Human

Adult T-cell leukemia (ATL) is a highly aggressive disease caused by malignant transformation and clonal proliferation of T-cells infected with human T-cell leukemia virus type-I (HTLV-1). Transition from a polyclonal to a monoclonal proliferation is one of the major events of ATL progression. Dissecting the mechanism responsible for clonal expansion of HTLV-1-infected cells requires extensive monitoring of provirus integration sites and the number of infected cells in each clone (clone size). We took advantage of next-generation sequencing technology, a tag system, and an in silico analysis pipeline to develop and internally validate a new high-throughput methodology for isolating integration sites and estimating the original number of cells in each clone. Initial analysis and validation was performed with DNA samples from HTLV-1-infected individuals having a different proviral load and disease status. We then used appropriate controls with known integration sites and clonality status to confirm the accuracy, sensitivity, and reproducibility of our system, which indeed had the least errors among the currently available techniques.