Multiplexed High Resolution Melting Assay for Versatile Sample Tracking in a Diagnostic and Research Setting Céline Helsmoortel, R. Frank Kooy, Geert.

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Multiplexed High Resolution Melting Assay for Versatile Sample Tracking in a Diagnostic and Research Setting Céline Helsmoortel, R. Frank Kooy, Geert Vandeweyer The Journal of Molecular Diagnostics Volume 18, Issue 1, Pages 32-38 (January 2016) DOI: 10.1016/j.jmoldx.2015.06.011 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Melting curves of the eight different high resolution melting (HRM) multiplexes for cohort 2 (34 SeqCap Choice samples). The specific single-nucleotide polymorphisms (SNPs) assayed in every multiplex are listed below the melting curves, in the order of the peaks. The Journal of Molecular Diagnostics 2016 18, 32-38DOI: (10.1016/j.jmoldx.2015.06.011) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Example set-up of one multiplex high resolution melting (HRM) reaction (SNP 12 – 14 – 19) with eight selected control samples representing every status of each single-nucleotide polymorphism (SNP) (homozygous reference, heterozygous, homozygous alternative). All three genotypes can be derived after subsequent application of the respective normalization temperatures (listed in Table 3). The Journal of Molecular Diagnostics 2016 18, 32-38DOI: (10.1016/j.jmoldx.2015.06.011) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Experimental and simulated (1000×) discriminative power distribution per data set of up to 40 samples [Experimental: discriminative power (DP), 7.8 ± 1.6; Simulation: DP = 8.3 ± 1.4; A] and 213 samples (Experimental: DP = 6.3 ± 1.4, Simulation: DP = 6.9 ± 1.3; B). A: The experimental data set was retrieved from analyzing the genotypes per experiment (of up to 40 samples) for the three different cohorts. B: All 213 experimental samples from the three cohorts were analyzed together. The left and right y axis represents the number of samples with a specific discriminative power for the experimental (black bars) and simulated (gray bars) samples, respectively. The Journal of Molecular Diagnostics 2016 18, 32-38DOI: (10.1016/j.jmoldx.2015.06.011) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S1 Proposed experimental design for a complete analysis of all eight multiplex HRM assays for 40 samples and eight control samples in a 384-well plate. The Journal of Molecular Diagnostics 2016 18, 32-38DOI: (10.1016/j.jmoldx.2015.06.011) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions