Deamination Effects in Formalin-Fixed, Paraffin-Embedded Tissue Samples in the Era of Precision Medicine Seokhwi Kim, Charny Park, Yongick Ji, Deok G.

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Deamination Effects in Formalin-Fixed, Paraffin-Embedded Tissue Samples in the Era of Precision Medicine Seokhwi Kim, Charny Park, Yongick Ji, Deok G. Kim, Hyunsik Bae, Michael van Vrancken, Duk-Hwan Kim, Kyoung-Mee Kim The Journal of Molecular Diagnostics Volume 19, Issue 1, Pages 137-146 (January 2017) DOI: 10.1016/j.jmoldx.2016.09.006 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Allele frequency and variant counts over sample count in samples. A: Relative allele frequency (y axis) of all possible nucleotide changes (x axis) in sample 1 through sample 6. Allele frequency of C:G>T:A transition in the normal samples 1 to 6 is clearly lower than that of the others. Most homozygous or heterozygous variant allele frequency from the normal samples is close to 0.5 or 1. However, the mean allele frequency of our samples' C:G>T:A transitions is <0.4, suggesting that C:G>T:A transition allele frequency includes artifacts before uracil DNA glycosylase (UDG) treatment. B: Variant counts divided by total sample counts for each nucleotide change. The Journal of Molecular Diagnostics 2017 19, 137-146DOI: (10.1016/j.jmoldx.2016.09.006) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Normalized mean coverage and 95% CI of the normal gastric tissue in sample 2 with various fixation conditions. D1, D2, D3, D4, and D5 indicate formalin fixation for days 1, 2, 3, 4, and 5, respectively. The Journal of Molecular Diagnostics 2017 19, 137-146DOI: (10.1016/j.jmoldx.2016.09.006) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Comparison of allele frequency before and after uracil DNA glycosylase (UDG) treatment. A and B: Result of t-test. C and D: Allele frequency of sample 2 and samples 3 to 6 (blue, pre-UDG treatment; red, post-UDG treatment). E and F: C:G>T:A count of sample 2 and samples 3 to 6 (blue, pre-UDG treatment; red, post-UDG treatment). Transition counts of sample 2 are summation of all 21 experimental conditions with different fixation times and pH conditions. The Journal of Molecular Diagnostics 2017 19, 137-146DOI: (10.1016/j.jmoldx.2016.09.006) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Normalized depth and single-nucleotide variant count of frequently identified genes before and after uracil DNA glycosylase (UDG) treatment for samples 2 to 6 (blue, pre-UDG treatment; red, post-UDG treatment). A and C: C:G>T:A changes. B and D: Other nucleotide changes, except C:G>T:A changes. The Journal of Molecular Diagnostics 2017 19, 137-146DOI: (10.1016/j.jmoldx.2016.09.006) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S1 Histogram of allele frequency in samples 1 and 2 with various fixation conditions. When calculating bayesian probability in samples 1 and 2, we set a quartile (25%, red line) as the low allele frequency threshold. The Journal of Molecular Diagnostics 2017 19, 137-146DOI: (10.1016/j.jmoldx.2016.09.006) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S2 Frequency distribution of root mean square (RMS) mapping quality according to filtering conditions for all samples (samples 1 to 6). Frequencies of C:G>T:A RMS mapping quality without (A) and with (B) filtering. Frequencies of other nucleotide changes without (C) and with (D) filtering. The Journal of Molecular Diagnostics 2017 19, 137-146DOI: (10.1016/j.jmoldx.2016.09.006) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions