TY - JOUR
T1 - Detection of low-frequency mutations and identification of heat-induced artifactual mutations using duplex sequencing
AU - Ahn, Eun Hyun
AU - Lee, Seung Hyuk
N1 - Funding Information:
This research was funded by grants from the National Institute of Environmental Health Sciences (NIEHS) P30 ES007033 sponsored-University of Washington (UW) Center for Exposures, Diseases, Genomics and Environment (EDGE) pilot grant (to EH Ahn), UW Office of Research Royalty Research Fund (to EH Ahn), and the National Cancer Institute (NCI) P30 CA015704-39 Fred Hutchinson Cancer Research Center-UW Cancer Consortium Support Grant (to EH Ahn), NCI of the National Institutes of Health (NIH) under award number R21 CA220111 (to EH Ahn), and NCI of NIH under award numbers P01 AG001751 and R33 CA181771 (to LA Loeb). The content is solely the responsibility of the authors, and does not necessarily represent the official views of NIH.
Funding Information:
Funding: This research was funded by grants from the National Institute of Environmental Health Sciences (NIEHS) P30 ES007033 sponsored-University of Washington (UW) Center for Exposures, Diseases, Genomics and Environment (EDGE) pilot grant (to EH Ahn), UW Office of Research Royalty Research Fund (to EH Ahn), and the National Cancer Institute (NCI) P30 CA015704-39 Fred Hutchinson Cancer Research Center-UW Cancer Consortium Support Grant (to EH Ahn), NCI of the National Institutes of Health (NIH) under award number R21 CA220111 (to EH Ahn), and NCI of NIH under award numbers P01 AG001751 and R33 CA181771 (to LA Loeb). The content is solely the responsibility of the authors, and does not necessarily represent the official views of NIH.
Publisher Copyright:
© 2019 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2019/1/1
Y1 - 2019/1/1
N2 - 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.
AB - 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.
KW - Duplex consensus sequence (DCS)
KW - Duplex sequencing
KW - Heat-induced mutations
KW - Human breast cells
KW - Mitochondrial dna
KW - Next-Generation sequencing (NGS)
KW - Oxidative DNA damage
KW - Rare mutations
KW - Sequencing error
KW - Single strand consensus sequence (SSCS)
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UR - http://www.scopus.com/inward/citedby.url?scp=85059796001&partnerID=8YFLogxK
U2 - 10.3390/ijms20010199
DO - 10.3390/ijms20010199
M3 - Article
C2 - 30625989
AN - SCOPUS:85059796001
SN - 1661-6596
VL - 20
JO - International journal of molecular sciences
JF - International journal of molecular sciences
IS - 1
M1 - 199
ER -