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. 2020 Sep 22;20:902. doi: 10.1186/s12885-020-07411-1

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 7 — Difference plot depicting the different concentrations of Ex21_std at 2.5 ng/μl & 0.25 ng/μl. a and b depicts HRM assays represented as difference plot, dF/dT (y-axis) is plotted against melting temperature range (x-axis). a shows peaks with distinct colors annotated to different MAF, and b shows corresponding peaks detected as mutant or wildtype by HRM. Samples in both a & b have a total DNA concentration of 2.5 ng/μl. MAF annotations in a are Blue (1%), Pink (0.5%), Red (0.25%), Green (0.125%), and Grey (wildtype). b shows the detection of mutation (Red) or wildtype (Blue) by HRM. Note that the HRM detected mutants consistently (as red peak in b) in blue (1%), pink (0.5%) and red (0.25%) peaks in a. c & d have a DNA concentration of 0.25 ng/μl. c shows the difference plot depicting different MAF of Ex21_std as Blue (10%), Pink (5%), Red (2.5%) and Green (wild-type). d depicts the corresponding MAF as variant (Red) or wildtype (Blue). Note that the HRM consistently identified as variants (as red peak in d) in only blue peak (10% MAF)