Methods for the Analysis of High Resolution Melt Curve Data

Abstract

The present application provides for various embodiments of methods for the analysis of high resolution melt (HRM) curve data; where statistical assay variations in melt curve data may result from system noise in an analysis system. Such system noise may arise from various sources, such as the thermal non-uniformity of a thermocycler block in a thermal cycler apparatus, a detection system, etc. Additionally, various methods for the analysis of HRM curve data may provide an identification of a sample without the need for a user inputted information.

1 . A method for analyzing melt curve data, the method comprising:
 providing melt curve data for at least one test sample deposited in a plurality of support regions of a sample support device in thermal cycler system, wherein the melt curve data is an experimental set of melt curve data; and   selecting a weighting function, wherein the weighing function selected is used for the construction of a dendrogram of the corrected experimental set of melt curve data;   constructing a dendrogram of the corrected experimental set of melt curve data, wherein the dendrogram creates a set of at least one cluster from the corrected experimental set of melt curve data; and   determining a cut level of the dendrogram, wherein the cut level determines a final number of clusters from the set of at least one cluster.   
     
     
         2 . The method of  claim 1 , wherein the at least one test sample is a plurality of test samples. 
     
     
         3 . The method of  claim 2 , wherein the weighting function utilizes all data points in each of a corrected experimental set of melt curve data for each test sample. 
     
     
         4 . The method of  claim 2 , further comprising:
 providing melt curve data for a calibration sample deposited in a plurality of support regions of a sample support device in a thermal cycler system, wherein the melt curve data is a calibration set of melt curve data; and   correcting the experimental set of melt curve data using the calibration set of melt curve data.   
     
     
         5 . The method of  claim 4 , wherein the correction is done on a derivative form of the melt curve data. 
     
     
         6 . The method of  claim 4 , wherein the correction of the experimental set of melt curve data using the calibration set of melt curve data is a correction for assay system noise. 
     
     
         7 . The method of  claim 6 , wherein the source of assay system noise is thermal non-uniformity. 
     
     
         8 . The method of  claim 6 , wherein the source of assay system noise is excitation source non-uniformity. 
     
     
         9 . The method of  claim 6 , wherein the source of assay system noise is detection noise. 
     
     
         10 . The method of  claim 2 , further comprising the step of scaling the corrected experimental set of melt curve data over an estimated temperature range. 
     
     
         11 . The method of  claim 10 , further comprising the step of fitting the scaled corrected experimental set of melt curve data to an estimated asymptote for a low temperature region of a melting region of the melt curve data.