Integration Site Analysis Services
Integration Site Analysis Services
NGS-based integration site analysis supports the development of gene-modified biologics, cell therapies, plasmid-based platforms, and engineered cell lines. These methods enable precise identification and genomic mapping of vector or transgene integration events within the host genome. By applying targeted enrichment approaches such as hybridization-based capture, biotinylated LM-PCR, Extension Primer Tag Selection (EPTS), and Whole Genome Sequencing (WGS), integration junctions can be localized and quantified to assess distribution, frequency, and genomic context. The resulting data support regulatory submissions, cell bank characterization, and evaluation of genome integrity across research and clinical development programs.
Our Expertise
Avance Biosciences has a long-standing interest in gene integration studies. Traditionally, we have utilized Southern blot analysis to determine the number of integration sites, supporting cell bank characterization for protein expression in GMP manufacturing. Building on our leadership in NGS applications for biologics development and manufacturing, we have established a series of advanced methods to help our clients understand integration sites. These methods are instrumental in characterizing CHO cell lines, autologous CAR-T cells, plasmid vaccines, among other applications. Through our comprehensive approach, we provide detailed insights that optimize vector design and delivery, ensuring precise and predictable gene integration for therapeutic development.
Integration Site Analysis Services
Methods
Description
Applications
Target Sequencing by Hybridization
Genomic DNA is fragmented and prepared as an NGS library. Target enrichment is performed using hybridization probes specific to vector or transgene sequences, followed by high-throughput sequencing.
Sensitive method for detecting integration junctions without reliance on LTR assumptions. Suitable for diverse integration constructs.
Biotinylated LM-PCR by NGS
Fragmented genomic DNA undergoes adapter ligation with UMI incorporation. Biotinylated primers targeting vector–genome junctions enable selective enrichment via streptavidin pull-down prior to NGS.
High-sensitivity integration site localization and frequency assessment for gene and cell therapy programs.
EPTS (Extension Primer Tag Selection) with NGS
A vector- or transgene-specific primer performs linear extension from integration-containing fragments, incorporating a capture tag that enables magnetic enrichment prior to library amplification and sequencing. Enrichment occurs through targeted linear amplification rather than exponential PCR or hybridization capture.
Targeted enrichment of defined transgene integrations with high specificity. Suitable for well-characterized constructs and structured integration analysis.
Genomic DNA is sequenced without targeted enrichment using short-read or long-read NGS platforms. Integration sites are identified through computational detection of split reads and structural variants.
Broad genome integrity assessment and clonal cell line characterization. Lower sensitivity for rare integration events compared to targeted enrichment methods.
Interesting Facts
- Our Target Sequencing by Hybridization method has been validated and demonstrated to have high specificity and sensitivity. It does not rely on the assumption that an LTR is present at the integration sites, although we have predesigned LTR-specific hybridization probes to facilitate off-the-shelf analysis.
- For our modified EPTS/LM-PCR method, after capturing fragments with integration sites and completing ligation, NGS amplicon sequencing is performed instead of nested PCR and primer walking with Sanger sequencing, improving assay sensitivity.
Technical Information
Explore the power of Next Generation Sequencing (NGS) for biologics development support with our latest whitepaper. Avance Biosciences is at the forefront of NGS testing, offering tailored solutions to support drug development initiatives and GMP manufacturing activities. Delve into real-world case studies, including CRISPR indel amplicon sequencing and insertion site studies, showcasing our expertise in assay development and validation...