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Related Experiment Video

Updated: Nov 26, 2025

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
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Genome-wide integration site detection using Cas9 enriched amplification-free long-range sequencing.

Joost van Haasteren1, Altar M Munis1, Deborah R Gill1

  • 1Gene Medicine Group, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

Nucleic Acids Research
|December 8, 2020
PubMed
Summary

A new Amplification-free Integration Site sequencing (AFIS-Seq) method uses long-read sequencing for accurate lentiviral integration site analysis. This advancement enhances the safety evaluation of gene therapies.

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Area of Science:

  • Gene and cell therapy
  • Molecular biology
  • Bioinformatics

Background:

  • Lentivirus vectors are crucial for gene therapy but require integration site (IS) analysis for safety.
  • Existing next-generation sequencing (NGS) methods for IS detection have limitations like short read lengths and PCR bias.

Purpose of the Study:

  • To introduce Amplification-free Integration Site sequencing (AFIS-Seq), a novel method for sequencing lentiviral integration sites.
  • To overcome the limitations of current IS detection methods.

Main Methods:

  • AFIS-Seq utilizes Cas9-mediated enrichment of high-molecular-weight DNA, avoiding PCR amplification.
  • The enriched DNA is sequenced using long-read Nanopore MinION technology.

Main Results:

  • AFIS-Seq enables rapid, high-certainty IS mapping within a single day.
  • Demonstrated up to 1600-fold signal enrichment in various cell models.
  • Achieved improved confidence in IS analysis for lentiviral vectors.

Conclusions:

  • AFIS-Seq offers an accessible, low-cost, and efficient approach for lentiviral integration site analysis.
  • The method can be extended for broader applications, including Cas9-mediated gene integration and in vivo studies.
  • AFIS-Seq significantly enhances safety evaluation for preclinical gene therapies.