title: A genome-wide analysis of lentivector integration sites using targeted sequence capture and next generation sequencing technology.
abstract: One application of next-generation sequencing (NGS) is the targeted resequencing of interested genes which has not been used in viral integration site analysis of gene therapy applications. Here, we combined targeted sequence capture array and next generation sequencing to address the whole genome profiling of viral integration sites. Human 293T and K562 cells were transduced with a HIV-1 derived vector. A custom made DNA probe sets targeted pLVTHM vector used to capture lentiviral vector/human genome junctions. The captured DNA was sequenced using GS FLX platform. Seven thousand four hundred and eighty four human genome sequences flanking the long terminal repeats (LTR) of pLVTHM fragment sequences matched with an identity of at least 98% and minimum 50 bp criteria in both cells. In total, 203 unique integration sites were identified. The integrations in both cell lines were totally distant from the CpG islands and from the transcription start sites and preferentially located in introns. A comparison between the two cell lines showed that the lentiviral-transduced DNA does not have the same preferred regions in the two different cell lines.
authors.affiliation: Department of Genetics, Istanbul University, Institute for Experimental Medicine, Istanbul 34093, Turkey. dustek@istanbul.edu.tr
authors.forename: Duran
authors.initials: D
authors.lastname: Ustek
authors.forename: Sema
authors.initials: S
authors.lastname: Sirma
authors.forename: Ergun
authors.initials: E
authors.lastname: Gumus
authors.forename: Muzaffer
authors.initials: M
authors.lastname: Arikan
authors.forename: Aris
authors.initials: A
authors.lastname: Cakiris
authors.forename: Neslihan
authors.initials: N
authors.lastname: Abaci
authors.forename: Jaicy
authors.initials: J
authors.lastname: Mathew
authors.forename: Zeliha
authors.initials: Z
authors.lastname: Emrence
authors.forename: Hulya
authors.initials: H
authors.lastname: Azakli
authors.forename: Fulya
authors.initials: F
authors.lastname: Cosan
authors.forename: Atilla
authors.initials: A
authors.lastname: Cakar
authors.forename: Mahmut
authors.initials: M
authors.lastname: Parlak
authors.forename: Olcay
authors.initials: O
authors.lastname: Kursun
mesh_terms: D002469:Cell Separation; D002877:Chromosomes, Human / Q000821:virology; D005434:Flow Cytometry; D052138:Genes, Neoplasm; D015894:Genome, Human; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000502:physiology*; D016254:Mutagenesis, Insertional; D017422:Sequence Analysis, DNA; D014162:Transfection; D016662:Virus Integration