Somatic mutations represent a significant health concern as they underlie many cancers and age-related disorders. Although technological advances have led to a deeper understanding of germline mutations, somatic mutations have eluded deeper investigation until more recently. The mutational process in germline versus somatic cells is likely similar, given both depend on many of the same parameters (e.g., rates of DNA polymerase errors occurring during replication and levels of DNA damage). In large multicellular organisms, however, somatic cells differentiate during development and accumulate mutations due to repeated rounds of cell division, thereby shifting these parameters across tissue types and over time. We propose to investigate the relative influence of germline mutation rates, location, and age on somatic mutation rates by performing whole genome sequencing on tissue-specific populations of cells from multiple time points across genotypes for which a wide range of germline mutation has already been observed. To our knowledge, this would be the first in organism comparison of somatic and germline mutations across genotypes within a species. Our research strategy for identifying somatic mutations will be PCR-free whole-genome DNA sequencing of cells sampled from brain and heart, from juveniles and adults, across 9 genotypes of Daphnia magna, an aquatic microcrustacean already in use for ageing research. Samples will be taken by sectioning animals and using laser capture microdissection to isolate populations of cells from specific tissues. High-throughput sequencing of multiplexed samples will allow us to efficiently obtain sufficient (10x) coverage to call somatic variants across the genome. Continued development of the D. magna system for investigating mutational processes allows us to (1) leverage our current knowledge of germline mutation rates and phenotypic declines in this species and (2) capitalize on our ability to quickly and easily target different tissue types in a transparent arthropod, a long-standing model system in biology and public health research and an emerging model system in genomics. Investigating the factors influencing variation in somatic mutation rates will fill a major gap in our current knowledge of how DNA errors and damage accumulates, providing insight into the frequency of mutation in different genomic backgrounds, across tissues, and over time.
The goal of this project is to investigate three major factors influencing somatic mutation rates: tissue type, developmental stage, and germline mutation rate (known to differ among genotypes) using the Daphnia model system. These data will be used to test hypotheses about the relationship between germline (heritable) mutation rates and those that occur in somatic tissue, to assess whether the genotoxic cellular environment differs among tissue types, and to determine if somatic mutation rates increase with age. Estimates of somatic mutation rates are important, as they are the cause of many cancers and age- related disorders. Understanding the co-variation in such rates with these critical factors represents the current frontier in mutation research.
