Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 109, Issue 36, Pages 14508-14513Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1208715109
Keywords
cancer; diagnostics; subclone; quasispecies; biomarker
Categories
Funding
- National Institutes of Health [R01 CA115802, R01 CA102029, P01 AG0751, T32 AG000057, F30 AG033485, F30 AG039173]
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Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of similar to 1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when deep sequencing genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage. We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.
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