Abstract: The invention relates to a method of generating a nucleic acid library for sequencing with error rates <1 error per 100 million base pairs and uses thereof. Provided are methods for detecting mutations (including driver mutations) present in single DNA molecules, such as a single DNA molecule from a polyclonal population of cells, and use of the methods in measuring somatic mutation rates, mutational signatures and driver mutations in vivo or in vitro. Also provided are methods of discovering somatic mutations involved in disease and detecting mutations from cells in culture, diagnosing disease and for detecting the presence and/or identity of a suspected mutation in a cell cultured in vitro and/or subjected to a mutagenic process in vitro. Further provided is a method of computational analysis of duplex sequencing data.