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J Vis Exp. 2017 Jun 14;(124). doi: 10.3791/55812.

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow.

Author information

1
UCLA AIDS Institute, University of California at Los Angeles (UCLA); Department of Microbiology, Immunology, & Molecular Genetics, University of California at Los Angeles (UCLA).
2
Departments of Biomathematics and Mathematics, University of California at Los Angeles (UCLA).
3
UCLA AIDS Institute, University of California at Los Angeles (UCLA).
4
Personalized Genomic Medicine Research Center, Division of Strategic Research Groups, Korea Research Institute of Bioscience and Biotechnology.
5
UCLA AIDS Institute, University of California at Los Angeles (UCLA); Department of Medicine, University of California at Los Angeles (UCLA); syuchen@mednet.ucla.edu.
6
Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University (OSU); kim.6477@osu.edu.

Abstract

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

PMID:
28654067
PMCID:
PMC5608418
DOI:
10.3791/55812
[Indexed for MEDLINE]
Free PMC Article

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