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. 2016 Dec 18:4:39-49.
doi: 10.1016/j.omtm.2016.11.002. eCollection 2017 Mar 17.

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

Eric Sherman  1 , Christopher Nobles  1 , Charles C Berry  2 , Emmanuelle Six  3 , Yinghua Wu  1 , Anatoly Dryga  1 , Nirav Malani  1 , Frances Male  1 , Shantan Reddy  1 , Aubrey Bailey  1 , Kyle Bittinger  1 , John K Everett  1 , Laure Caccavelli  4 , Mary J Drake  1 , Paul Bates  1 , Salima Hacein-Bey-Abina  4 , Marina Cavazzana  4 , Frederic D Bushman  1
Affiliations

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

Eric Sherman et al. Mol Ther Methods Clin Dev. .

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.

Keywords: SCID-X1; gammaretrovirus; gene therapy; insertional mutagenesis; lentivirus; mutagenesis; recombination; retrovirus; vector; vector driving.

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Figures

Figure 1
Figure 1
Diagram of the Biochemical Method for Isolating and Sequencing Sites of New DNA Integration Genomic DNA containing an integrated retrovirus or retroviral vector (top) is sheared and DNA linkers are ligated onto the resulting ends (middle). The molecules are then subjected to two rounds of PCR amplification and then Illumina paired-end sequencing (bottom). The color code for sequence elements is summarized on the right. Black spheres indicate DNA 5′ ends. -NH2 indicates an amino-modifier group, which prevents polymerase extension from the modified 3′ end.
Figure 2
Figure 2
Use of a Locked Oligonucleotide to Block Recovery of the Internal Fragment to Improve Integration Site Yield (A) Diagram of the method. The BNA-containing blocking oligonucleotide is shown in black; DNA polymerase is shown in gray. Other markings are as in Figure 1. (B) Quantification of recovery of the internal fragment. Twelve replicates were compared for each sample. (C) Increase in yield as a result of use of the locked oligonucleotide blocking primer. Error bars are SD.
Figure 3
Figure 3
Estimating Abundance Using the SonicAbundance Method Cells harboring integrated vectors are shown at the top. One cell clone has expanded to comprise 4/6 cells (flanking DNA colored cyan). DNA is then purified and cleaved and linkers are ligated. Note that the cyan expanded clone is present as four distinct fragment lengths. A stacked bar graph (bottom) summarizes the differences seen based on summing the abundance of different length fragments.
Figure 4
Figure 4
Interpretation of Paired Read Data (A) Unique integration site. In this case, the two reads are within a short distance of one another on the chromosome and are correctly oriented on opposite DNA strands. (B) An artifactual chimera. In this case, the two reads are on two distinct chromosomes, or found implausibly far apart on the same chromosome, and so are judged to be artifacts formed during construction of the library for sequencing. (C) Multihit. In this case, both reads in the pair have equally good alignments at multiple distinct locations.
Figure 5
Figure 5
Results of a Control Study of Synthetic Mixtures of DNA from Cell Lines with Known Integration Site Distributions The expected abundance based on the composition of the mixture is shown on the x axis, and the observed abundance (over 4 replicates) is shown on the y axis. Three cell mixtures were compared. In set 1, the clones were mixed in equal amounts. In set 2, the composition was 20% clone 1, 10% clone 2, 45% clone 3, 15% clone 4, and 10% clone 5. In set 3, the composition was 9% clone 1, 15% clone 2, 1% clone 3, 10% clone 4, and 65% clone 5. Error bars are SD.
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