期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 20, 期 1, 页码 -出版社
MDPI
DOI: 10.3390/ijms20010199
关键词
duplex sequencing; duplex consensus sequence (DCS); single strand consensus sequence (SSCS); next-Generation sequencing (NGS); sequencing error; rare mutations; oxidative DNA damage; heat-induced mutations; mitochondrial dna; human breast cells
资金
- National Institute of Environmental Health Sciences (NIEHS) [P30 ES007033]
- UW Office of Research Royalty Research Fund
- National Cancer Institute (NCI) [P30 CA015704-39]
- NCI of the National Institutes of Health (NIH) [R21 CA220111]
- NCI of NIH [P01 AG001751, R33 CA181771]
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 x 10(-4). The frequency is reduced to 1.3 x 10(-4) with SSCS and is further reduced to 1.0 x 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.
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