title: Detecting ultralow-frequency mutations by Duplex Sequencing.
abstract: Duplex Sequencing (DS) is a next-generation sequencing methodology capable of detecting a single mutation among >1 × 10(7) wild-type nucleotides, thereby enabling the study of heterogeneous populations and very-low-frequency genetic alterations. DS can be applied to any double-stranded DNA sample, but it is ideal for small genomic regions of <1 Mb in size. The method relies on the ligation of sequencing adapters harboring random yet complementary double-stranded nucleotide sequences to the sample DNA of interest. Individually labeled strands are then PCR-amplified, creating sequence 'families' that share a common tag sequence derived from the two original complementary strands. Mutations are scored only if the variant is present in the PCR families arising from both of the two DNA strands. Here we provide a detailed protocol for efficient DS adapter synthesis, library preparation and target enrichment, as well as an overview of the data analysis workflow. The protocol typically takes 1-3 d.
authors.affiliation: Department of Pathology, University of Washington, Seattle, USA.
authors.forename: Scott R
authors.initials: SR
authors.lastname: Kennedy
authors.affiliation: Department of Medicine, University of Washington, Seattle, USA.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Schmitt
authors.affiliation: Department of Pathology, University of Washington, Seattle, USA.
authors.forename: Edward J
authors.identifier: 000000023740651X
authors.initials: EJ
authors.lastname: Fox
authors.affiliation: Department of Biology, Portland State University, Portland, Oregon, USA.
authors.forename: Brendan F
authors.initials: BF
authors.lastname: Kohrn
authors.affiliation: Department of Medicine, University of Washington, Seattle, USA.
authors.forename: Jesse J
authors.initials: JJ
authors.lastname: Salk
authors.affiliation: Department of Pathology, University of Washington, Seattle, USA.
authors.forename: Eun Hyun
authors.initials: EH
authors.lastname: Ahn
authors.affiliation: Department of Pathology, University of Washington, Seattle, USA.
authors.forename: Marc J
authors.initials: MJ
authors.lastname: Prindle
authors.affiliation: Department of Pathology, University of Washington, Seattle, USA.
authors.forename: Kawai J
authors.initials: KJ
authors.lastname: Kuong
authors.affiliation: Department of Pathology, University of Washington, Seattle, USA.
authors.forename: Jiang-Cheng
authors.initials: JC
authors.lastname: Shen
authors.affiliation: Department of Pathology, University of Washington, Seattle, USA.
authors.forename: Rosa-Ana
authors.initials: RA
authors.lastname: Risques
authors.affiliation: 1] Department of Pathology, University of Washington, Seattle, USA. [2] Department of Biochemistry, University of Washington, Seattle, USA.
authors.forename: Lawrence A
authors.initials: LA
authors.lastname: Loeb
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D004272:DNA, Mitochondrial; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D059645:Mutation Rate; D016133:Polymerase Chain Reaction / Q000379:methods; D057188:Workflow
chemical_list: D004272:DNA, Mitochondrial