title: INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes.
abstract: 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.
authors.affiliation: Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6076, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Sherman
authors.affiliation: Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6076, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Nobles
authors.affiliation: Department of Family Medicine and Public Health, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Charles C
authors.initials: CC
authors.lastname: Berry
authors.affiliation: Imagine Institute, Paris Descartes-Sorbonne Paris Cité University, 75014 Paris, France; Laboratory of Human Lymphohematopoiesis, INSERM 24, 75014 Paris, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Six
authors.affiliation: Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6076, USA.
authors.forename: Yinghua
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6076, USA.
authors.forename: Anatoly
authors.initials: A
authors.lastname: Dryga
authors.affiliation: Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6076, USA.
authors.forename: Nirav
authors.initials: N
authors.lastname: Malani
authors.affiliation: Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6076, USA.
authors.forename: Frances
authors.initials: F
authors.lastname: Male
authors.affiliation: Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6076, USA.
authors.forename: Shantan
authors.initials: S
authors.lastname: Reddy
authors.affiliation: Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6076, USA.
authors.forename: Aubrey
authors.initials: A
authors.lastname: Bailey
authors.affiliation: Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6076, USA.
authors.forename: Kyle
authors.initials: K
authors.lastname: Bittinger
authors.affiliation: Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6076, USA.
authors.forename: John K
authors.initials: JK
authors.lastname: Everett
authors.affiliation: Biotherapy Department, Necker Children's Hospital, Assistance Publique-Hôpitaux de Paris, 75014 Paris, France; Biotherapy Clinical Investigation Center, Groupe Hospitalier Universitaire Ouest, Assistance Publique-Hôpitaux de Paris, INSERM, 75014 Paris, France.
authors.forename: Laure
authors.initials: L
authors.lastname: Caccavelli
authors.affiliation: Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6076, USA.
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Drake
authors.affiliation: Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6076, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Bates
authors.affiliation: Biotherapy Department, Necker Children's Hospital, Assistance Publique-Hôpitaux de Paris, 75014 Paris, France; Biotherapy Clinical Investigation Center, Groupe Hospitalier Universitaire Ouest, Assistance Publique-Hôpitaux de Paris, INSERM, 75014 Paris, France.
authors.forename: Salima
authors.initials: S
authors.lastname: Hacein-Bey-Abina
authors.affiliation: Biotherapy Department, Necker Children's Hospital, Assistance Publique-Hôpitaux de Paris, 75014 Paris, France; Biotherapy Clinical Investigation Center, Groupe Hospitalier Universitaire Ouest, Assistance Publique-Hôpitaux de Paris, INSERM, 75014 Paris, France.
authors.forename: Marina
authors.initials: M
authors.lastname: Cavazzana
authors.affiliation: Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6076, USA.
authors.forename: Frederic D
authors.initials: FD
authors.lastname: Bushman
keywords: SCID-X1; gammaretrovirus; gene therapy; insertional mutagenesis; lentivirus; mutagenesis; recombination; retrovirus; vector; vector driving