Impact of Mutation Type and Amplicon Characteristics on Genetic Diversity Measures Generated Using a High-Resolution Melting Diversity Assay

Abstract

We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutation type, position, and concentration on HRM scores. The impact of amplicon length and G/C content on HRM scores was also evaluated. Different mutation types affected HRM scores to varying degrees (1-bp deletion< 1-bp change < 3-bp insertion < 9-bp insertion). The impact of mutations on HRM scores was influenced by amplicon length and the position of the mutation within the amplicon. Mutations were detected at concentrations of 5% to 95%, with the greatest impact at 50%. The G/C content altered melting temperature values of amplicons but had no impact on HRM scores. These data are relevant to the design of assays that measure genetic diversity using HRM technology.

HIV Diversity as a Biomarker for HIV Incidence Estimation: Including a High-Resolution Melting Diversity Assay in a Multiassay Algorithm

“…The HRM diversity assay was performed as previously described (12,16) using the LightScanner HR-1 (BioFire Diagnostics, Inc., Salt Lake City, UT). Briefly, a region of the HIV genome is amplified, followed by nested amplification of a smaller region in the presence of a fluorescent duplex-dependent DNA dye (LCGreen Plus, BioFire Diagnostics, Inc.).…”

“…Of the 114 samples from the HIVNET 001 cohort, 97 samples were from men (95 MSM, 2 injection drug users) and 17 samples were from women (including injection drug users). The HRM diversity assay was performed for eight regions of the HIV genome (GAG1, GAG2, POL, ENV1, ENV2, ENV3, ENV4, and ENV5) (16) for each of the 124 samples (for a total of 992 diversity measures). A higher proportion of samples failed HRM diversity assay analysis for the ENV4 and ENV5 regions (see Table S1 in the supplemental material); therefore, the composite variable (6-region HRM score sum ϭ sum of HRM scores from the GAG1, GAG2, POL, ENV1, ENV2, and ENV3 regions) did not include HRM scores from those two regions.…”

“…This assay is based on high-resolution melting (HRM) and can be used to measure HIV diversity in different regions of the HIV genome. The level of genetic diversity in each region is reported as a single numeric HRM score that corresponds to the width of the DNA melting peak (12,(15)(16)(17). HRM scores correlate highly with diversity measures obtained from next-generation sequencing data (18).…”

Use of a High Resolution Melting Assay to Analyze HIV Diversity in HIV-infected Ugandan Children

“…In the HRM diversity assay, more diverse DNA populations (e.g., amplicons from more diverse HIV populations in clinical samples) melt over a broader range of temperatures, which is reflected in higher HRM scores. Our recent work demonstrates that the HRM diversity assay can detect single base mutations that are present at levels as low as 5% in plasmid mixtures, and can also detect insertions and deletions at low levels [7]. The HRM diversity assay is highly reproducible [8], and HRM scores are significantly associated with sequence-based measures of HIV diversity obtained from analysis of cloned HIV DNA [6] and next generation sequencing [9].…”

Analysis of HIV Diversity in HIV-Infected Black Men Who Have Sex with Men (HPTN 061)

“…We developed a high resolution melting (HRM) diversity assay that quantifies viral diversity without sequencing; the level of genetic diversity is reported as a single numeric HRM score [13,14]. A previous study provides detailed information about the impact of mutations on HRM scores [15]. In a separate study that included analysis of HIV env and gag diversity in 220 samples, HRM scores were closely correlated with sequence-based diversity measures (genetic diversity, genetic complexity, and Shannon entropy) obtained by analyzing data from next-generation sequencing [16].…”