HRM analysis for hotspot mutations of IDH2 and BRAF.

<p>First column: Sequence-wild-type DNA results, including no heteroduplex-derived peak in either derivative curve. Second column: Clear heteroduplex-derived peaks are seen in both derivative curves of the sequence-mutant DNA. C) Distribution plots of HRM-MI for 52 DNA samples analyzed for <i>BRAF</i>^<i>V600E</i>. HRM-MI values completely match the sequence results.</p

Primer sequences for high resolution melting analysis.

<p>Primer sequences for high resolution melting analysis.</p

Discrepant results between a duplicate HRM analyses.

<p>Difference plots of discriminated wild-type calls and variant (i.e., mutant) calls from the first (left upper) and second (right upper) 96 runs, which are displayed as light blue and light red curves, respectively. A representative discrepancy for a duplicate HRM analysis, i.e., one with a wild-type call in the first run and a variant call in the second run, is shown as a black curve in both difference plots. Negative derivative curves of this discrepancy were similar to those of less interpretable plots (left lower, first run; right lower, second run).</p

HRM-MI assay.

<p>A) Representation of the HRM-Mutation Index (HRM-MI), which is defined as the difference between the low-temperature melting transition and high-temperature melting transition. B) Representative -<i>d</i>² curves of mutated and wild-type DNA samples. The <i>x</i>-axis indicates the position relative to <i>Tm</i>. A single asterisk shows <i>Z</i>_HTMT, and double asterisk shows <i>Z</i>_LTMT. Mutated DNA samples presented positive values of HRM-MIs owing to heteroduplex-derived peaks (upper graph). Conversely, wild-type DNA samples showed simple ascending curves between <i>Z</i>_HTMT and <i>Z</i>_LTMT, and negative HRM-MI values (lower graph). The colors represent different samples. C) Distribution plots of HRM-MI for 192 DNA samples analyzed for <i>IDH1</i>^<i>R132</i>. All-HRM-MI values of sequence-mutant (seq-mut) DNA samples were distributed in the positive range (black plots). The corresponding values for all wild-type (seq-wt) DNA samples were negatively dispersed (red plots), except for six values (blue plots) obtained from the duplicated results of three DNA samples.</p

Differential calculus analysis of HRM data.

<p>A) Representative results of differential calculus analyses of HRM for <i>IDH1</i>^<i>R132</i>. First row: Sanger sequencing; second row: fluorescence intensity curve; third row: -<i>d</i>¹ curve; fourth row: -<i>d</i>² curve. First column: A result of sequence-wild type DNA containing no heteroduplex-derived peaks in either the -<i>d</i>¹ or -<i>d</i>² curves. Second column: A result of sequence-mutant DNA. Whereas the heteroduplex-derived peak is recognized in the -<i>d</i>¹ curve as a slight change of shape, the -<i>d</i>² curve demonstrates a more distinct peak formation. B) Distributions of run-to-run variability in the low-temperature melting transition (LTMT) (blue) and high-temperature melting transition (HTMT) (red) positions from <i>Tm</i>. Bars indicate the number of observations within the bins of width 0.01°C. The curved black line shows the approximate normal distributions of LTMT and HTMT, in which the standard deviations were 0.076°C and 0.048°C, respectively.</p

HRM analysis using different amplicon lengths.

<p>Multiple amplicon lengths (90, 129, and 212 bp) were tested for mutation analyses in order to optimize the HRM analysis. First row: -<i>d</i>¹ curve; second row: -<i>d</i>² curve. The 90-bp amplicon showed the most interpretable heteroduplex-derived peaks.</p

Analysis of IDH1 mutation in an inappropriate tissue sample.

<p>Analysis of a tumor tissue showing a sampling discrepancy. Whereas wild-type calls were obtained by SS and conventional HRM for the first sampling, the mutation was detected using our present method based on a heteroduplex-derived curve that presented a positive HRM-MI value (first column). The second sampling confirmed the mutation, with consistent results among the three approaches (second column).</p

HRM analysis using mixed samples.

<p>A) Distribution plots of HRM-MI results obtained using an HRM analysis with mixed samples. The <i>x</i>-axis shows the fraction of tumor DNA in the mixture. The <i>y</i>-axis shows the HRM-MI value. Each dot represents the HRM-MI value for each assay, performed with multiple replicate across the 6 different mixing ratios. Green and blue lines indicate mean and SD HRM-MI values, respectively. B) Mutation detection ratios for HRM-MI (red line) and conventional HRM analyses (blue line). At a mixing ratio of 20% (i.e., 20% tumor DNA and 80% nontumor DNA), the sensitivities of the conventional HRM method and HRM-MI were 30% and 100%, respectively. C) The representative -<i>d</i>² curves of samples with tumor-to-nontumor DNA ratios. Even the 10% tumor DNA sample presented an interpretable heteroduplex-derived peak.</p