Project Summary Transposable elements are major sources of heritable human genetic variation and can contribute to disease by causing somatic mutations. This application focuses on the development of genomic tools and technologies to assess the contributions of these transposons to somatic mosaicism in normal tissues. Specifically, we will develop tools and technologies to detect variants in long interspersed element-1 (LINE-1, L1) sequences. L1 is the only active self-propagating protein-coding transposon in humans. We each inherit a distinct complement of active L1 loci, each of which is typically repressed through methylation of its internal promoter. Here, we propose to develop single cell mapping approaches to detect de novo L1 insertions and integrate these with long-read sequencing approaches to reveal the epigenetic status of source elements. We will also pioneer highly innovative approaches to detect intermediates of the target primed reverse transcription (TPRT) reaction that gives rise to L1 insertion events. Our multidisciplinary team brings together investigators with expertise in retrotransposon biology and genetic variation; innovation in the field of epigenetics; and experience with molecular biology and computational genomics methods development. Our shared goal in this UG3/UH3 project is to provide enabling tools and technologies to the Somatic Mosaicism across Human Tissues (SMaHT) Network.