Integration Site Analysis

What is INTEGRATION SITE ANALYSIS and why is it important?

Classical integration site analysis (ISA) methods such as fluorescence based karyology or southern blotting can give information on the integration location in the genome, but is still very low resolution. Sequencing the integration site provides single nucleotide resolution on the integration site, however can be challenging, especially if the transgene is randomly integrated into the host genome. Methods such as long range PCR or Sanger sequencing are also employed, but are cumbersome and also require a priori knowledge of the integration site. More recently, NGS whole genome sequencing (WGS) has been used to characterize the integration site.  However, WGS can be complex for cells with unstable genomes such as CHO. In addition, WGS tends to provide a lower level of sequence coverage, making conclusive identification of the integration site and its epigenetic status difficult.

A targeted ISA short read NGS method utilizes whole genome crosslinking to generate circular DNA fragments up to 100kb either side of a known primer site from the gene of interest. This method does not require any a priori sequence information of the insertion site, and good information is obtained on the junction site. However, due to the nature of short read sequencing this approach can struggle to identify concatemers, truncations and inverted repeats.

PathoQuest has validated an enhanced ISA approach that uses Cas9 targeted DNA cleavage combined with long read nanopore sequencing. By targeting Cas9 to cleave regions within the transgene expression cassette, both the transgene and the junction site can be comprehensively sequenced. Long read nanopore sequencing has the advantage over short-read ISA methods as it better characterize concatamers, truncations and inverted repeats.