High-throughput mapping and clonal quantification of retroviral integration sites

Nicolas A. Gillet, Anat Melamed, Charles R.M. Bangham

    Research output: Contribution in Book/Catalog/Report/Conference proceedingChapter (peer-reviewed)peer-review

    Abstract

    We describe here a method to identify the position of retroviral insertion sites and simultaneously to quantify the absolute abundance of each clone, i.e., the number of cells having the provirus inserted at a given place in the host genome. The method is based on random shearing of the host cell DNA, followed by a linker-mediated PCR to amplify the genomic regions flanking the proviruses, and high-throughput sequencing of the amplicons. The quantification of the abundance of each infected clone allowed us to develop two new metrics: i. the oligoclonality index, which quantifies the nonuniformity of the distribution of clone abundance, and ii. an estimator of the total number of clones in the body of the host. These new tools are valuable for the study of retroviral infections and can also be adapted for the tracking of gene-edited cells.

    Original languageEnglish
    Title of host publicationHuman T-Lymphotropic Viruses
    Subtitle of host publicationMethods and Protocols
    EditorsClaudio Casoli
    PublisherSpringer Verlag
    Pages127-141
    Number of pages15
    DOIs
    Publication statusPublished - 1 Jan 2017

    Publication series

    NameMethods in Molecular Biology
    PublisherHumana Press Inc.
    Volume1582
    ISSN (Print)1064-3745

    Keywords

    • Clonal distribution
    • Clonal expansion
    • Clonality
    • Gene therapy
    • Gene-edited cells
    • Insertion sites
    • Number of clones
    • Off-target insertion
    • Oligoclonal
    • Retroviral integration sites

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