642          0.076008
  Chr. 2               2.43 × 10^8^       1,344,151          171,652       23,708         147,944          0.06996
  Chr. 3               1.98 × 10^8^       985,756            134,430       19,312         115,118          0.067181
  Chr. 4               1.91 × 10^8^       763,084            101,024       27,548         73,476           0.051384
  Chr. 5               1.81 × 10^8^       878,501            107,805       12,905         94,900           0.059238
  Chr. 6               1.71 × 10^8^       978,370            143,424       13,186         130,238          0.083435
  Chr. 7               1.59 × 10^8^       1,044,242          136,171       13,449         122,722          0.085094
  Chr. 8               1.46 × 10^8^       762,738            93,417        14,827         78,590           0.063299
  Chr. 9               1.41 × 10^8^       599,234            65,741        9779           55,962           0.046138
  Chr. 10              1.36 × 10^8^       769,055            110,259       27,795         82,464           0.079924
  Chr. 11              1.35 × 10^8^       863,578            2,006,583     126,939        1,879,644        1.488781
  Chr. 12              1.34 × 10^8^       736,429            115,138       21,172         93,966           0.084807
  Chr. 13              1.15 × 10^8^       412,243            58,815        10,367         48,448           0.050038
  Chr. 14              1.07 × 10^8^       455,809            53,252        4762           48,490           0.049338
  Chr. 15              1.03 × 10^8^       490,545            348,653       17,775         330,878          0.338605
  Chr. 16              90,354,753         563,883            114,919       48,517         66,402           0.122922
  Chr. 17              81,195,210         524,407            106,805       25,583         81,222           0.128115
  Chr. 18              78,077,248         394,380            52,617        7057           45,560           0.067025
  Chr. 19              59,128,983         447,625            53,263        7119           46144            0.089332
  Chr. 20              63,025,520         474,048            68,184        5948           62,236           0.107619
  Chr. 21              48,129,895         254,738            32,117        4913           27,204           0.0658
  Chr. 22              51,304,566         253,101            29,371        3049           26,322           0.05685
  Chr. X               1.55 × 108         674,067            73,717        8491           65,226           0.047146
  Chr. Y               59,373,566         100,605            8838          8018           820              0.013505
  Chr. MT              16,569             14,178             4540          172            4368             27.45869
  HBV                  3215               3213               1,784,734     448,408        1,336,326        53,732.01

Chr., chromosome (Hg19); MT, mitochondria; HBV, hepatitis B virus (AB014378) showing 90% (2915/3220) homology to full-length HBV sequence (PLC-HBV).

genes-11-00661-t002_Table 2

###### 

HBV and human genomes in PLC/PRF/5 cells determined by HBV sequence capture-based next-generation sequencing.

  Human Chromosome (Chr.)   Human Junction Nucleotide Position   Gene Name   HBV Fragment Start Position   HBV Fragment End Position   HBV Genome   Number of Total Reads \*
  ------------------------- ------------------------------------ ----------- ----------------------------- --------------------------- ------------ --------------------------
  Chr.3                     131170552                            NA          1044                          1406                        P, S, X      3673
  Chr.3                     131171957                            NA          1415                          1914                        C, P, S, X   3589
  Chr.4                     181507570                            NA          96                            432                         P, S         1548
  Chr.4                     181508764                            NA          235                           387                         P, S         4377
  Chr.5                     1297593                              NA          1175                          1364                        P, S         3680
  Chr.8                     35304663                             UNC5D       2389                          2862                        C, P, S      4625
  Chr.11                    64808415                             SNX15       1313                          1575                        P, S, X      5911
  Chr.11                    64808432                             SAC3D1      2575                          2851                        P, S         6616
  Chr.12                    110012332                            MVK         692                           1379                        P, S, X      10,130
  Chr.13                    33662251                             NA          1897                          2109                        C, P, S      4060
  Chr.13                    33662698                             NA          783                           1612                        P, S, X      1494
  Chr.17                    80063662                             CCDC57      428                           2586                        P, S         7699
  Chr.17                    80065497                             CCDC57      2062                          2420                        C, P, S      6918

Chr., chromosome (Hg19); NA, not available; UNC5D, unc-5 netrin receptor D; SNX15, sorting nexin 15; SAC3D1, SAC3 domain-containing 1; MVK, mevalonate kinase; CCDC57, coiled-coil domain-containing 57; HBV, hepatitis B virus; C, core; P, polymerase; S, surface antigen; X, HBx; \*, confirmed by HBV sequence capture-based next-generation sequencing.