Detection of low-frequency mutations and identification of heat-induced artifactual mutations using duplex sequencing

Eun Hyun Ahn, Seung Hyuk Lee

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


We present a genome-wide comparative and comprehensive analysis of three different sequencing methods (conventional next generation sequencing (NGS), tag-based single strand sequencing (e.g., SSCS), and Duplex Sequencing for investigating mitochondrial mutations in human breast epithelial cells. Duplex Sequencing produces a single strand consensus sequence (SSCS) and a duplex consensus sequence (DCS) analysis, respectively. Our study validates that although high-frequency mutations are detectable by all the three sequencing methods with the similar accuracy and reproducibility, rare (low-frequency) mutations are not accurately detectable by NGS and SSCS. Even with conservative bioinformatical modification to overcome the high error rate of NGS, the NGS frequency of rare mutations is 7.0 × 10−4. The frequency is reduced to 1.3 × 10−4 with SSCS and is further reduced to 1.0 × 10−5 using DCS. Rare mutation context spectra obtained from NGS significantly vary across independent experiments, and it is not possible to identify a dominant mutation context. In contrast, rare mutation context spectra are consistently similar in all independent DCS experiments. We have systematically identified heat-induced artifactual variants and corrected the artifacts using Duplex Sequencing. Specific sequence contexts were analyzed to examine the effects of neighboring bases on the accumulation of heat-induced artifactual variants. All of these artifacts are stochastically occurring rare mutations. C > A/G > T, a signature of oxidative damage, is the most increased (170-fold) heat-induced artifactual mutation type. Our results strongly support the claim that Duplex Sequencing accurately detects low-frequency mutations and identifies and corrects artifactual mutations introduced by heating during DNA preparation.

Original languageEnglish (US)
Article number199
JournalInternational journal of molecular sciences
Issue number1
StatePublished - Jan 1 2019
Externally publishedYes


  • Duplex consensus sequence (DCS)
  • Duplex sequencing
  • Heat-induced mutations
  • Human breast cells
  • Mitochondrial dna
  • Next-Generation sequencing (NGS)
  • Oxidative DNA damage
  • Rare mutations
  • Sequencing error
  • Single strand consensus sequence (SSCS)

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Spectroscopy
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry


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