Integration site analysis of viral vectors with (nr)LAM-PCR
The identification of viral vector flanking genomic sequences is performed with linear amplification mediated PCR (LAM-PCR) as described in Schmidt et. al 2007. Optionally, non-restrictive variant of LAM-PCR (nrLAM-PCR) is applied (Gabriel et al. 2009, Paruzynski et al. 2010).
For LAM-PCR, flanking sequences are amplified by linear PCR using biotinylated primers hybridizing to vector sequences (e.g. 3-prime region of the long terminal repeat (LTR) of the vector). Subsequent steps involve magnetic capture of the biotinylated PCR products, hexanucleotide priming by Klenow polymerase for double strand DNA synthesis and restriction digest using e.g. MluCI and MseI. After digestion, a double-stranded sequence adaptor (linker cassette) carrying a molecular barcode is ligated to the restricted DNA. Also for nrLAM-PCR two linear PCR amplification steps with a vector specific biotinylated primer is used. Subsequent steps involve magnetic capture of the biotinylated PCR products and ligation of a single stranded linker cassette carrying a molecular barcode. For both methods, LAM-PCR and nrLAM-PCR, the ligated PCR product is used as template in an exponential PCR using biotinylated vector- and adaptor-specific primers. Magnetic capture of the biotinylated PCR-products is performed before reamplification with nested vector- and adaptor-specific primers in a second exponential PCR step.
LAM-PCR amplicons are regularly sequenced on the MiSeq instrument (Illumina) after sample preparation for high-throughput sequencing. Therefore, an additional PCR with special fusion-primers carrying MiSeq specific sequencing adaptors is performed. DNA barcoding is used to allow parallel sequencing of multiple samples in a single sequencing run.
Raw sequence data are trimmed according to sequence quality (Phred 30). Only sequences carrying correct barcodes (linker cassette barcode, sequencing adapter barcodes) are further analyzed. Our (semi-) automated bioinformatical data mining pipeline is used to analyze the data according to your needs (Arens et al. 2012 and unpublished).
- Schmidt M, Schwarzwaelder K, Bartholomae CC, Zaoui K, Ball C, Pilz I, Braun S, Glimm H, von Kalle C: High-Resolution Insertion Site Analysis by Linear Amplification-Mediated PCR (LAM-PCR). Nature Methods. 4, 1051-1057, 2007.
- Gabriel R, Eckenberg R, Paruzynski A, Bartholomae C, Nowrouzi A, Arens A, Howe SJ, Recchia A, Cattoglio C, Wang W, Faber K, Schwarzwaelder K, Kirsten R, Deichmann A, Ball CR, Balaggan KS, Yáñez-Muñoz RJ, Ali RR, Gaspar HB, Biasco L, Aiuti A, Cesana D, Montini E, Naldini L, Cohen-Haguenauer O, Mavilio F, Thrasher AJ, Glimm H, von Kalle C, Saurin W, Schmidt M. Comprehensive genomic access to vector integration in clinical gene therapy. Nature Medicine. 15, 1431-1436, 2009.
- Paruzynski A, Arens A, Gabriel R, Bartholomae CC, Scholz S, Wang W, Wolf S, Glimm H, Schmidt M, von Kalle C: Genome-wide high-throughput integrome analyses by nrLAM-PCR and next-generation sequencing. Nature Protocols. 5, 1379-1395, 2010.
- Arens A, Appelt JU, Bartholomae CC, Gabriel R, Paruzynski A, Gustafson D, Cartier N, Aubourg P, Deichmann A, Glimm H, von Kalle C, Schmidt M: Bioinformatic clonality analysis of next-generation sequencing-derived viral vector integration sites. Human Gene Therapy Methods. 23, 111-118, 2012.