Project Summary The per-generation de novo mutation rate spans more than an order of magnitude among eukaryotes and at least a two-fold range among primates. While many studies focus on those mutations that arise during gametogenesis, experiments have shown that the rate of mutation is higher during embryogenesis, and that half of all mutations are already present in the germline at puberty. In this proposal we carry out multiple experiments using pedigrees from the model nonhuman primate, rhesus macaques (Macaca mulatta), to quantify the number and types of mutations produced during embryogenesis and gametogenesis. First, we detect embryogenic mutations in parents by sequencing the genomes of multiple of their offspring. Transmitted mutations produced during parental embryogenesis will appear mosaically, enabling us to detect them in some, but not all, offspring. By sequencing multiple siblings within a family, we will be able to measure the embryogenic mutation rate in an unbiased manner. Second, we will quantify male germline mosaicism with pooled sperm sequencing. The frequency of mosaic mutations reveals the timing of their genesis in development. By deeply sequencing sperm collected from individual sires of the same pedigrees, we will quantify the frequency of embryogenic mutations and they stage in which they arose. In addition, pooled sequencing of sperm from offspring will help to estimate the fraction of embryogenic mutations missed by pedigree studies. Both of these experiments together will lead to more accurate estimates of the mutation rate during embryogenesis. Third, we will uncover early embryogenic mutations in the offspring via paired comparisons with placenta. Mutations arising very early in an individual's development will appear in almost all cells. To quantify these early embryogenic mutations, we will sequence placentas from the same sequenced offspring. The placenta separates from the lineage leading to the embryo shortly after fertilization. Comparing mutations found in the placenta with those from blood samples of the developed embryo will allow us to delineate the timing of mutations. This experiment has many of the same advantages as studies of monozygotic twins, but with tissues that are readily available from a singleton birth.