The identification of mutations that are present in a small fraction of 
DNA templates is essential for progress in several areas of biomedical research. Though 
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. One example of this approach, called “Safe-SeqS” for (Safe-Sequencing 
System) includes (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 truly 
mutant (“super-mutants”) 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.