Detection of Low-Frequency Mutations and Identification of Heat-Induced Artifactual Mutations Using Duplex Sequencing.

Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE

Original Publication: Basel, Switzerland : MDPI, [2000-

Hot Temperature*; High-Throughput Nucleotide Sequencing/*methods ; Point Mutation/*genetics; Adult ; Artifacts ; Cell Line ; Consensus Sequence ; DNA, Mitochondrial/genetics ; Genome, Mitochondrial ; Humans ; Reproducibility of Results ; Stochastic Processes ; Young Adult

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.

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P01 AG001751 United States CA NCI NIH HHS; P01 CA077852 United States CA NCI NIH HHS; P30 ES007033 United States ES NIEHS NIH HHS; R21 CA220111 United States CA NCI NIH HHS; . UW Office of Research Royalty Research Fund; R33 CA181771 United States CA NCI NIH HHS; P30 CA015704-39 United States CA NCI NIH HHS

Keywords: 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)

0 (DNA, Mitochondrial)

Date Created: 20190111 Date Completed: 20190426 Latest Revision: 20231005

20231215

PMC6337315

10.3390/ijms20010199

30625989