INSERT-seq enables high-resolution mapping of genomically integrated DNA using Nanopore sequencing.

Comprehensive characterisation of genome engineering technologies is relevant for their development and safe use in human gene therapy. Short-read based methods can overlook insertion events in repetitive regions. We develop INSERT-seq, a method that combines targeted amplification of integrated DNA, UMI-based correction of PCR bias and Oxford Nanopore long-read sequencing for robust analysis of DNA integration. The experimental pipeline improves the number of mappable insertions at repetitive regions by 4.8-7.3% and larger repeats are processed with a computational peak calling pipeline. INSERT-seq is a simple, cheap and robust method to quantitatively characterise DNA integration in diverse ex vivo and in vivo samples.