Analysis of HIV Using a High Resolution Melting (HRM) Diversity Assay: Automation of HRM Data Analysis Enhances the Utility of the Assay for Analysis of HIV Incidence

<div><h3>Background</h3><p>HIV diversity may be a useful biomarker for discriminating between recent and non-recent HIV infection. The high resolution melting (HRM) diversity assay was developed to quantify HIV diversity in viral populations without sequencing. In this assay, HIV diversity is expressed as a single numeric HRM score that represents the width of a melting peak. HRM scores are highly associated with diversity measures obtained with next generation sequencing. In this report, a software package, the HRM Diversity Assay Analysis Tool (DivMelt), was developed to automate calculation of HRM scores from melting curve data.</p> <h3>Methods</h3><p>DivMelt uses computational algorithms to calculate HRM scores by identifying the start (T1) and end (T2) melting temperatures for a DNA sample and subtracting them (T2–T1 = HRM score). DivMelt contains many user-supplied analysis parameters to allow analyses to be tailored to different contexts. DivMelt analysis options were optimized to discriminate between recent and non-recent HIV infection and to maximize HRM score reproducibility. HRM scores calculated using DivMelt were compared to HRM scores obtained using a manual method that is based on visual inspection of DNA melting curves.</p> <h3>Results</h3><p>HRM scores generated with DivMelt agreed with manually generated HRM scores obtained from the same DNA melting data. Optimal parameters for discriminating between recent and non-recent HIV infection were identified. DivMelt provided greater discrimination between recent and non-recent HIV infection than the manual method.</p> <h3>Conclusion</h3><p>DivMelt provides a rapid, accurate method of determining HRM scores from melting curve data, facilitating use of the HRM diversity assay for large-scale studies.</p> </div

DivMelt Graphic User Interface (GUI).

<p>DivMelt displays various options when the package is opened. (A) The initial GUI window displays the “File Menu,” “Settings Menu,” and “Main Menu.” Sub-windows relating to file management may be opened from the “File Menu” window. These include (B) the “Input Options” window and (C) the “Output Options” window. From the “Settings Menu” window, additional options can be selected. These include (D) the “Plotting Options” window, (E) the “Analysis Options” window, and (F) the “Show Settings” window. The “Main Menu” is used to initiate analyses. Clicking “Run Analysis” opens the “Review Settings” window (similar to F). See the user manual for a description of the various tools accessible from the GUI. At the left of each row for items B–E, the “<i>i</i>” may be clicked to open a comment box that describes the function of the tool in question. These windows are shown as they display in Windows 7. Note that the windows will display slightly differently in other operating systems.</p

Correlation between HRM scores calculated with the optimal DivMelt analysis protocols and HRM scores calculated using the manual method ^a.

a<p>Pearson’s correlation coefficient with manual HRM score. The region used to optimize each protocol is shown in the column on the left; the regions analyzed are shown in the headers for the six other columns.</p>b<p>Detailed region-specific analysis protocol descriptions are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051359#pone-0051359-t001" target="_blank">Table 1</a>.</p>c<p>HIV genome regions are described in the footnote of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051359#pone-0051359-t001" target="_blank">Table 1</a>.</p

Optimal DivMelt analysis protocols for discrimination between recent and non-recent HIV infection.

<p>Definitions and abbreviations: T1– temperature when melting began; T2– temperature when melting was complete; Theta 1– theta angle (°) for selection of T1; Theta 2– theta angle (°) for selection of T2; HI – hold interval (°C); SW – slope window (°C); SH - Shoulder ht threshold (%) tool (“off” indicates that the shoulder height threshold tool was not in use).</p>a<p>HIV genome regions (amplicons examined) have been described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051359#pone.0051359-Cousins1" target="_blank">[5]</a>. Briefly, the amplicons and the proteins coded by the corresponding HIV genomic regions are as follows: GAG1, p7; GAG2, p6 and transframe; POL, protease and reverse transcriptase; ENV1, HR1 region of gp41; ENV2, immunodominant region of gp41; and ENV3, HR2 region of gp41.</p

Percentage of samples excluded by the internal quality control for each of the optimal DivMelt analysis protocols^a.

a<p>All values are reported as percentages. The region used to optimize each protocol is shown in the column on the left; the regions analyzed are shown in the headers for the six other columns.</p>b<p>Detailed region-specific analysis protocol descriptions are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051359#pone-0051359-t001" target="_blank">Table 1</a>.</p>c<p>HIV genome regions are described in the footnote of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051359#pone-0051359-t001" target="_blank">Table 1</a>.</p

Generation of melting curves, melting peaks, and HRM scores.

<p>Melting curves (top panel) are generated by graphing Fluorescence against Temperature. Fluorescence declines as the DNA melts. DNA melting is visualized through the use of a saturating duplex-dependent DNA intercalating dye (LCGreen Plus). As the DNA melts, the dye is released; unbound dye does not fluoresce. Melting peaks (bottom panel) are generated by taking the negative derivative of Fluorescence with respect to Temperature and graphing these values against Temperature (−dF/dT vs T). T1 and T2 are identified on the melting peak, and these values are used to calculate the HRM score (T2–T1).</p

Methods for selection of T1 and T2 values.

<p>The slope of a line tangent to the melting peak is calculated for each point along the melting peak through the use of a sliding window (slope window). The user can define the width of this window. Each slope is compared with a user-defined Theta 1 value for selection of T1 and Theta 2 value for selection of T2. When the angle meets or exceeds Theta 1, T1 is identified. When the angle meets or exceeds Theta 2, T2 is identified.</p

Fluorescence vs. Temperature and -dFluorescence/Temperature vs. Temperature plots.

<p>DivMelt generates PDF plots to allow visualization of the melting data. The plate file name is given at the top of each plot. Below the file name is the sample location information. This information includes the sample well number (X1; ranging 1–96), the row letter (X2; ranging A–H), and the column designation (X3; ranging 1–12). The sample name is presented in bold above the Fluorescence vs. Temperature plot (top panel). The Fluorescence vs. Temperature plot tracks the decline in fluorescence as the DNA melts in response to the temperature increase. To the right of this panel, the settings used in the analysis are noted. The lower panel consists of the melting peak or –dFluorescence/dT vs. Temperature plot. To the right of the lower panel are the analysis results (marked on the melting peak in equivalent colors). These include the T1 and T2 values (in blue) and the temperature that corresponds to the peak (in purple). Up to three alternative T1 and T2 values may be given if they are present in the analysis (in green). These alternatives are additional values that met the theta value criteria for T1 and T2 identification but were not selected by DivMelt. The HRM score (Score), the diversity output of the assay, is noted between the top and bottom panels of the figure. This value is calculated by subtracting T1 from T2 and corresponds to the peak width.</p

Description of the shoulder height threshold tool.

<p>The shoulder height threshold tool allows selective inclusion or exclusion of small peaks that are connected to the principal melting peak (shoulders). The image indicates the variation in T1 selection based on the specifications of the shoulder height threshold tool and the height of the shoulder.</p

Tests for enrichment of signature sites in biologically-defined subsets compared to all other sites.

<p>For each of the six pre-specified site sets, we compared the number of the 12 vaccine immunogen signature sites in Env that are in the set to the number that are outside of the set, to test whether membership in the set is independent of the “signature site” designation. For example, 7 of the 12 signature sites are in the <i>Hotpots</i> site set, while 89 of the 441 tested non-signature sites are in that set.</p><p>Tests for enrichment of signature sites in biologically-defined subsets compared to all other sites.</p