High-throughput mapping and clonal quantification of retroviral integration sites

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

doi:10.1007/978-1-4939-6872-5_10

High-throughput mapping and clonal quantification of retroviral integration sites

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

Unit of Research in Plant Cellular and Molecular Biology

Research output: Contribution in Book/Catalog/Report/Conference proceeding › Chapter (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 language	English
Title of host publication	Human T-Lymphotropic Viruses
Subtitle of host publication	Methods and Protocols
Editors	Claudio Casoli
Publisher	Springer Verlag
Pages	127-141
Number of pages	15
DOIs	https://doi.org/10.1007/978-1-4939-6872-5_10
Publication status	Published - 1 Jan 2017

Publication series

Name	Methods in Molecular Biology
Publisher	Humana Press Inc.
Volume	1582
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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10.1007/978-1-4939-6872-5_10

Cite this

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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.",

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High-throughput mapping and clonal quantification of retroviral integration sites. / Gillet, Nicolas A.; Melamed, Anat; Bangham, Charles R.M.
Human T-Lymphotropic Viruses : Methods and Protocols. ed. / Claudio Casoli. Springer Verlag, 2017. p. 127-141 (Methods in Molecular Biology; Vol. 1582).

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

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T1 - High-throughput mapping and clonal quantification of retroviral integration sites

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AU - Melamed, Anat

AU - Bangham, Charles R.M.

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N2 - 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.

AB - 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.

KW - Clonal distribution

KW - Clonal expansion

KW - Clonality

KW - Gene therapy

KW - Gene-edited cells

KW - Insertion sites

KW - Number of clones

KW - Off-target insertion

KW - Oligoclonal

KW - Retroviral integration sites

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BT - Human T-Lymphotropic Viruses

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PB - Springer Verlag

ER -

High-throughput mapping and clonal quantification of retroviral integration sites

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Keywords

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