Detecting ultralow-frequency mutations by Duplex Sequencing

Scott R. Kennedy, Michael W. Schmitt, Edward J. Fox, Brendan F. Kohrn, Jesse J. Salk, Eun Hyun Ahn, Marc J. Prindle, Kawai J. Kuong, Jiang Cheng Shen, Rosa Ana Risques, Lawrence A. Loeb

Research output: Contribution to journalArticlepeer-review

190 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)2586-2606
Number of pages21
JournalNature protocols
Issue number11
StatePublished - Nov 27 2014
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)


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