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      Detection of ultra-rare mutations by next-generation sequencing.

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          Abstract

          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 ~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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          Analysis of genetic inheritance in a family quartet by whole-genome sequencing.

          We analyzed the whole-genome sequences of a family of four, consisting of two siblings and their parents. Family-based sequencing allowed us to delineate recombination sites precisely, identify 70% of the sequencing errors (resulting in > 99.999% accuracy), and identify very rare single-nucleotide polymorphisms. We also directly estimated a human intergeneration mutation rate of approximately 1.1 x 10(-8) per position per haploid genome. Both offspring in this family have two recessive disorders: Miller syndrome, for which the gene was concurrently identified, and primary ciliary dyskinesia, for which causative genes have been previously identified. Family-based genome analysis enabled us to narrow the candidate genes for both of these Mendelian disorders to only four. Our results demonstrate the value of complete genome sequencing in families.
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            Author and article information

            Journal
            Proc Natl Acad Sci U S A
            Proceedings of the National Academy of Sciences of the United States of America
            Proceedings of the National Academy of Sciences
            1091-6490
            0027-8424
            Sep 04 2012
            : 109
            : 36
            Affiliations
            [1 ] Departments of Pathology, Genome Sciences, and Biochemistry, University of Washington School of Medicine, Seattle, WA 98195, USA.
            Article
            1208715109
            10.1073/pnas.1208715109
            3437896
            22853953
            c5e99318-c768-4f2e-afa3-fd50828eca12
            History

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