Automated analysis of viral integration sites in gene therapy research using the SeqMap web resource

Peters, Brandon; Dirscherl, Sara; Dantzer, Jessica; Nowacki, Jonathan; Cross, Scott; Li, Xiaoman; Cornetta, Kenneth; Dinauer, Mary C.; Mooney, Sean D.

doi:10.1038/gt.2008.99

Cited by 12 publications

(13 citation statements)

References 14 publications

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“…For further details, see Supplemental Materials and Methods. Retroviral-genomic junction sequences were submitted to SeqMap, a web-based tool for mapping retroviral integration sites (RIS) 30. RIS were initially identified by mapping to the UCSC (mm6 assembly) and Ensembl (version38.35) genome databases.…”

Section: Methodsmentioning

confidence: 99%

Retroviral vector integration in post-transplant hematopoiesis in mice conditioned with either submyeloablative or ablative irradiation

et al. 2009

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X-linked chronic granulomatous disease (X-CGD) is an inherited immunodeficiency with absent phagocyte NADPH oxidase activity caused by defects in the gene encoding gp91phox. Here we evaluated strategies for less intensive conditioning for gene therapy of genetic blood disorders without selective advantage for gene correction, such as might be used in a human X-CGD protocol. We compared submyeloablative with ablative irradiation as conditioning in murine X-CGD, examining engraftment, oxidase activity and vector integration in mice transplanted with marrow transduced with a gamma-retroviral vector for gp91phox expression. The frequency of oxidase-positive neutrophils in the donor population was unexpectedly higher in many 300 cGy-conditioned mice compared to lethally irradiated recipients, as was the fraction of vector-marked donor secondary CFU-S12. Vector integration sites in marrow, spleen, and secondary CFU-S12 DNA from primary recipients were enriched for cancer-associated genes, including Evi1, and integrations in or near cancer-associated genes were more frequent in marrow and secondary CFU-S12 from 300 cGy-conditioned mice compared to fully ablated mice. These findings support the concept that vector integration can confer a selection bias, and suggest that intensity of the conditioning regimen may further influence the effects of vector integration on clonal selection in post-transplant engraftment and hematopoiesis.

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Section: Methodsmentioning

confidence: 99%

Retroviral vector integration in post-transplant hematopoiesis in mice conditioned with either submyeloablative or ablative irradiation

et al. 2009

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“…The Illumina platform has the advantages of allowing paired-end sequencing and providing larger data volumes. Several reports have described methods for analysis of these data 9, 29, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. However, to date, none have taken full advantage of all types of paired reads, dealt comprehensively with integration in repeated sequences, or provided a statistical framework for quantitative inference of cell abundances based on integration site data.…”

Section: Introductionmentioning

confidence: 99%

INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes

Sherman

Nobles

Berry

et al. 2017

Molecular Therapy - Methods & Clinical Development

120

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Integration of new DNA into cellular genomes mediates replication of retroviruses and transposons; integration reactions have also been adapted for use in human gene therapy. Tracking the distributions of integration sites is important to characterize populations of transduced cells and to monitor potential outgrow of pathogenic cell clones. Here, we describe a pipeline for quantitative analysis of integration site distributions named INSPIIRED (integration site pipeline for paired-end reads). We describe optimized biochemical steps for site isolation using Illumina paired-end sequencing, including new technology for suppressing recovery of unwanted contaminants, then software for alignment, quality control, and management of integration site sequences. During library preparation, DNAs are broken by sonication, so that after ligation-mediated PCR the number of ligation junction sites can be used to infer abundance of gene-modified cells. We generated integration sites of known positions in silico, and we describe optimization of sample processing parameters refined by comparison to truth. We also present a novel graph-theory-based method for quantifying integration sites in repeated sequences, and we characterize the consequences using synthetic and experimental data. In an accompanying paper, we describe an additional set of statistical tools for data analysis and visualization. Software is available at https://github.com/BushmanLab/INSPIIRED.

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“…Contemporary methods take advantage of Illumina paired-end sequencing to report the location of newly integrated DNA 13, 14, 15, 16. Multiple reports have described methods for quantifying and analyzing such data, but optimal methods for statistical analysis, data reduction, and data visualization are the topics of ongoing development 13, 14, 16, 17, 18, 19. Here, we describe a suite of tools for integration site analysis and visualization that are useful for characterizing samples from human gene therapy and other applications.…”

Section: Introductionmentioning

confidence: 99%

INSPIIRED: Quantification and Visualization Tools for Analyzing Integration Site Distributions

Berry

Nobles

Six

et al. 2017

Molecular Therapy - Methods & Clinical Development

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Analysis of sites of newly integrated DNA in cellular genomes is important to several fields, but methods for analyzing and visualizing these datasets are still under development. Here, we describe tools for data analysis and visualization that take as input integration site data from our INSPIIRED pipeline. Paired-end sequencing allows inference of the numbers of transduced cells as well as the distributions of integration sites in target genomes. We present interactive heatmaps that allow comparison of distributions of integration sites to genomic features and that support numerous user-defined statistical tests. To summarize integration site data from human gene therapy samples, we developed a reproducible report format that catalogs sample population structure, longitudinal dynamics, and integration frequency near cancer-associated genes. We also introduce a novel summary statistic, the UC50 (unique cell progenitors contributing the most expanded 50% of progeny cell clones), which provides a single number summarizing possible clonal expansion. Using these tools, we characterize ongoing longitudinal characterization of a patient from the first trial to treat severe combined immunodeficiency-X1 (SCID-X1), showing successful reconstitution for 15 years accompanied by persistence of a cell clone with an integration site near the cancer-associated gene CCND2. Software is available at https://github.com/BushmanLab/INSPIIRED.

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Automated analysis of viral integration sites in gene therapy research using the SeqMap web resource

Cited by 12 publications

References 14 publications

Retroviral vector integration in post-transplant hematopoiesis in mice conditioned with either submyeloablative or ablative irradiation

Retroviral vector integration in post-transplant hematopoiesis in mice conditioned with either submyeloablative or ablative irradiation

INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes

INSPIIRED: Quantification and Visualization Tools for Analyzing Integration Site Distributions

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