INSPIIRED: Quantification and Visualization Tools for Analyzing Integration Site Distributions

Abstract

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.

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

“…Alignment information is then paired between the reads, and the integration site position and DNA fragment breakpoints (linker positions) are returned and stored in the IntSiteDB database (described in detail in our accompanying paper 52 ). Read 1 and read 2 are joined based on location in the sequencing flow cell (encoded as the read name).…”

“…These include (1) interactive heatmaps summarizing relationships of integration site data to genomic and epigenetic features; (2) reproducible reports on gene-corrected patient samples summarizing numerous features of integration site populations, including expansion of clones with integration sites near cancer-related genes; and (3) data frames suitable for statistical analysis based on the SonicAbundance method. These tools are described in our accompanying paper 52 and are available at https://github.com/BushmanLab/INSPIIRED.…”

“…For the studies reported here, the h18 draft of the human genome was used, to allow direct use of chromatin immunoprecipitation sequencing (ChIP-seq) data that was originally analyzed on this background (see our accompanying paper 52 ). However, the organism and draft genome used for analysis can be selected by users and analyzed with any suitable annotation tracks compiled on that sequence.…”

“…Performance was then tested over several datasets ranging from experimental infections to human gene therapy samples, allowing analysis of the influence of repeated sequences on site capture. In our accompanying paper in this issue of Molecular Therapy: Methods & Clinical Development , 52 we describe a suite of analytical tools that draws on the data products described here.…”

An analytical pipeline for identifying and mapping the integration sites of HIV and other retroviruses

“…If a DNA fragment that carries a particular integration site is rare (present only once or a few times in an Illumina dataset), it could be the result of contamination, and it is important not only to be very careful with the interpretation of the data, but, as has already been discussed, to design the workspace and amplification procedures to reduce contamination artifacts to a minimum. There are other published methods that give good advice for avoiding or reducing PCR/sequencing artifacts associated with the retroviral integration site analysis [43,44].…”