Mobile elements (MEs) compose more than half of the human genome, and their transposition and rearrangement have been directly implicated in causing more than 100 genetic diseases. Because of these mutagenic properties, MEs are important drivers of genome evolution and innovation, accounting for as much as 20-30% of structural variation in the human genome. However, the mechanisms through which they exert their effects are almost completely unknown. In this renewal application, we propose to exploit new high- throughput sequencing technologies to address a series of fundamental questions about the biology of MEs and their genomic impact: how often and when in development do ME insertions occur, and how does their activity vary among human populations (Aim 1)? How, and under which circumstances, do MEs create structural genomic variation through non-allelic homologous recombination (NAHR) (Aim 2)? How do MEs contribute to changes in gene expression within humans and in primate evolution (Aim 3)? In Aim 1, we will assess ME insertion activity in 3,000 whole-genome sequences sampled from multigenerational Utah families and in a set of more than 300 whole-genome sequences from individuals from 125 different worldwide human populations. (These sequencing studies, both carried out at 30-50x coverage per genome, are already under way under separate funding mechanisms, and the sequences are available to us at no cost.) These genomic resources, in combination with sequence data from the 1000 Genomes Project, will also be used to identify the largest existing collection of ME-mediated NAHR events, enabling us to identify the genomic factors that promote or inhibit the genomic instability mediated by MEs (Aim 2). In addition, we will apply the ME-Scan technology developed in our last funding cycle to a collection of sperm and blood cells from 90 males in order to assess, for the first time the timing of mobile element activity during spermatogenesis. Finally, in Aim 3, we will test the hypothesis that polymorphic and lineage-specific MEs make a substantial contribution to gene expression changes within humans and between primate species, respectively. Intraspecific effects will be assessed by testing for association between ME insertion polymorphisms and gene expression variation across 421 humans. To explore interspecific effects, we will capitalize on comparative RNA sequencing of the same cell types across a set of species representing all major anthropoid primate lineages. In particular, these data will provide us with an unprecedented opportunity to evaluate the role of MEs in the evolution and regulation of long noncoding RNAs, which have emerged as an abundant and important class of genomic regulators.