Detection of Low-Frequency Mutations and Identification of Heat-Induced Artifactual Mutations Using Duplex Sequencing
Abstract
:1. Introduction
2. Results
2.1. Homoplasmic Mutations are Detectable by All Three Methods (Conventional NGS, Tag-Based Single Strand Sequencing, and Duplex Sequencing) with the Similar Accuracy and Reproducibility
2.2. Rarely Occurring Mutations are Neither Accurately Detectable by Conventional NGS Methods nor Tag-Based Single Strand DNA Sequencing, but are Accurately Detectable by Duplex Sequencing
2.3. Duplex Sequencing Identifies and Corrects the Heat-Induced Artifactual Variants Introduced During DNA Sample Preparation
2.4. Duplex Sequencing Identifies the Specific Mutation Spectra of Heat-Induced Artifacts
2.5. Independent Experiments of Duplex Sequencing Reproducibly Identify the Heat-Induced Artifactual Mutaiton Profiles
2.6. All Identified Heat-Induced Artifactual Variants are Stochastically Occurring Variants Throughout the Whole Mitochondrial Genome
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. DNA Extraction, Adapter Synthesis, and Library Preparation
4.3. Heat-Induced DNA Damage
4.4. Data Processing
4.4.1. Conventional NGS Datasets and conventional NGS, SSCS and DCS Data Processing
4.4.2. Base Quality and PCR Duplicates
4.4.3. Comparison of Variant Positions
4.4.4. Counting Mutations
4.5. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
DCS | Duplex Consensus Sequence |
DS | Duplex Sequencing |
Mt | Mitochondrial |
NGS | Next-generation Sequencing |
SSCS | Single Strand Consensus Sequence |
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Ahn, E.H.; Lee, S.H. Detection of Low-Frequency Mutations and Identification of Heat-Induced Artifactual Mutations Using Duplex Sequencing. Int. J. Mol. Sci. 2019, 20, 199. https://doi.org/10.3390/ijms20010199
Ahn EH, Lee SH. Detection of Low-Frequency Mutations and Identification of Heat-Induced Artifactual Mutations Using Duplex Sequencing. International Journal of Molecular Sciences. 2019; 20(1):199. https://doi.org/10.3390/ijms20010199
Chicago/Turabian StyleAhn, Eun Hyun, and Seung Hyuk Lee. 2019. "Detection of Low-Frequency Mutations and Identification of Heat-Induced Artifactual Mutations Using Duplex Sequencing" International Journal of Molecular Sciences 20, no. 1: 199. https://doi.org/10.3390/ijms20010199