The LAM-PCR Method to Sequence LV Integration Sites.
Integrating viral gene transfer vectors are commonly used gene delivery tools in clinical gene therapy trials providing stable integration and continuous gene expression of the transgene in the treated host cell. However, integration of the reverse-transcribed vector DNA into the host genome is a potentially mutagenic event that may directly contribute to unwanted side effects. A comprehensive and accurate analysis of the integration site (IS) repertoire is indispensable to study clonality in transduced cells obtained from patients undergoing gene therapy and to identify potential in vivo selection of affected cell clones. To date, next-generation sequencing (NGS) of vector-genome junctions allows sophisticated studies on the integration repertoire in vitro and in vivo. We have explored the use of the Illumina MiSeq Personal Sequencer platform to sequence vector ISs amplified by non-restrictive linear amplification-mediated PCR (nrLAM-PCR) and LAM-PCR. MiSeq-based high-quality IS sequence retrieval is accomplished by the introduction of a double-barcode strategy that substantially minimizes the frequency of IS sequence collisions compared to the conventionally used single-barcode protocol. Here, we present an updated protocol of (nr)LAM-PCR for the analysis of lentiviral IS using a double-barcode system and followed by deep sequencing using the MiSeq device.
Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Im Neuenheimer Feld 581, 69120, Heidelberg, Germany.
Wei
W
Wang
Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Im Neuenheimer Feld 581, 69120, Heidelberg, Germany.
Cynthia C
CC
Bartholomae
Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Im Neuenheimer Feld 581, 69120, Heidelberg, Germany.
Richard
R
Gabriel
Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Im Neuenheimer Feld 581, 69120, Heidelberg, Germany.
Annette
A
Deichmann
Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Im Neuenheimer Feld 581, 69120, Heidelberg, Germany. manfred.schmidt@nct-heidelberg.de.
Manfred
M
Schmidt
D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D016662:Virus Integration / Q000235:genetics*
(nr)LAM-PCR; Clonality; Double-barcoding strategy; Gene therapy; Integration sites; Lentiviral vector; Next-generation sequencing (NGS); Safety