A method and system for correcting fluorescence spectral crosstalk of DNA

Abstract

The application discloses a DNA fluorescence spectrum crosstalk correction method and system, and aims to solve the fluorescence signal crosstalk problem caused by the multicolor fluorescence parallel electrophoresis technology in genetic analysis. The method obtains the fluorescence signal curves of the first dye and the second dye through a spectrum correction algorithm, collects main peak height and crosstalk part data points, determines the crosstalk proportion and deviation by using a fitting function, and constructs a relationship expression to identify and correct the crosstalk part. The application significantly reduces the crosstalk influence between the fluorescence signals, and ensures the reliability and accuracy of the DNA test results.

Description

Technical Field

[0001] This invention relates to a method for correcting crosstalk in DNA fluorescence spectroscopy, and also to a system for correcting crosstalk in DNA fluorescence spectroscopy, belonging to the field of genetic material detection technology. Background Technology

[0002] In the field of genetic analysis, the application of multicolor parallel fluorescence electrophoresis has made it possible to simultaneously detect multiple short tandem repeat (STR) loci, thus significantly improving detection efficiency. However, this technology faces a challenge: crosstalk can occur between different fluorescence signals, causing infiltration in the fluorescence curves after spectral interpretation, which in turn interferes with the peak data of dye fluorescence signals in different color channels. This interference affects the accurate genotyping of STR loci.

[0003] Chinese invention patent ZL 202110274608.3 discloses a method for dynamically correcting DNA fluorescence spectra using multicolor fluorescent internal standards. This method includes identifying the STR loci to be detected, planning the arrangement of each fluorescence color channel, designing multicolor fluorescent internal standards, identifying these internal standards during each DNA fragment detection analysis, and dynamically constructing a spectral correction matrix to correct the DNA fluorescence spectrum. This technical solution eliminates the need for dedicated spectral correction reagents and independent correction steps, simplifies the workflow of DNA testing technicians, reduces the application complexity of short tandem repeat sequence detection technology, and reduces testing costs.

[0004] In DNA genetic analysis instruments, multicolor fluorescent labeling technology is crucial for the simultaneous detection and analysis of multiple target sequences or loci. This not only reduces the number of repeated experiments but also improves detection efficiency and sample processing capabilities. In practice, a laser emitted from a laser illuminates fluorescent dyes in a capillary tube through a focusing lens. The resulting fluorescence signal is filtered by a filter and then enters a grating. The dispersed fluorescence beam is imaged on a CCD to form a spectrum. Then, the fluorescence signals of the multicolor fluorescent dyes in the capillary tube are analyzed using a spectral correction matrix to obtain the spectral data for each dye. Nevertheless, this method may still face crosstalk issues between different fluorescence spectral data after analysis, which could affect the accuracy of STR genotyping. Summary of the Invention

[0005] The primary technical problem to be solved by this invention is to provide a method for correcting crosstalk in DNA fluorescence spectroscopy.

[0006] Another technical problem to be solved by the present invention is to provide a system for correcting crosstalk in DNA fluorescence spectroscopy.

[0007] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution:

[0008] According to a first aspect of the present invention, a method for correcting crosstalk in DNA fluorescence spectroscopy is provided, comprising the following steps:

[0009] S1: Set the fluorescence signal of the first dye to cause crosstalk to the fluorescence signal of the second dye, obtain the fluorescence signal curves of the first dye and the second dye through the spectral correction algorithm, and collect the peak height of the main peak of the fluorescence signal of the first dye and the data points of the crosstalk part in the fluorescence signal of the second dye.

[0010] S2: Use a fitting function to fit the data points of the crosstalk part to obtain the penetration ratio and deviation;

[0011] S3: Construct a relationship expression between the crosstalk component in the fluorescence signal of the second dye and the fluorescence signal of the first dye, in order to identify the crosstalk component in the fluorescence signal of the second dye;

[0012] S4: Subtract the identified crosstalk portion from the fluorescence signal of the second dye to correct the fluorescence signal of the second dye.

[0013] Preferably, step S1 includes the following sub-steps:

[0014] S11: Set the first dye to penetrate into the second dye;

[0015] S12: Obtain the fluorescence signal curves of the first dye and the second dye through a spectral correction algorithm;

[0016] S13: Based on the fluorescence signal curve of the first dye, the peak height of the main peak is obtained through a peak detection algorithm;

[0017] S14: Set the fluorescence signal curve of the first dye and the fluorescence signal curve of the second dye as a cell array, and sort the cell array according to the value of the fluorescence signal of the first dye.

[0018] S15: Set the number of iterations and execute the iterations until the predetermined number of iterations is reached;

[0019] S16: Traverse the cell array set in step S14. During the traversal, if the value of the second dye fluorescence signal is less than the value of the first dye fluorescence signal, record and collect the data point of the crosstalk part at that point.

[0020] Preferably, in step S3, the crosstalk component of the second dye fluorescence signal and the linear function relationship between them and the first dye fluorescence signal are expressed as follows:

[0021] P B =k·H A +m

[0022] Among them, PB This refers to the crosstalk component in the fluorescence signal of the second dye; H A denoted as , where is the peak height of the main peak of the fluorescence signal of the first dye; k is the penetration ratio; and m is the deviation.

[0023] Preferably, in step S3, the quadratic function relationship between the crosstalk component of the second dye fluorescence signal and the first dye fluorescence signal is expressed as follows:

[0024]

[0025] Where a and b are both permeation ratios; c is the deviation.

[0026] Preferably, in step S3, the cubic function relationship between the crosstalk component of the second dye fluorescence signal and the first dye fluorescence signal is expressed as follows:

[0027]

[0028] Where d, e, and f are all permeation ratios; g is the deviation.

[0029] Preferably, in step S4, the fluorescence signal of the second dye is corrected using the following formula:

[0030] S B,corrected =S B -P B

[0031] Among them, S B,corrected The corrected fluorescence signal of the second dye; S B The fluorescence signal of the original second dye; P B This refers to the crosstalk component.

[0032] Preferably, the first dye is a LiZ dye and the second dye is a SiD dye.

[0033] According to a second aspect of the present invention, a system for correcting crosstalk in DNA fluorescence spectroscopy is provided, comprising a processor and a memory; wherein the memory is coupled to the processor and is used to store a computer program that, when executed by the processor, causes the processor to implement the method described above.

[0034] Compared with existing technologies, this invention, by exploring the crosstalk relationship function between different fluorescence signals, can effectively reduce the influence of crosstalk signals, reduce the crosstalk effect between fluorescence signals, improve the accuracy of analysis, and ensure the reliability and precision of short tandem repeat sequence test results. This improves the detection effect of the capillary electrophoresis genetic analysis platform and provides important technical support for forensic short tandem repeat sequence test and other genetic analysis applications. Attached Figure Description

[0035] Figure 1 A flowchart of a method for correcting crosstalk in DNA fluorescence spectroscopy provided in an embodiment of the present invention;

[0036] Figure 2 This is a flowchart illustrating the extraction of crosstalk data points and the peak height of the main peak in an embodiment of the present invention.

[0037] Figure 3A , Figure 3B , Figure 3C This is the original electrophoresis image of SID dye and LIZ dye in an embodiment of the present invention;

[0038] Figure 4 This is a schematic diagram of the permeation point marking in an embodiment of the present invention;

[0039] Figure 5 This is a schematic diagram of the fitting of the penetration point of a linear function in an embodiment of the present invention;

[0040] Figure 6 This is a diagram illustrating the penetration effect of a linear function in an embodiment of the present invention.

[0041] Figure 7 This is a schematic diagram of the original main peak in an embodiment of the present invention;

[0042] Figure 8 This is a schematic diagram of the quadratic function penetration point fitting in an embodiment of the present invention;

[0043] Figure 9 This is a diagram illustrating the penetration effect of a quadratic function in an embodiment of the present invention.

[0044] Figure 10 This is a schematic diagram of a DNA fluorescence spectral crosstalk correction system provided in an embodiment of the present invention. Detailed Implementation

[0045] The technical content of the present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0046] First Embodiment

[0047] like Figure 1 As shown, the first embodiment of the present invention provides a method for correcting crosstalk in DNA fluorescence spectroscopy, which includes at least the following steps:

[0048] S1: Set the first dye A to permeate into the second dye B, obtain the fluorescence signal curve SA of the first dye A and the fluorescence signal curve SB of the second dye B through the spectral correction algorithm, and collect the peak height of the main peak of the first dye A and the data points (a,b) of the crosstalk part in the fluorescence signal curve of the second dye B.

[0049] S2: Fit the data points (a,b) of the crosstalk part using a fitting function to obtain the penetration ratio and deviation.

[0050] S3: Establish the relationship expression between the crosstalk part in the second dye B and the first dye A to obtain the crosstalk part in the second dye B.

[0051] In one embodiment of the present invention, the relational expression is:

[0052] P B =k·H A +m

[0053] Among them, P B This refers to the crosstalk portion in the second dye B. A denoted as peak height of the first dye A. k represents the penetration ratio. m represents the deviation.

[0054] It should be noted that the above expression is a linear function. If, after correction, the crosstalk of the second dye B is not accurate enough due to the linear function, the expression will be adjusted to a more complex polynomial relationship, such as a quadratic or cubic function.

[0055] In one embodiment of the present invention, the expression for the quadratic function is:

[0056]

[0057] Where a and b are both permeation ratios, and c is the deviation.

[0058] In one embodiment of the present invention, the expression for the cubic function is:

[0059]

[0060] Where d, e, and f are all permeation ratios. g is the deviation.

[0061] S4: Correct the fluorescence signal of the second dye B by subtracting the crosstalk portion in the second dye B.

[0062] In one embodiment of the present invention, the correction formula is:

[0063] S B,corrected =S B -P B

[0064] Among them, S B,corrected This represents the corrected fluorescence signal of the second dye, B. B This represents the original fluorescence signal of the second dye B, i.e., the fluorescence signal curve of the second dye B. P B This refers to the crosstalk component.

[0065] This invention uses given array A and given array B, and assumes that the signal in array A will cause upseeping or downseeping effects on the signal in array B. Next, it calculates the magnitude of the effect y of the value x in array A on the signal in array B, and subtracts the crosstalk part from array B to finally obtain a fluorescence signal with higher accuracy.

[0066] like Figure 2 As shown, in one embodiment of the present invention, step S1, which involves collecting data points (a, b) of the crosstalk portion and the peak height of the main peak of the first dye A, includes the following sub-steps:

[0067] S11: Set the first dye A to permeate into the second dye B.

[0068] S12: Obtain the fluorescence signal curves of the first dye A and the second dye B through a spectral correction algorithm.

[0069] S13: Based on the fluorescence signal curve of the first dye A, the peak height of its main peak is obtained through a peak detection algorithm.

[0070] S14: Set the fluorescence signal curve of the first dye A and the fluorescence signal curve of the second dye B into a cell array, and sort the cell array according to the value of the fluorescence signal curve of the first dye A.

[0071] S15: Set the number of iterations N and execute the iteration loop until the number of iterations is reached.

[0072] S16: Traverse the cell array set in step S14. During the traversal, if the value of the fluorescence signal curve of the second dye B is less than the value of the fluorescence signal curve of the first dye A, then record and collect the data point (a,b) of the crosstalk part.

[0073] The above method will now be described in detail through different embodiments.

[0074] In the first embodiment, the relationship between the crosstalk component in the fluorescence signal of the second dye and that of the first dye was explored. This relationship can be expressed by a linear function. The specific steps are as follows:

[0075] First, electrophoretic curves of LIZ dye (i.e., the first dye) and SID dye (i.e., the second dye) were obtained using a spectroscopic algorithm. These curves show the changes in fluorescence signals of the two dyes during electrophoresis, and analysis of these curves can determine whether crosstalk occurs. Figure 3A , Figure 3B and Figure 3C As shown, it was determined that the enhancement of the fluorescence signal of LIZ dye leads to the decrease of the fluorescence signal of SID dye, i.e., there is a downsinking effect.

[0076] It should be noted that LIZ dye is a commercially available mixture of fluorescent dyes manufactured by Applied Biosystems. It is commonly used in DNA sequencing and fragment analysis, particularly as a size standard or internal standard in capillary electrophoresis. LIZ dyes contain a variety of dyes with different fluorescence properties, producing a continuous range of fluorescence signals for calibrating and comparing the sizes of different DNA fragments. Due to the complex nature of LIZ dyes, they provide a wide range of fluorescence signals, making them ideal for determining DNA fragment sizes. SID dye is a single fluorescent dye, also used in DNA sequencing and fragment analysis. It is typically used as a marker in capillary electrophoresis to indicate the migration front of DNA fragments or as a reference point during the electrophoresis process. The single fluorescence property of SID dye makes it very useful in specific applications, especially where a single, well-defined signal is required.

[0077] Next, scatter plots of LIZ and SID dyes were drawn (see...). Figure 4 The scatter points in the figure were marked sequentially. These scatter points represent the actual relationship between the fluorescence signals of the two dyes, providing a basis for subsequent data analysis and fitting.

[0078] Then, a linear equation is fitted based on the scatter plot of these markers. Figure 5 The distribution of these scattered points is shown. It can be observed that there is no obvious distribution pattern among the scattered points. Therefore, a linear function was chosen for fitting to simplify the model and facilitate calculation.

[0079] After fitting, the fluorescence signal of the LIZ dye was corrected to eliminate the influence of crosstalk. Figure 6 The corrected results are shown. By subtracting the calculated crosstalk, the final processed fluorescence signal is obtained, thereby improving the accuracy and reliability of DNA genetic analysis.

[0080] In the second embodiment, the relationship between the crosstalk component in the fluorescence signal of the second dye and that of the first dye is considered, and this relationship can be expressed by a quadratic function. The specific steps are as follows:

[0081] First, from Figure 7 The original main peak plot shown extracted the crosstalk portion of the second dye and the peak value of the main peak in the first dye. These data provide quantitative information about the relationship between the two dye signals, providing a basis for subsequent data analysis and fitting.

[0082] like Figure 8 As shown, these scatter points clearly exhibit a quadratic function distribution. Therefore, a quadratic function is used to fit them, establishing the relationship between the crosstalk component in the fluorescence signal of the second dye and that of the first dye:

[0083] y = a·x 2 +b·x+c

[0084] Where y is the crosstalk part in the fluorescence signal of the second dye, x is the peak height of the main peak of the fluorescence signal of the first dye, and a and b are the penetration ratios.

[0085] After fitting is complete, result correction is performed, i.e.:

[0086] x'=xy

[0087] Where x' is the corrected fluorescence signal of the second dye, x is the original fluorescence signal of the second dye, and y is the permeation fitting result of the second dye calculated from the first dye.

[0088] Based on these data points, a quadratic function curve was fitted. Compared with a linear function, the quadratic function can more accurately describe the complex relationship between the two dye signals, especially when crosstalk is complex.

[0089] Finally, after fitting was completed, the fluorescence signal of the second dye was corrected to eliminate the influence of crosstalk. For example... Figure 9 As shown, by subtracting the calculated crosstalk, the final processed fluorescence signal can be obtained, further improving the accuracy and reliability of DNA genetic analysis.

[0090] Second Embodiment

[0091] Based on the above method, a second embodiment of the present invention provides a system for correcting crosstalk in DNA fluorescence spectroscopy. For example... Figure 10 As shown, the system includes a processor and a memory; wherein the memory is coupled to the processor and is used to store a computer program, which, when executed by the processor, enables the processor to implement the above-mentioned method for correcting DNA fluorescence spectral crosstalk.

[0092] The processor controls the overall operation of the system to complete all or part of the steps described above. This processor can be a central processing unit (CPU), graphics processing unit (GPU), field-programmable gate array (FPGA), application-specific integrated circuit (ASIC), digital signal processing (DSP) chip, etc. The memory stores various types of data to support the operation of the system. This data may include, for example, instructions for any application or method operating on the system, as well as application-related data. The memory can be implemented using any type of volatile or non-volatile storage device or a combination thereof, such as static random access memory (SRAM), electrically erasable programmable read-only memory (EEPROM), erasable programmable read-only memory (EPROM), programmable read-only memory (PROM), read-only memory (ROM), magnetic storage, flash memory, etc.

[0093] In one exemplary embodiment, the system may be implemented by a computer or microprocessor, or by a product with certain functions, to perform the methods described above and achieve the same technical effects as those methods described above. Specifically, the computer may be, for example, a personal computer, a laptop computer, an in-vehicle human-machine interface device, a cellular phone, a camera phone, a smartphone, a personal digital assistant, a media player, a navigation device, an email device, a game console, a tablet computer, a wearable device, or any combination of these devices.

[0094] In another exemplary embodiment, the present invention also provides a computer-readable storage medium including program instructions that, when executed by a processor, implement the steps of the method in any of the above embodiments. For example, the computer-readable storage medium may be the memory including the program instructions described above, which can be executed by a processor to complete the above method and achieve the same technical effects as the above method.

[0095] It should be noted that the above embodiments are merely illustrative examples. The technical solutions of each embodiment can be combined, and all are within the protection scope of this invention.

[0096] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0097] The method and system for DNA fluorescence spectral crosstalk correction provided by this invention have been described in detail above. Any obvious modifications made by those skilled in the art without departing from the essence of this invention will constitute an infringement of the patent rights of this invention and will incur corresponding legal liability.

Claims

1. A method for correcting crosstalk in DNA fluorescence spectroscopy, characterized in that... Includes the following steps: S1: The fluorescence signal of the first dye is set to cause crosstalk to the fluorescence signal of the second dye. The fluorescence signal curves of the first dye and the second dye are obtained through a spectral correction algorithm. The fluorescence signal curves of the first dye and the second dye are set as a cell array. The cell array is sorted according to the value of the fluorescence signal of the first dye. The cell array is traversed. If the value of the fluorescence signal of the second dye is less than the value of the fluorescence signal of the first dye, the data point of the crosstalk part is recorded and collected. The peak height of the main peak of the fluorescence signal of the first dye is also collected. S2: Use a fitting function to fit the data points of the crosstalk part to obtain the penetration ratio and deviation; S3: Based on the penetration ratio and deviation, construct an expression relating the crosstalk component in the fluorescence signal of the second dye to the peak height of the main peak of the fluorescence signal of the first dye, in order to identify the crosstalk component in the fluorescence signal of the second dye. S4: Subtract the identified crosstalk portion from the fluorescence signal of the second dye to correct the fluorescence signal of the second dye.

2. The method as described in claim 1, characterized in that... Step S1 includes the following sub-steps: S11: Set the first dye to penetrate into the second dye; S12: Obtain the fluorescence signal curves of the first dye and the second dye through a spectral correction algorithm; S13: Based on the fluorescence signal curve of the first dye, the peak height of the main peak is obtained through a peak detection algorithm; S14: Set the fluorescence signal curve of the first dye and the fluorescence signal curve of the second dye as a cell array, and sort the cell array according to the value of the fluorescence signal of the first dye. S15: Set the number of iterations and execute the iterations until the predetermined number of iterations is reached; S16: Traverse the cell array set in step S14. During the traversal, if the value of the second dye fluorescence signal is less than the value of the first dye fluorescence signal, record and collect the data point of the crosstalk part at that point.

3. The method as described in claim 1, characterized in that... In step S3, the linear function relationship between the crosstalk component of the second dye fluorescence signal and the first dye fluorescence signal is expressed as follows: Among them, P B This refers to the crosstalk component in the fluorescence signal of the second dye; H A The peak height of the main peak of the fluorescence signal of the first dye is denoted by k; k is the penetration ratio. This is a deviation.

4. The method as described in claim 1, characterized in that... In step S3, the quadratic function relationship between the crosstalk component of the second dye fluorescence signal and the first dye fluorescence signal is expressed as follows: Where a and b are both permeation ratios; c is the deviation.

5. The method as described in claim 1, characterized in that... In step S3, the cubic function relationship between the crosstalk component of the second dye fluorescence signal and the first dye fluorescence signal is expressed as follows: Where d, e, and f are all permeation ratios; g is the deviation.

6. The method as described in claim 1, characterized in that... In step S4, the fluorescence signal of the second dye is corrected using the following formula: Among them, S B,corrected The corrected fluorescence signal of the second dye; S B The fluorescence signal of the original second dye; P B This refers to the crosstalk component.

7. The method according to any one of claims 1 to 6, characterized in that: The first dye is LIZ dye, and the second dye is SID dye.

8. A system for correcting crosstalk in DNA fluorescence spectroscopy, characterized in that... It includes a processor and a memory; wherein the memory is coupled to the processor and is used to store a computer program that, when executed by the processor, causes the processor to implement the method described in any one of claims 1 to 7.

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