Abstract
Background and Objectives
As next-generation sequencing (NGS) becomes a major sequencing platform in clinical diagnostic laboratories, it is critical to identify artifacts that constitute baseline noise and may interfere with detection of low-level gene mutations. This is especially critical for applications requiring ultrasensitive detection, such as molecular relapse of solid tumors and early detection of cancer. We recently observed a ~10-fold higher frequency of C:G > T:A mutations than the background noise level in both wild-type peripheral blood and formalin-fixed paraffin-embedded samples. We hypothesized that these might represent cytosine deamination events, which have been seen using other platforms.
Methods
To test this hypothesis, we pretreated samples with uracil N-glycosylase (UNG). Additionally, to test whether some of the cytosine deamination might be a laboratory artifact, we simulated the heat associated with polymerase chain reaction thermocycling by subjecting samples to thermocycling in the absence of polymerase. To test the safety of universal UNG pretreatment, we tested known positive samples treated with UNG.
Results
UNG pretreatment significantly reduced the frequencies of these mutations, consistent with a biologic source of cytosine deamination. The simulated thermocycling-heated samples demonstrated significantly increased frequencies of C:G > T:A mutations without other baseline base substitutions being affected. Samples with known mutations demonstrated no decrease in our ability to detect these after treatment with UNG.
Conclusion
Baseline noise during NGS is mostly due to cytosine deamination, the source of which is likely to be both biologic and an artifact of thermocycling, and it can be reduced by UNG pretreatment.
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Acknowledgments
The authors wish to acknowledge Dr. James Stivers (Johns Hopkins University School of Medicine) for helpful discussions. Grant support was received from the Women’s Board of The Johns Hopkins Hospital (to Ming-Tseh Lin) and through National Institutes of Health grant numbers R21HG005745 (to Christopher D. Gocke) and R21CA164592 (to James R. Eshleman), and the Pancreatic Cancer Action Network Innovation Award (to James R. Eshleman).
Conflicts of Interest
Guoli Chen, Stacy Mosier, Christopher D. Gocke, Ming-Tseh Lin, and James R. Eshleman have no conflicts of interest that are directly relevant to the content of this study.
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Chen, G., Mosier, S., Gocke, C.D. et al. Cytosine Deamination Is a Major Cause of Baseline Noise in Next-Generation Sequencing. Mol Diagn Ther 18, 587–593 (2014). https://doi.org/10.1007/s40291-014-0115-2
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DOI: https://doi.org/10.1007/s40291-014-0115-2