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Nat Protoc. 2014 Nov;9(11):2586-606. doi: 10.1038/nprot.2014.170. Epub 2014 Oct 9.

Detecting ultralow-frequency mutations by Duplex Sequencing.

Author information

1
Department of Pathology, University of Washington, Seattle, USA.
2
Department of Medicine, University of Washington, Seattle, USA.
3
Department of Biology, Portland State University, Portland, Oregon, USA.
4
1] Department of Pathology, University of Washington, Seattle, USA. [2] Department of Biochemistry, University of Washington, Seattle, USA.

Erratum in

  • Nat Protoc. 2014 Dec;9(12):2903.

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.

PMID:
25299156
PMCID:
PMC4271547
DOI:
10.1038/nprot.2014.170
[Indexed for MEDLINE]
Free PMC Article

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