BACKGROUND

Field of the Invention

The present invention relates to methods for the analysis of nucleic acids present in biological samples, and more specifically to normalize a high resolution melt curve to assist in the identification of one or more properties of the nucleic acids.

Description of Related Art

The detection and identification of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. A number of high throughput approaches to performing polymerase chain reactions (PCR) and other amplification reactions to identify and detect characteristics of nucleic acids have been developed. One example involves amplification reactions in microfluidic devices, as well as methods for detecting and analyzing amplified nucleic acids in or on the devices. These devices enable further characterization of the amplified DNA molecules contained therein. One method of characterizing the DNA is to examine the DNA's dissociation behavior as the DNA transitions from double stranded DNA (dsDNA) to single stranded DNA (ssDNA). The process of causing DNA to transition from dsDNA to ssDNA with increasing temperature is sometimes referred to as a “high-resolution temperature (thermal) melt (HRTm)” process, or simply a “high-resolution melt” process.

Melting profile analysis enables characterization of nucleic acids, e.g. genotyping or determination whether a mutation is present or absent. A sample melting profile is usually obtained by denaturing double stranded nucleic acids into single stranded nucleic acids. To monitor the sample melt, a fluorescent dye that indicates whether the two DNA strands are bound or not is used. Examples of such indicator dyes include non-specific binding dyes such as SYBR® Green I, whose fluorescence efficiency depends strongly on whether the DNA is double stranded or single stranded. As the temperature of the mixture is raised, a reduction in fluorescence from the dye indicates that the nucleic acid molecule has melted, i.e., unzipped, partially or completely. Thus, by measuring the dye fluorescence as a function of temperature, information is gained regarding the length of the duplex, the GC content or even the exact sequence.

Some nucleic acid assays have been designed so that it permits differentiation between different genotypes. Generally, for thermal melt analysis, a simple visual inspection of a thermal melt profile allows determination of the melting temperature of the nucleic acid in the sample. However, this inspection task may be challenging for some nucleic acid assays that have been designed for the identification of a single nucleotide change where the difference in melting temperature (Tm) between the wild type nucleic acid and a mutant nucleic acid is quite small (e.g. less than 0.25° C.). A sample melting curve is generally presented with the fluorescence information on the Y-axis versus the temperature on the X-axis. The negative derivative of melting curve is also frequently derived since providing an ease of determination of the sample characteristic (i.e., genotyping), knowing that each potential genotype is revealed by a unique signature or curve profile. The determination of a sample genotype is usually performed by visually comparing its melting profile with that of reference or baseline samples which genotypes are known. Examples of genotyping classification include identifying the genotype as homozygous wildtype (WT), heterozygous (HET) or homozygous mutant (HOM) depending on the alleles that make up the DNA.

A melting curve of a DNA sequence is obtained by measuring the amount of double-stranded DNA in the sample while the temperature of the product is sequentially increased. In exemplary operation, an amount of double-stranded DNA contained in a biological sample may be obtained during melt processing by measuring a fluorescence level of a dye bounded to double-stranded DNA during the Polymerase Chain Reaction (PCR)/amplification process which precedes the melt processing. The dye molecules bound to the double stranded DNA are actively fluorescing (e.g. emitting detectable light signatures) on the doubled-stranded DNA (dsDNA) and become inactive when the double stranded DNA or portions thereof are denatured into single-stranded DNA (ssDNA). The shape and relative position on the temperature scale of the resulting melting curve provides critical information relative to either the DNA type, or presence or absence of a mutation. To acquire the melting curve, an optical signal emitted by the sample is detected as the temperature of the sample continuously increases. The optical signal may be detected by a photo diode or an imagining system.

DNA melting curves generated on any existing instrument are typically not normalized on the fluorescence scale. The melt curves generated include not only information associated with the DNA, but also information associated on the remaining unused primers, dyes, and other reagents or instrumental component interaction that are necessary for the PCR/amplification of the DNA sample.FIG. 1depicts an exemplary melt curve10along with a non-template control (NTC) curve11. The melt curve10ofFIG. 1illustrates two types of regions based on the slope of the curve. A first type of region12having a slow downward slope is present at low and high temperatures is mostly produced by the melt of the remaining unused primers and other reagents in the product. A second type of region14, shown here between the lines in the central region of the curve presenting a rapid downward slope is mostly revealing the DNA melt component.

Numerous methods have been suggested for identification of background information and subtraction of the background information from a sample melting curve. These methods are often referred as melting curve normalization methods. Conventional curve analysis and workflow are performed so that underlying genotyping of each sample curve be decided upon either in a manual or semi-manual manner. This may typically include a step of normalization followed by a step of comparing/differentiating pairs of melting curves, using either a baseline or well-known reference curve, or simply all possible pairs of melting curves generated for a plate or cartridge. It is sometimes beneficial for the normalization step to determine a melt region in melting curves. Accordingly, there is a need for an algorithm that allows for automatically determining start and end temperatures defining the melt region. After a differentiation step is performed, a classification step follows which assigns a given sample to a certain genotype or classify the sample as having a mutation or not. Furthermore, there is a need for interactive melting curve analysis involving computational operations over a cloud based web application. In addition, there is a need for rapid, alternative methods of determining copy number variations (CNVs) through melting curve analysis.

SUMMARY OF THE INVENTION

The present invention relates to a method and system for determining a copy number variation (CNV) in a genomic nucleic acid test sample comprising a target nucleic acid and a reference nucleic acid. Specifically, a temperature control system is provided to amplify the target nucleic acid and the reference nucleic acid in the test sample to generate a plurality of target and reference amplicons. An optical system is used to measure a DNA melting curve while providing a temperature gradient to the test sample. Specifically, to generate the melting curve, a signal emitted by the test sample is detected by the optical system. Furthermore, a processor is provided to execute instructions stored in memory of a computing device. The instructions include fitting a mathematical model to melting curve data and calculating fitting parameters of the mathematical model. Specifically, the mathematical model describes a plurality of reactions, including target amplicons, reference amplicons, and background melting reactions. The CNV in the test sample is determined based on the fitting parameters calculated for the target and reference amplicons melting reactions.

In one embodiment, the mathematical model is a sum of reaction terms, each of the reaction terms corresponding to a single melting reaction that goes into the sum with a mixture coefficient. The fitting parameters used for determining the CNV in the test sample are the mixture coefficients. In yet another embodiment of the present invention, the CNV in the test sample is determined based on the ratio of a mixture coefficient for the target amplicons melting reaction to a mixture coefficient for the reference amplicons melting reaction. The reference nucleic acid may be a synthetic sequence or a portion of the genomic DNA.

In another aspect of the invention a training is performed to estimate a correspondence of mixture coefficients to CNV. Specifically, for sequences with a known copy number, the mixture coefficients are experimentally obtained and calculated. Then, the distribution of the mixture coefficients given the copy number is estimated along with the likelihood of the observed mixture coefficients given each possible copy number. By way of example and without limitation, the mathematical model may be the Van't Hoff mixture model. In one embodiment, melting curve derivatives having a target melt region and a reference melt region are shifted and rescaled to align all target peaks to a specific target temperature and all reference peaks to a specific reference target temperature. The computing device according to the present invention may be a server communicating with a client computer over a network, wherein the melting curve is communicated to the server through a user interface local to the client computer.

In another aspect of the invention, a method for determining a copy number variation (CNV) in a genomic nucleic acid test sample is provided. The test sample comprises a target nucleic acid and a reference nucleic acid. A temperature control system is used to amplify the target nucleic acid and the reference nucleic acid in the test sample to generate a plurality of target and reference amplicons. An optical system is used to measure a melting curve while providing a temperature gradient to the test sample. Instructions stored in a memory of a computing device are executed by a processor and are directed to shifting and rescaling in temperature the melting curves. Then, difference curves reflecting the difference between the shifted and rescaled melting curves and one or more reference curves having a known copy number are used to determine CNV. The melting curves derivatives having only a reference melt region are shifted to align all reference peaks to a reference target temperature.

