High-throughput mapping and clonal quantification of retroviral integration sites

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

Research output: Contribution to journalArticlepeer-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
Pages (from-to)127-141
Number of pages15
JournalMethods in Molecular Biology
Volume1582
DOIs
Publication statusPublished - 1 Jan 2017

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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