DNA fluorescence spectrum crosstalk correction method and system

Abstract

The invention discloses a DNA fluorescence spectrum crosstalk correction method and system, and aims to solve the problem of fluorescence signal crosstalk caused by a multicolor fluorescence parallel electrophoresis technology in genetic analysis. According to the method, fluorescence signal curves of a first dye and a second dye are obtained through a spectrum correction algorithm, the peak height of a main peak and data points of a crosstalk part are collected, the crosstalk proportion and deviation are determined through a fitting function, and a relational expression is constructed to recognize and correct the crosstalk part. According to the method, the crosstalk influence between fluorescence signals is remarkably reduced, and the reliability and the accuracy of a DNA detection result are ensured.

Description

Technical Field

[0001] The invention relates to a method for correcting DNA fluorescence spectrum crosstalk, and also relates to a system for correcting DNA fluorescence spectrum crosstalk, belonging to the technical field of genetic material detection. Background Art

[0002] In the field of genetic analysis, the application of multicolor fluorescence parallel electrophoresis technology makes it possible to detect multiple short tandem repeat (STR) loci at the same time, thus significantly improving the detection efficiency. However, this technology faces a challenge: crosstalk occurs between different fluorescent signals, resulting in penetration of the fluorescence curve after spectrum decomposition, and then interference between the spectrum peak data of the dye fluorescence signal in different color channels. This interference affects the accurate typing of STR loci.

[0003] In the Chinese invention patent with patent number ZL 202110274608.3, a method for dynamically correcting the DNA fluorescence spectrum using a multicolor fluorescent internal standard is disclosed. The method includes determining the STR loci to be detected, planning the arrangement of each fluorescent color channel, designing a multicolor fluorescent internal standard, and identifying these internal standards during each DNA fragment detection and 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 links, simplifies the workflow of DNA detection technicians, reduces the application complexity of short tandem repeat sequence detection technology, and reduces detection costs.

[0004] In DNA genetic analysis instruments, multicolor fluorescent labeling technology is essential for the simultaneous detection and analysis of multiple target sequences or sites, which not only reduces the number of repeated experiments, but also improves detection efficiency and sample processing capabilities. In specific operations, the laser emitted by the laser irradiates the fluorescent dye in the capillary through a focusing mirror, and the generated fluorescent signal is filtered by a filter and enters the grating. The split fluorescence beam is imaged on the CCD to form a spectrum. Then, the fluorescence signal of the multicolor fluorescent dye in the capillary is deconvoluted by the spectral correction matrix to obtain the spectral data of each dye. Nevertheless, this method may still face the problem of crosstalk penetration between different fluorescence spectral data after deconvolution, which may affect the accuracy of STR typing. Summary of the invention

[0005] The primary technical problem to be solved by the present invention is to provide a method for correcting DNA fluorescence spectrum crosstalk.

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

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

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

[0009] S1: setting the fluorescence signal of the first dye to produce crosstalk to the fluorescence signal of the second dye, obtaining the fluorescence signal curves of the first dye and the second dye through a spectral correction algorithm, and collecting the main peak height 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 the fitting function to fit the data points of the crosstalk part to obtain the penetration ratio and deviation;

[0011] S3: constructing a relationship expression between the crosstalk portion in the second dye fluorescence signal and the first dye fluorescence signal to identify the crosstalk portion in the second dye fluorescence signal;

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

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

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

[0015] S12: obtaining a fluorescence signal curve of the first dye and a fluorescence signal curve of the second dye through a spectrum correction algorithm;

[0016] S13: according to the fluorescence signal curve of the first dye, obtaining the main peak height by using a peak detection algorithm;

[0017] S14: setting the fluorescence signal curve of the first dye and the fluorescence signal curve of the second dye into a unit array, and sorting the unit array according to the value of the fluorescence signal of the first dye;

[0018] S15: setting the number of iteration cycles, and executing the iteration cycle until the predetermined number is reached;

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

[0020] Preferably, in step S3, the linear function relationship between the crosstalk part in the second dye fluorescence signal and the first dye fluorescence signal is expressed as:

[0021] P B = k·H A +m

[0022] Among them, PB is the crosstalk part of the second dye fluorescence signal; H A is the main peak height of the first dye fluorescence signal; k is the penetration ratio; m is the deviation.

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

[0024]

[0025] Among them, a and b are penetration ratios; c is the deviation.

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

[0027]

[0028] Among them, d, e, and f are penetration ratios; g is the deviation.

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

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

[0031] Among them, S B,corrected is the corrected fluorescence signal of the second dye; S B is the original fluorescence signal of the second dye; P B For the crosstalk part.

[0032] Preferably, the first dye is a LI Z dye, and the second dye is a S ID dye.

[0033] According to a second aspect of an embodiment of the present invention, a system for correcting DNA fluorescence spectral crosstalk is provided, comprising a processor and a memory; wherein the memory is coupled to the processor and is used to store a computer program, and when the computer program is executed by the processor, the processor implements the above method.

[0034] Compared with the prior art, the present invention can effectively reduce the impact of crosstalk signals and the crosstalk impact between fluorescent signals by exploring the crosstalk relationship function between different fluorescences, thereby improving the accuracy of analysis and ensuring the reliability and accuracy of short tandem repeat sequence test results, thereby improving the detection effect of the capillary electrophoresis genetic analysis platform and providing important technical support for forensic short tandem repeat sequence testing and other genetic analysis applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A flow chart of a method for correcting DNA fluorescence spectrum crosstalk provided by an embodiment of the present invention;

[0036] Figure 2 A flowchart for extracting crosstalk data points and main peak height in an embodiment of the present invention;

[0037] Figure 3A , Figure 3B , Figure 3C 1 is the original electrophoresis diagram of SID dye and L IZ dye in the embodiment of the present invention;

[0038] Figure 4 A schematic diagram of a penetration point mark in an embodiment of the present invention;

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

[0040] Figure 6 This is a penetration effect diagram 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 It is a schematic diagram of quadratic function penetration point fitting in an embodiment of the present invention;

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

[0044] Fig.10 A schematic diagram of a system for correcting DNA fluorescence spectrum crosstalk provided by an embodiment of the present invention. DETAILED DESCRIPTION

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

[0046] First embodiment

[0047] like Figure 1 As shown, a method for correcting DNA fluorescence spectrum crosstalk provided by the first embodiment of the present invention at least comprises the following steps:

[0048] S1: Set the first dye A to penetrate 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 data points (a, b) of the main peak height of the first dye A and 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 through the fitting function to obtain the penetration ratio and deviation.

[0050] S3: Establish a relationship expression between the crosstalk part in the second dye B and the first dye A, and 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 is the crosstalk portion in the second dye B. A is the peak height of the first dye A. k is the penetration ratio. m is the deviation.

[0054] It should be noted that the above expression is a linear function. If after correction, the crosstalk part of the second dye B is expressed as a linear function and cannot meet its accuracy, the expression is adjusted to a more complex polynomial relationship, such as a quadratic function or a cubic function.

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

[0056]

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

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

[0059]

[0060] Among them, d, e, and f are penetration ratios, and 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 is the corrected fluorescence signal of the second dye B. B is the original fluorescence signal of the second dye B, that is, the fluorescence signal curve of the second dye B. B For the crosstalk part.

[0065] The present invention is based on a given array A and a given array B, and assumes that the signal in array A will cause an upward or downward influence on the signal in array B. Next, the magnitude of the influence y on array B caused by the value x in array A is calculated, and the crosstalk part is subtracted from array B, so as to finally obtain a fluorescent signal with higher accuracy.

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

[0067] S11: Setting the first dye A to penetrate into the second dye B.

[0068] S12: Obtaining a fluorescence signal curve of the first dye A and a fluorescence signal curve of the second dye B through a spectrum correction algorithm.

[0069] S13: According to the fluorescence signal curve of the first dye A, the main peak height is obtained by a peak detection algorithm.

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

[0071] S15: Set the number of iteration cycles N, and execute the iteration cycle until the number of iteration cycles is reached.

[0072] S16: traverse the cell array set in step S14. During the traversal process, 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, record and collect the point as the data point (a, b) of the crosstalk part.

[0073] The above method is described in detail below through different embodiments.

[0074] In the first embodiment, the relationship between the crosstalk part in the second dye fluorescence signal and the first dye is discussed, and the relationship can be expressed by a linear function. The specific steps are as follows:

[0075] First, the electrophoresis curves of the LZ dye (i.e., the first dye) and the SID dye (i.e., the second dye) were obtained by using a spectral algorithm. These curves show the changes in the fluorescence signals of the two dyes during the electrophoresis process. By analyzing these curves, it can be determined whether there is crosstalk. Figure 3A , Figure 3B and Figure 3C As shown, it was determined that the enhancement of the fluorescence signal of the LIZ dye would lead to the decrease of the fluorescence signal of the SID dye, that is, there was a permeation effect.

[0076] It should be noted that L IZ dye is a commercial fluorescent dye mixture produced by Applied Biosystems. It is commonly used in DNA sequencing and fragment analysis, especially as a size standard or internal standard in capillary electrophoresis. LIZ dye contains multiple dyes with different fluorescent properties, which can produce a continuous range of fluorescent signals for calibrating and comparing the sizes of different DNA fragments. Due to the complex nature of L IZ dye, it can provide a wide range of fluorescent signals, making it ideal for determining the size of DNA fragments. S ID dye is a single fluorescent dye that is also used for DNA sequencing and fragment analysis. It is usually used as a marker in capillary electrophoresis to indicate the migration front of DNA fragments or as a reference point during electrophoresis. The single fluorescent property of S ID dye makes it very useful in specific applications, especially when a single, clear signal identification is required.

[0077] Next, the scatter plots of the LIZ dye and the SID dye were plotted (see Figure 4 ), and marked the scattered points in the figure in turn. These scattered 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 these marked scatter points. Figure 5 The distribution of these scattered points is shown. It can be observed that there is no obvious distribution pattern for the scattered points. Therefore, a linear function is selected for fitting in order to simplify the model and facilitate calculation.

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

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

[0081] First, from Figure 7 The original main peak graph shown extracts the crosstalk portion of the second dye and the peak 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 scattered points are obviously distributed in a quadratic function, so a quadratic function is used to fit them and establish the relationship expression between the crosstalk part of the second dye fluorescence signal and the first dye:

[0083] y=a·x 2 +b·x+c

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

[0085] After the fitting is completed, the result correction is performed, namely:

[0086] x'=xy

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

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

[0089] Finally, after the fitting is completed, the fluorescence signal of the second dye is corrected to eliminate the influence of crosstalk. Fig. 9 As shown, by subtracting the calculated crosstalk part, 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, the second embodiment of the present invention provides a system for correcting DNA fluorescence spectrum crosstalk. Fig.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. When the computer program is executed by the processor, the processor implements the above-mentioned method for correcting DNA fluorescence spectrum crosstalk.

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

[0093] In an exemplary embodiment, the system can be implemented by a computer or a microprocessor, or by a product with a certain function, for executing the above method and achieving the same technical effect as the above method. Specifically, the computer can be, for example, a personal computer, a laptop computer, a vehicle-mounted human-computer interaction device, a cellular phone, a camera phone, a smart phone, a personal digital assistant, a media player, a navigation device, an email device, a game console, a tablet computer, a wearable device, or a combination of any of these devices.

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

[0095] It should be noted that the above embodiments are only examples, and the technical solutions of the various embodiments can be combined, all within the protection scope of the present invention.

[0096] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0097] The above is a detailed description of a method and system for correcting DNA fluorescence spectrum crosstalk provided by the present invention. For those skilled in the art, any obvious changes made to it without departing from the essence of the present invention will constitute an infringement of the patent right of the present invention and will bear corresponding legal responsibilities.

Claims

1. A method for correcting DNA fluorescence spectrum crosstalk, characterized in that The steps include: S1: setting the fluorescence signal of the first dye to produce crosstalk to the fluorescence signal of the second dye, obtaining the fluorescence signal curves of the first dye and the second dye through a spectral correction algorithm, and collecting the main peak height of the fluorescence signal of the first dye and the data points of the crosstalk part in the fluorescence signal of the second dye; S2: Use the fitting function to fit the data points of the crosstalk part to obtain the penetration ratio and deviation; S3: constructing a relationship expression between the crosstalk portion in the second dye fluorescence signal and the first dye fluorescence signal to identify the crosstalk portion in the second dye fluorescence signal; S4: Subtracting the identified crosstalk portion from the fluorescence signal of the second dye to correct the fluorescence signal of the second dye.

2. The method according to claim 1, characterized in that The step S1 includes the following sub-steps: S11: Setting the first dye to penetrate into the second dye; S12: obtaining a fluorescence signal curve of the first dye and a fluorescence signal curve of the second dye through a spectrum correction algorithm; S13: according to the fluorescence signal curve of the first dye, obtaining the main peak height by using a peak detection algorithm; S14: setting the fluorescence signal curve of the first dye and the fluorescence signal curve of the second dye into a unit array, and sorting the unit array according to the value of the fluorescence signal of the first dye; S15: setting the number of iteration cycles, and executing the iteration cycle until the predetermined number is reached; S16: traverse the cell array set in step S14. During the traversal process, if the value of the second dye fluorescence signal is less than the value of the first dye fluorescence signal, record and collect the point as a data point of the crosstalk part.

3. The method according to claim 1, characterized in that In step S3, the linear function relationship between the crosstalk part in the second dye fluorescence signal and the first dye fluorescence signal is expressed as: P B =k·H A +m Among them, P B is the crosstalk part of the second dye fluorescence signal; H A is the main peak height of the first dye fluorescence signal; k is the penetration ratio; m is the deviation.

4. The method according to claim 3, characterized in that In step S3, the quadratic function relationship between the crosstalk part in the second dye fluorescence signal and the first dye fluorescence signal is expressed as: Among them, a and b are penetration ratios; c is the deviation.

5. The method according to claim 4, characterized in that In step S3, the cubic function relationship between the crosstalk part in the second dye fluorescence signal and the first dye fluorescence signal is expressed as: Among them, d, e, and f are penetration ratios; g is the deviation.

6. The method according to claim 1, characterized in that In step S4, the fluorescence signal of the second dye is corrected by the following formula: S B,corrected =S B -P B Among them, S B,corrected is the corrected fluorescence signal of the second dye; S B is the original fluorescence signal of the second dye; P B For the crosstalk part.

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

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

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