Bioinformatic Clonality Analysis of Next-Generation Sequencing-Derived Viral Vector Integration Sites

Abstract

Clonality analysis of viral vector-transduced cell populations represents a convincing approach to dissect the physiology of tissue and organ regeneration, to monitor the fate of individual gene-corrected cells in vivo, and to assess vector biosafety. With the decoding of mammalian genomes and the introduction of next-generation sequencing technologies, the demand for automated bioinformatic analysis tools that can rapidly process and annotate vector integration sites is rising. Here, we provide a publicly accessible, graphical user interface-guided automated bioinformatic high-throughput integration site analysis pipeline. Its performance and key features are illustrated on pyrosequenced linear amplification-mediated PCR products derived from one patient previously enrolled in the first lentiviral vector clinical gene therapy study. Analysis includes trimming of vector genome junctions, alignment of genomic sequence fragments to the host genome for the identification of integration sites, and the annotation of nearby genomic elements. Most importantly, clinically relevant features comprise the determination of identical integration sites with respect to different time points or cell lineages, as well as the retrieval of the most prominent cell clones and common integration sites. The resulting output is summarized in tables within a convenient spreadsheet and can be further processed by researchers without profound bioinformatic knowledge.

Efficiency and Safety of O6-Methylguanine DNA Methyltransferase (MGMTP140K)-MediatedIn VivoSelection in a Humanized Mouse Model

“…This step was performed using cross_match (Phil Green, University of Washington; www.phrap.org). Subsequent to these preprocessing steps, the sequences were uploaded to the freely available High Throughput Insertion Site Analysis Pipeline (HISAP) (Arens et al, 2012) for insertion-site analysis. The integration sites retrieved from HISAP analysis were ranked according to the read counts for each sample.…”

“…After removal of the linker-and vector-specific sequences, 486 unique sequences were mapped to the human genome using the HISAP (Arens et al, 2012) and assigned to the following samples: 231 in 2 pretransplant samples (pre-TX; from reduced chemotherapy and GFP control experiments), 144 in treated (10 BM and 20 PB; samples from all dose groups), 54 in NT (4 BM and 8 PB), and 57 in control (3 BM and 6 PB) samples. Irrespective of the vector or treatment, all groups exerted similar insertion-site profiles (Fig.…”

Presence of a trs -Like Motif Promotes Rep-Mediated Wild-Type Adeno-Associated Virus Type 2 Integration

“…PCRs were performed using linker-specific primers and primers binding to the 3= and 5= ends of the viral genome. Bioinformatic data mining of LAM-PCR and nrLAM-PCR sequences was performed using HISAP (15). We analyzed a total of 255,772 454 GS FLX (Roche Diagnostics) and 10,398,424 MiSeq (Illumina) sequencing reads derived from LAM-PCR products, thereby determining a total of 4,340 unique ISs (HeLa, 3,060 ISs; HDFs, 1,280 ISs) (Fig.…”

Linear Amplification Mediated PCR – Localization of Genetic Elements and Characterization of Unknown Flanking DNA

“…High-throughput sequencing of (nr)LAM-PCR products and mapping of retrieved raw sequences to the corresponding genome allows characterizing unknown flanking DNA or identifying the exact localization of vector-genome junctions 30 . By introducing barcode sequences into the fusionprimers several hundreds of LAM-and nrLAM-PCR products can be sequenced in one sequencing run 30 .…”