Impact of Mutation Type and Amplicon Characteristics on Genetic Diversity Measures Generated Using a High-Resolution Melting Diversity Assay  Matthew M. Cousins, Deborah Donnell, Susan H. Eshleman  The Journal of Molecular Diagnostics  Volume 15, Issue 1, Pages 130-137 (January 2013) DOI: 10.1016/j.jmoldx.2012.08.008 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Regions of the HIV genome analyzed using the HRM diversity assay. The relevant regions of the HIV genome are shown. Numbers at the ends of each genomic segment correspond to coordinates in HXB2 (GenBank accession number K03455). The amplicons used for HRM diversity analysis (regions analyzed) are indicated by shaded boxes. A: Gag amplicons: The GAG3 amplicon contains a portion of the coding region of gag p24. The GAG1 amplicon includes a portion of the coding regions for gag p7 and gag p1. The GAG2 amplicon includes the coding regions for gag p1 and gag p6 and extends into the coding region for HIV protease; this amplicon also corresponds to the transframe protein. B: Pol amplicons: The POL amplicon spans the junction between the coding regions of HIV protease and HIV reverse transcriptase. The POL2 and POL3 amplicons each include a portion of the region that codes the integrase enzyme. C: Env amplicons: The ENV4 and ENV5 amplicons each encompass the V4 and V5 hypervariable loop regions of gp120, respectively. The ENV1 amplicon includes the coding region for heptad repeat 1 (HR1) of gp41 and portions of the coding regions to either side of HR1. The ENV2 amplicon includes the coding region for immunodominant region (IDR) cluster I of gp41 and portions of the coding regions for HR1 and HR2.24 The ENV3 amplicon includes the coding region for heptad repeat 2 (HR2) and portions of the coding regions to either side of HR2. D: Nef amplicon: The NEF amplicon includes a portion of the region that codes the nef accessory protein. Figure adapted from Kuiken et al (Eds.).23 The Journal of Molecular Diagnostics 2013 15, 130-137DOI: (10.1016/j.jmoldx.2012.08.008) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 The impact of mutation type on the ability to detect a mutation in a mixed population, including WT and mutant amplicons. Pure plasmid populations (Pure; n = 21; consisting of each WT, single-base mutant plasmid, and insertion/deletion mutant plasmid from each of the three regions as pure populations) and plasmid mixtures containing single-base changes (SBC; n = 18), 1-bp deletions (1 bp Del; n = 3), 3-bp insertions (3 bp Ins; n = 3), and 9-bp insertions (9 bp Ins; n = 3) were evaluated. A: Different types of mutations had varied impacts on the HRM score. Region-specific differences in the sequence surrounding the mutations influenced HRM score. B: The impact of mutations on HRM scores was expressed as the difference between the HRM score for the mixed population and the weighted average of HRM scores for each plasmid in a mixture when the plasmids were assayed as pure populations (difference, see Materials and Methods). Circle, GAG; square, ENV; triangle, POL. The Journal of Molecular Diagnostics 2013 15, 130-137DOI: (10.1016/j.jmoldx.2012.08.008) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 The impact of amplicon length on HRM score. The impact of amplicon length on difference in HRM score is shown for the following mutations: single-base changes (n = 18) (A), 1-bp deletions (n = 3) (B), 3-bp insertions (n = 3) (C), and 9-bp insertions (n = 3) (D). Circle, GAG; square, ENV; triangle, POL. The Journal of Molecular Diagnostics 2013 15, 130-137DOI: (10.1016/j.jmoldx.2012.08.008) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 The impact of mutation location on HRM score. The impact of mutation location (the position of the mutation within the amplicon) on the ability to detect HRM score differences is shown for the following mutations: single-base changes (n = 18) (A), 1-bp deletions (n = 3) (B), 3-bp insertions (n = 3) (C), and 9-bp insertions (n = 3) (D). Circle, GAG; square, ENV; triangle, POL. The Journal of Molecular Diagnostics 2013 15, 130-137DOI: (10.1016/j.jmoldx.2012.08.008) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 The impact of mutant plasmid concentration on HRM score. The impact of mutant plasmid concentration on the ability to detect HRM score differences is shown for the following mutations: single-base changes (n = 9) (A), 1-bp deletions (n = 3) (B), 3-bp insertions (n = 3) (C), and 9-bp insertions (n = 3) (D). Circle, GAG; square, ENV; triangle, POL. The Journal of Molecular Diagnostics 2013 15, 130-137DOI: (10.1016/j.jmoldx.2012.08.008) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 The impact of G/C content on duplex melting. The impacts of amplicon G/C content on duplex melting are shown: T1 (P < 0.0001; R2 = 0.7964, y = 64.8175 + 0.4313x) (A), T2 (P < 0.0001; R2 = 0.8551, y = 68.1143 + 0.4524x) (B), peak Tm (P < 0.0001; R2 = 0.8518, y = 67.1294 + 0.4295x) (C), and HRM score (P = 0.0955; R2 = 0.0212, y = 3.2968 + 0.0211x) (D). These values are obtained by analyzing 12 amplicons for each of the 11 plasmids. The HIV-derived plasmid inserts consist of nearly complete HIV genomes. The Journal of Molecular Diagnostics 2013 15, 130-137DOI: (10.1016/j.jmoldx.2012.08.008) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions