A novel High-Throughput Ligase-Independent Mapping method to detect Viral Integration Sites

Integrated DNA elements are central to virology, functional genomics, and gene therapy, but current insertion-site mapping methods often rely on restriction digestion, ligation, or complex sequencing workflows that introduce bias and limit recovery. Here, we present Terminal Mapping, a high-throughput method that identifies host–insert junctions without restriction enzymes or DNA ligation. The workflow combines linear amplification from a known terminal sequence, enrichment of single-stranded products, terminal transferase-mediated poly-A tailing, and PCR amplification for Illumina or Oxford Nanopore sequencing. Applied to Jurkat T cells transduced with HIV-1- and SIVmac251-derived vectors, Terminal Mapping recovered more HIV-1 insertion sites than inverse PCR, reproduced known integration biases, and showed improved robustness with long-read sequencing. It revealed shared but quantitatively distinct HIV-1 and SIVmac251 hotspots, as well as substantial differences between Jurkat cell sources. Comparison of 5′ and 3′ LTR-derived reads also provided an internal control for unintegrated viral DNA. Terminal Mapping therefore offers a rapid and flexible platform for profiling integrated genetic elements across vectors and cellular contexts.