A genome-wide analysis of lentivector integration sites using targeted sequence capture and next generation sequencing technology

被引:11
|
作者
Ustek, Duran [1 ]
Sirma, Sema [1 ]
Gumus, Ergun [2 ]
Arikan, Muzaffer [1 ]
Cakiris, Aris [1 ]
Abaci, Neslihan [1 ]
Mathew, Jaicy [1 ]
Emrence, Zeliha [1 ]
Azakli, Hulya [1 ]
Cosan, Fulya [1 ]
Cakar, Atilla [1 ]
Parlak, Mahmut [1 ]
Kursun, Olcay [2 ]
机构
[1] Istanbul Univ, Inst Expt Med, Dept Genet, TR-34093 Istanbul, Turkey
[2] Istanbul Univ, Dept Comp Engn, TR-34320 Istanbul, Turkey
关键词
Lentivector integration; Next generation sequencing; Targeted DNA capture; Virus; GENE-THERAPY; SELECTION; DNA; PCR; HIV;
D O I
10.1016/j.meegid.2012.05.001
中图分类号
R51 [传染病];
学科分类号
100401 ;
摘要
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. (c) 2012 Elsevier B.V. All rights reserved.
引用
收藏
页码:1349 / 1354
页数:6
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