Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 108, Issue 23, Pages 9530-9535Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1105422108
Keywords
diagnostics; early diagnosis; biomarkers; genetics; cancer
Categories
Funding
- Lustgarten Foundation for Pancreatic Cancer Research
- Virginia and D. K. Ludwig Fund for Cancer Research
- Sol Goldman Center for Pancreatic Cancer Research
- Joseph L. Rabinowitz Fund for Pancreatic Cancer Research
- National Cancer Institute Division of Cancer Prevention [N01-CN-43302]
- National Institutes of Health [CA62924, CA43460, CA57345]
- United Negro College Fund-Merck Graduate Fellowship
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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 templatemolecule, (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.
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