Abstract High-throughput DNA sequencing has fundamentally changed practice standards in clinical oncology. We can now systematically examine the dysfunctional genes and pathways of individual tumors and can often pair these with targeted therapies. Moreover, NGS has improved our ability to resolve molecular heterogeneity, such as results from evolving subclones within a tumor or admixtures of cancer-derived and normal DNA in liquid biopsies. However, this remains imperfect. With standard NGS, sequencing errors create a background that obscures variants present at less than 1-2%. Methods of tag-based error correction improve accuracy, but most rely on hemimolecular tagging and are thus still limited by certain types of errors caused by DNA damage. Our group pioneered Duplex Sequencing, an error correction technology that independently genotypes and compares both single-stranded halves of individual DNA duplexes. This approach improves sequencing accuracy by >100,000-fold and allows detection of a single mutation among ten million unmutated nucleotides. We carried out Duplex Sequencing on uterine lavages from women with and without high-grade serous ovarian cancer to determine the sensitivity for identifying tumor-derived TP53 mutations as a screening test. Under blinded conditions we detected tumor-derived mutations in 80% of cancer cases. However, in addition, we found that nearly all women, including those without cancer, carried very low frequency TP53 mutations that did not originate from a tumor. These could not be explained by technical errors, as they exhibited a strong signature of positive selection, indicated by higher-than-random non-synonymous fraction, substantial clustering in cancer-associated hotspots and a spectrum, exonic distribution and predicted pathogenicity closely mirroring TP53 mutations in cancer databases. Furthermore, abundance of these “biological background” mutations increased with age. To more broadly investigate the extent of clonal evolution in normal aging, we used Duplex Sequencing to examine tissue specimens from two individuals without cancer, differing in age by 101 years. We assessed relative mutation load in 22 cancer-associated genes and one non-cancer associated gene in 19 different tissues. Initial results indicate that the extent of age-associated mutation accumulation varies by organ and the pattern of apparent clonal selection is influenced by both tissue type and genetic context. Our findings illustrate the near-universal presence of cancer-like mutations arising from age-associated processes in multiple non-neoplastic tissues. Somatic clonal evolution appears to be a ubiquitous part of human life. Beyond many biological implications, this highlights an important specificity challenge that must be accounted for when using ultra-accurate sequencing technologies for cancer screening and other sensitive diagnostics. Citation Format: Jesse J. Salk, Elisabeth Maritschnegg, Paul Speiser, Robert Zeillinger, Rosana Risques, Lawrence A. Loeb. Duplex Sequencing detects cancer-associated mutations arising during normal aging: Clonal evolution over a century of human lifetime [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3041. doi:10.1158/1538-7445.AM2017-3041