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Lentiviral/Retroviral Integration Site Sequencing

Lentiviral/Retroviral Integration Site Sequencing

Overview

CD Genomics' Lentiviral/Retroviral Integration Site Sequencing provides detailed mapping of integration sites within the host genome using advanced sequencing technologies. We deliver high-resolution data and thorough bioinformatics analysis to accurately identify integration locations, offering insights into viral vector interactions with the genome. Our service supports both lentiviral and retroviral vectors, providing dependable results for research and application development.

Our Advantages:

Multiplex samples for cost-effective results.
Effective workflow and fast turnaround time.
Sequencing scale: 4 million PE reads per sample.
Comprehensive bioinformatics analysis.
Compatible with nearly all commercially available retrovirus vectors.

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What is Integration Site Analysis of Lentivirus

Integration site analysis of lentiviruses delves into the precise identification and characterization of where lentiviral vectors incorporate their genetic material within the host genome. Lentiviruses, such as HIV-1, are often leveraged as gene delivery systems owing to their remarkable ability to achieve stable integration into the host genome.

The integration process initiates with the conversion of lentiviral RNA genomes into DNA through reverse transcription. Once this new DNA is synthesized, it embeds into the host's genome. While the integration process is generally random, the specific genomic context of the host cell holds sway over where integration may occur. The crux of these integration sites lies in their potential to considerably influence gene expression and cellular phenotype, underscoring the necessity to thoroughly assess their safety and efficacy within the context of gene therapies.

What is the Process of Retroviral Integration

Retroviral integration is a complex and intricately regulated process, characterized by several pivotal phases:

Reverse Transcription: The retrovirus's RNA genome is converted into complementary DNA (cDNA) by reverse transcriptase.
Integration into Host Genome: The cDNA is transported into the nucleus and inserted into the host genome by the viral integrase enzyme.
Integration Site Selection: Integration sites are chosen based on interactions with host chromatin, often near active genes or regulatory regions.
Stabilization and Transcription: The integrated viral DNA is transcribed along with the host DNA, facilitating the expression of the transgene.

Applications

Safety Assessment
Gene Function Studies
Cell Line Development
Regulatory Compliance

Service Specifications

Introduction to Our Lentiviral Integration Sites Solution

Our cutting-edge platform delivers exceptionally precise sequencing of lentiviral and retroviral integration sites via the Illumina platform. Through the robust capabilities of next-generation sequencing (NGS) paired with targeted sequence capture, we achieve exact pinpointing and thorough analysis of viral vector integration within infected cells. This all-encompassing service covers every step of the process—from primer design and library construction to paired-end sequencing and intricate bioinformatics analysis—guaranteeing that the data we generate is of superior quality and utmost reliability.

What Can We Do:

1. Identification of lentiviral/retroviral integration sites in your lentiviral/retroviral infected cells.

2. Quantification of the numbers and types of gene-modified cell clones.

3. Monitor experiments using insertional mutagens and mechanistic studies of DNA integration.

Note: Our service is for research use only, not for disease diagnosis or treatment.

Detectable Objects:

Any retroviral infected cells.

Workflow of Our Lentiviral Integration Sites Sequencing

The Workflow of Fungal ITS Sequencing. Figure 1. The high-throughput sequencing analysis process.

Technical Parameters

Sequencing Platform	Sequencing Strategy	Data Volume
Illumina Hiseq/NovaSeq	PE150	For LM-PCR method (suitable for samples with known LTR sequences): construct 400~600 bp insert size library; 1Gb data output For WGS method (only suitable for monoclonal cell line): construct 350bp library, ~50X coverage

Bioinformatics Analysis

Our bioinformatics analysis pipeline is flexible to your needs.

Pipeline	Analysis Content
Read mapping	Sequence quality filtering, trimming of DNA sequences, align sequence data to the genome of interest, identification of junction reads.
Integration site calling	Identification and annotation of lentiviral integration sites, visualization of integration site datasets.
Estimation of clonal abundance	Estimation of clonal abundance using the IntSiteDB database that contains genomic locations of integration sites, PCR breakpoints, and their counts.
Custom analyses	Other bioinformatics analyses are available upon your request, such as the comparison with oncogene annotation.

Note: The above content includes only a portion of the bioinformatics analysis. For more information or to customize the analysis, please contact us directly.

The Workflow of Fungal ITS Sequencing.

Sample Requirement

Sample Type	Quantity	Concentration	OD260/OD280
Genomic DNA	≥100 ng	≥10 ng/μl	1. 8 - 2. 0

Note:

Ensure the DNA is purified, not degraded.
Must provide gDNA for infected cell lines and control (non-infected) cell lines.
Transport nucleic acid samples with sufficient ice packs or dry ice.
If you wish to obtain more accurate and detailed information regarding sample requirements, please feel free to contact us directly.

Deliverables

Raw sequencing data
Quality-control data
Statistic data
Your designated bioinformatics report.

Demo

Partial results of our Lentiviral/Retroviral Integration Site Sequencing service are shown below:

The Fungal ITS Sequencing Results Display Figure.

FAQs

Lentiviral Integration Sites Sequencing FAQ

1. What differentiates lentiviral and retroviral integration?
- Lentiviruses can integrate into both dividing and non-dividing cells, offering a broader range of applications due to this flexibility. On the other hand, retroviruses predominantly target dividing cells, restricting their usage to specific scenarios.
2. How does CD Genomics ensure the accuracy of integration site analysis?
- CD Genomics employs sophisticated methods such as LAM-PCR, targeted sequence capture, and NGS to deliver precise and reliable data on integration sites. Our protocols are fine-tuned to optimize both sensitivity and specificity, ensuring the highest accuracy in integration site analysis.
3. What are the safety considerations associated with lentiviral vectors?
- Safety concerns with lentiviral vectors include the risk of insertional mutagenesis, which may lead to adverse effects or oncogenesis. By providing comprehensive information on integration patterns, CD Genomics' integration site analysis helps mitigate these potential risks.
4. Can integration site analysis be used for both lentiviral and retroviral vectors?
- Yes, the advanced technologies used by CD Genomics are applicable to both lentiviral and retroviral vectors, offering extensive data for various types of viral vectors.

Case Study

Joint profiling of chromatin accessibility and CAR-T integration site analysis at population and single-cell levels
Journal: Proceedings of the National Academy of Sciences
Impact factor: 9.661
Published: February 24, 2020

Find out more

Background

Cancer immunotherapy with CAR-T cells often faces challenges with T cell persistence and efficacy. The EpiVIA assay addresses this by simultaneously mapping lentiviral integration sites and chromatin accessibility at single-cell resolution. It uses Tn5 transposase to insert sequencing adapters into viral-host DNA fragments, revealing integration sites with high accuracy. This method shows CAR-T integrations favoring specific genomic regions and provides insights into T cell behavior and treatment durability.

Materials & Methods

Sample preparation:

Clonal Cells

Method:

ATAC-Seq
scATAC-Seq
Integration site analysis

Data Analysis:

Identification of integration site
Local chromatin state of integration sites
Combined analysis of host chromatin state, integration site, and provirus accessibility.

Results

EpiVIA detects CAR-T integration sites at the single-cell level effectively. In a study of 5,000 CD8+ T cells, it identified 193 unique integration sites. Higher sequencing depth improved detection sensitivity, with more fragments per cell enhancing the ability to identify integration events.

EpiVIA's analysis of CAR-T integration sites reveals that 60% occur in transposable elements, predominantly Alu repeats, and are enriched in intronic regions. Integration sites identified by EpiVIA closely align with known HIV-1 integration sites and recurrent integration genes (RIGs). The analysis confirms EpiVIA's accuracy and highlights its ability to detect integration hotspots at a single-cell resolution.

Figure 1. Principal coordinates analysis (PCoA) of fungal community structure based on weighted UniFrac distance across three groups. (Gu et al., 2022) Fig 1. Genomic features of EpiVIA identified integration sites in single cells.

Conclusions

EpiVIA is a tool for profiling both epigenomes and lentiviral integration sites at bulk and single-cell levels. It accurately maps integration sites, cell fate, and chromatin accessibility, overcoming limitations of existing methods.

References

Berry C C, Nobles C, Six E, et al. INSPIIRED: quantification and visualization tools for analyzing integration site distributions. Molecular Therapy-Methods & Clinical Development, 2017, 4: 17-26.
Wang W, Fasolino M, Cattau B, et al. Joint profiling of chromatin accessibility and CAR-T integration site analysis at population and single-cell levels. Proceedings of the National Academy of Sciences. 2020, 117(10):5442-52.

* For Research Use Only. Not for use in diagnostic procedures or other clinical purposes.