title: Detection and quantification of rare mutations with massively parallel sequencing.
abstract: The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Although massively parallel sequencing instruments are in principle well suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called the Safe-Sequencing System ("Safe-SeqS"), are (i) assignment of a unique identifier (UID) to each template molecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are considered mutant ("supermutants") only if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.
authors.affiliation: The Ludwig Center for Cancer Genetics and Therapeutics and The Howard Hughes Medical Institute, Johns Hopkins Kimmel Cancer Center, Baltimore, MD 21231, USA.
authors.forename: Isaac
authors.initials: I
authors.lastname: Kinde
authors.forename: Jian
authors.initials: J
authors.lastname: Wu
authors.forename: Nick
authors.initials: N
authors.lastname: Papadopoulos
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Kinzler
authors.forename: Bert
authors.initials: B
authors.lastname: Vogelstein
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D051176:beta Catenin / Q000235:genetics
chemical_list: C495270:CTNNB1 protein, human; D051176:beta Catenin