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.