DNA short-chain hybridization fluorescence signal threshold calculation and discrimination method

Abstract

The invention discloses a DNA short chain hybridization fluorescence signal threshold calculation and discrimination method, and relates to the technical field of DNA storage, and the method comprises the following steps: obtaining fluorescence signal intensity data of DNA coding chains in a plurality of micro-pools to form a data set; calculating at least three statistics according to the data set, wherein the statistics comprise a strong signal mean value and a weak signal mean value; calculating a dynamic threshold based on the statistics; comparing the fluorescence signal intensity data with the dynamic threshold value, and judging the existence state of the DNA coding chain in each micro-pool; and generating binary information according to the existence state combination. Based on the obtained fluorescence intensity data, a reasonable fluorescence threshold value is dynamically set, and effective signals and error interference signals can be effectively distinguished; and on the basis of actual data distribution characteristics, a fluorescence threshold is dynamically calculated, and whether a signal exists or not is scientifically judged, so that DNA information is accurately and reliably read under various complex and abnormal environment conditions.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of DNA storage, in particular to a DNA short chain hybridization fluorescence signal threshold calculation and discrimination method. BACKGROUND

[0002] In the DNA information storage technology, the storage micropools are used to store DNA coding chain information, and each micropool may contain coding chains hybridized with multiple different fluorescent probes. Therefore, by adding multiple different fluorescent probes to each micropool for hybridization detection, the type of information stored in each storage micropool can be determined. When detecting the fluorescence information, the presence or absence of the corresponding fluorescence information in the micropool is often determined by detecting the corresponding fluorescence signal intensity, i.e., the fluorescence signal value.

[0003] However, when detecting the fluorescence signal, the fluorescence intensity value collected by the system is dynamically changing due to the interference and influence of the number of probes, the type of probes, fluorescence decay, and various external factors. The values of different probes differ greatly, and the numbers detected at different time periods for the same type of probe also have great differences. In addition, the fluorescence intensity values also have great differences for the same type of probe in different micropools due to the different number of probes. SUMMARY

[0004] In order to effectively read the information, based on the obtained fluorescence intensity data, a reasonable fluorescence threshold is dynamically set to distinguish the effective signal (indicating the presence of coding chain) and the error interference signal (indicating the absence of coding chain). The present application provides a DNA short chain hybridization fluorescence signal threshold calculation and discrimination method, and the specific technical scheme is as follows:

[0005] A DNA short chain hybridization fluorescence signal threshold calculation and discrimination method, comprising the following steps:

[0006] Obtaining fluorescence signal intensity data of DNA coding chains in multiple micropools to form a data set;

[0007] Calculating at least three statistics according to the data set, and the statistics include strong signal mean and weak signal mean;

[0008] Calculating a dynamic threshold based on the statistics;

[0009] Comparing the fluorescence signal intensity data with the dynamic threshold to determine the presence state of the DNA coding chain in each micropool;

[0010] Generating a binary information according to the presence state combination.

[0011] Preferably, the calculation of at least three statistics specifically includes:

[0012] Calculating the mean of all fluorescence signals Wherein xi is the fluorescence signal intensity of the i th micro-pool, and n is the total number of micro-pools;

[0013] calculating the mean of strong signals where S2 = {x i | x i > M1} ;

[0014] calculating the mean of weak signals where S3 = {x i | x i ≤ M1} ;

[0015] The calculating the dynamic threshold value specifically comprises: calculating the threshold value according to the formula T = a x M2 + b x M3, wherein a and b are predetermined weights, a + b = 1 and 0.2 ≤ a ≤ 0.4 are satisfied.

[0016] Preferably, the predetermined weights a = 0.382 and b = 0.618 are satisfied.

[0017] Preferably, the calculating at least three statistical quantities specifically comprises:

[0018] calculating the mean of all fluorescence signals where x i is the fluorescence signal intensity of the i th micro-pool, and n is the total number of micro-pools;

[0019] calculating the mean of strong signals where S2 = {x i | x i > M1} ;

[0020] calculating the mean of weak signals where S3 = {x i | x i ≤ M1} ;

[0021] The calculating the dynamic threshold value specifically comprises: calculating the threshold value according to the variance σ total and the separation index of the data set. The optimization weight selects a as the adaptive weight when S(a) is maximum, b = 1-a, and the threshold value T = a x M2 + b x M3 is calculated.

[0022] Preferably, the optimization weight specifically comprises: traversing a in the range of [0.2, 0.4] with a step length of 0.001, calculating the corresponding S(a) value, and selecting a = arg max S(a).

[0023] Preferably:

[0024] The judging the presence state of the DNA coding chain in each micro-pool specifically comprises: if the fluorescence signal intensity x iIf T, it is determined that the DNA coding chain exists in the corresponding micro-pool, otherwise it is determined that the DNA coding chain does not exist;

[0025] The generated binary information specifically includes: combining the existence states in a predetermined order, wherein the existence states are derived from the detection results of one or more colors of fluorescent probes and are decoded into text, picture, audio or video data through a predetermined encoding table.

[0026] Preferably, the method is applied to an n-well plate, and m different colors of fluorescent probes can be added to the micro-pools, and the fluorescent signal intensity data corresponds to the detection results of the m fluorescent probes, wherein n is an integer multiple of 4, and m is a natural number greater than 1.

[0027] Preferably, the combining the existence states in a predetermined order specifically includes: if it is determined that the DNA coding chain exists in the micro-pool, it is recorded as a binary value of 1, otherwise it is recorded as a binary value of 0; and the binary values of the micro-pools are arranged in sequence according to the predetermined order of the micro-pools to form a binary information sequence.

[0028] Preferably:

[0029] The data set is obtained by washing and detecting after the fluorescent probes hybridize with the DNA coding chain;

[0030] The predetermined encoding table includes an internal code or an ASCII code.

[0031] Preferably, the obtaining of the fluorescent signal intensity data of the DNA coding chain in the plurality of micro-pools specifically includes the following steps:

[0032] a. adding a plurality of groups of fluorescent probes to the micro-pools in stages to allow each group of fluorescent probes to hybridize with the corresponding DNA coding chain, wherein each group of fluorescent probes corresponds to one coding unit, and the hybridization reaction specifically includes: before hybridization, treating the micro-pools with a pre-hybridization buffer to reduce non-specific binding, and the hybridization temperature is 30-50℃ and the hybridization time is 0.5-2h;

[0033] b. washing the hybridized micro-pools to remove unbound fluorescent probes, wherein the washing includes using a washing solution containing SSC buffer and SDS, and the washing temperature is 30-50℃ and the washing times is 1-5 times;

[0034] c. using a multi-channel fluorescence detection technology to simultaneously detect the signals of a plurality of fluorescent probes in the micro-pools, correcting the fluorescence interference through a spectral separation algorithm to generate the fluorescent signal intensity of each probe, and forming the data set.

[0035] The DNA short chain hybridization fluorescence signal threshold calculation and discrimination method provided by the application has the following beneficial effects:

[0036] 1. Based on the obtained fluorescence intensity data, a reasonable fluorescence threshold is dynamically set, which can effectively distinguish effective signals from error interference signals;

[0037] 2. Based on the actual data distribution characteristics, the fluorescence threshold is dynamically calculated, and the presence of signals is scientifically determined, so that the reading of DNA information is accurate and reliable under various complex abnormal environmental conditions. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiment or prior art description will be briefly introduced as follows.

[0039] Figure 1 The algorithm flowchart of the DNA short chain hybridization fluorescence signal threshold calculation and discrimination method provided in the present embodiment;

[0040] Figure 2 The fluorescence signal value threshold discrimination schematic diagram provided in the present embodiment;

[0041] Figure 3 The software interface for applying automatic threshold calculation in the present embodiment;

[0042] Figure 4 The intermediate interface of the software for applying automatic threshold calculation in the present embodiment to restore the text. DETAILED DESCRIPTION

[0043] In order to make those skilled in the art better understand the technical solutions of the present application, the present application will be described in detail below with reference to the drawings, and the description in this part is only exemplary and explanatory, and should not have any limiting effect on the protection scope of the present application.

[0044] The present embodiment provides a DNA short chain hybridization fluorescence signal threshold calculation and discrimination method, including the following steps:

[0045] Obtain fluorescence signal intensity data of DNA coding chains in a plurality of micro-pools to form a data set.

[0046] Calculate at least three statistics from the data set, and the statistics include strong signal mean and weak signal mean.

[0047] Calculate a dynamic threshold based on the statistics.

[0048] Compare the fluorescence signal intensity data with the dynamic threshold to determine the presence state of the DNA coding chains in each micro-pool.

[0049] Generate binary information according to the presence state combination.

[0050] The DNA short chain hybridization fluorescence signal threshold calculation and discrimination method comprises fluorescence signal analysis, threshold calculation and stored information discrimination; when reading the DNA information, the fluorescence probe is added to hybridize with the DNA short chain in the micro-pool, and after washing, the corresponding fluorescence value is obtained by detecting the fluorescence signal intensity, then the corresponding fluorescence threshold is set, the fluorescence value in the micro-pool is compared with the threshold, and whether the coding chain corresponding to the fluorescence probe in the micro-pool exists is determined, so as to identify the state of each decoding chain in the micro-pool, calculate the combination state of the coding chain, and then decode into binary information.

[0051] Based on the obtained fluorescence intensity data, the reasonable fluorescence threshold is dynamically set to distinguish the effective signal (indicating the existence of the coding chain) and the error interference signal (indicating the non-existence of the coding chain). If the fluorescence value is higher than the threshold, it is determined that the coding chain exists; if the fluorescence value is lower than the threshold, it is determined that the coding chain does not exist.

[0052] In order to improve the accuracy and robustness of reading, the embodiment designs an algorithm for dynamically calculating the fluorescence threshold and scientifically discriminating the existence of the signal based on the actual data distribution characteristics, so that the reading of the DNA information is accurate and reliable under various complex abnormal environmental conditions.

[0053] The DNA short chain hybridization fluorescence signal threshold calculation and discrimination method provided by the embodiment has the following beneficial effects:

[0054] 1. Based on the obtained fluorescence intensity data, the reasonable fluorescence threshold is dynamically set, and the effective signal and the error interference signal can be effectively distinguished.

[0055] 2. Based on the actual data distribution characteristics, the fluorescence threshold is dynamically calculated, and the existence of the signal is scientifically discriminated, so that the reading of the DNA information is accurate and reliable under various complex abnormal environmental conditions.

[0056] Further, calculating at least three statistical quantities specifically comprises:

[0057] Calculating the mean value of all fluorescence signals Wherein x i is the fluorescence signal intensity of the ith micro-pool, and n is the total number of micro-pools.

[0058] Calculating the mean value of strong signals Wherein S2={x i | x i >M1}.

[0059] Calculating the mean value of weak signals Wherein S3={x i | x i ≤M1}.

[0060] Calculating the dynamic threshold specifically includes: calculating the threshold according to the formula T=a×M2+b×M3, where a and b are predetermined weights, satisfying a+b=1 and 0.2≤a≤0.4.

[0061] Furthermore, the predetermined weights are a=0.382, b=0.618.

[0062] In the process of fluorescence signal detection, the system detects and collects the fluorescence signal intensity values ​​in each micro-cell on the data storage medium to form a data set. Assume that in a fluorescence signal detection process, the fluorescence detection value set collected is S = {x1, x2, ..., x n}, where x i Represents the fluorescence signal intensity value of the i-th micro-pool; in order to improve scientificity and accuracy, three key statistics are defined according to the needs of data analysis.

[0063] 1. M1 is the mean of the global fluorescence intensity values, that is, the average of all fluorescence detection values, which is used to determine the overall distribution center of the data.

[0064] in:

[0065] 2. M2 is the strong signal mean, that is, the average of all fluorescence values ​​greater than the global mean M1, which is used to reflect the characteristics of strong signals.

[0066] in: S2={x i ∈S|x i >M1}.

[0067] 3. M3 is the weak signal mean, which represents the average value of all fluorescence values ​​less than the global mean M1 and is used to reflect the characteristics of weak signals.

[0068] in: S3={x i ∈S|x i <M1}.

[0069] By analyzing the distribution characteristics of the fluorescence signal, the threshold T for determining whether the micro-pool has or does not have a fluorescence signal is defined as:

[0070] T=0.382×M2+0.618×M3.

[0071] Furthermore, calculating the at least three statistics may also include:

[0072] Calculate the mean of all fluorescence signals where x i is the fluorescence signal intensity of the i-th micropool, and n is the total number of micropools.

[0073] Calculate the mean of strong signals wherein S2 = {x i |x i > M1}.

[0074] calculating the mean of weak signals wherein S3 = {x i |x i M1}.

[0075] calculating the dynamic threshold value specifically comprises: calculating the variance σ total and the separation index optimizing the weight, selecting a that maximizes S(a) as the adaptive weight, b = 1-a, and calculating the threshold value T = a x M2 + b x M3.

[0076] Further, the optimization of the weight specifically comprises: traversing a in the range of [0.2, 0.4] with a step size of 0.001, calculating the corresponding S(a) value, and selecting a = arg max S(a).

[0077] wherein the adaptive weight dynamically adjusts the threshold value T according to the data distribution, maximizes the separation of strong signals and weak signals, and in a complex environment, the probability of correctly identifying the existence of the coding chain is higher, further improving the reading accuracy. If the signal distribution of a batch of micro-pools is biased towards weak signals, the improved algorithm can reduce the value of a, increase the weight of b, and ensure that the threshold value is reasonable. Thus, under different experimental conditions, the algorithm maintains stable performance and avoids misjudgment caused by environmental factors.

[0078] Further:

[0079] judging the existence state of the DNA coding chain in each micro-pool specifically comprises: if the fluorescence signal intensity x i > T, it is determined that the DNA coding chain exists in the corresponding micro-pool, otherwise it is determined that it does not exist.

[0080] generating binary information specifically comprises: combining the existence states in a predetermined order, wherein the existence states are derived from the detection results of one or more colors of fluorescent probes, and are decoded into text, picture, audio or video data through a predetermined encoding table.

[0081] Further, the method is applied to an n-well plate, and m different colors of fluorescent probes can be added to the micro-pools, and the fluorescence signal intensity data corresponds to the detection results of the m fluorescent probes, wherein n is an integer multiple of 4, and m is a natural number greater than 1.

[0082] Specifically, the n-well plate can be a 96-well plate, a 128-well plate, a 256-well plate, a 512-well plate, or the like standard specification or customized specification, and the number of wells on the well plate is usually the number of rows x the number of columns, wherein the number of rows and the number of columns are mostly multiples of 2. Standard microwell plates (such as 96-well plates) usually adopt an 8-row x 12-column layout, and the number of rows and the number of columns of the well plate can be a combination of 2, 4, 8, 16, 32, etc., to facilitate the processing of automated equipment.

[0083] Further, the combination of the presence states in the predetermined order specifically includes: if it is determined that the DNA coding chain exists in the micro-pool, it is recorded as a binary value 1, otherwise it is recorded as a binary value 0; and the binary values of each micro-pool are arranged in sequence according to the predetermined order of the micro-pool to form a binary information sequence.

[0084] Further,

[0085] The data set is obtained by washing and detecting after the hybridization of the fluorescent probe and the DNA coding chain;

[0086] The predetermined encoding table includes an internal code or an ASCII code.

[0087] Specifically, the algorithm uses the statistical characteristics of the fluorescence signal intensity distribution to judge the signal intensity value of each micro-pool by a threshold T. For the fluorescence signal value x of any micro-pool i , if x i > T, it is determined that there is an effective signal in the micro-pool; otherwise, it is determined to be no signal, as shown in Figure 2 . This weighting method can maintain good discrimination under different experimental conditions, so that the threshold can consider the contribution of strong signals and combine the influence of weak signals, ensuring the adaptability and stability of the discrimination.

[0088] The implementation steps of the algorithm can be as shown in Figure 1 .

[0089] Further, the acquisition of the fluorescence signal intensity data of the DNA coding chain in the plurality of micro-pools specifically includes the following steps:

[0090] a. Add a plurality of groups of fluorescent probes to the micro-pools in stages to allow each group of fluorescent probes to hybridize with the corresponding DNA coding chain, wherein each group of fluorescent probes corresponds to one coding unit, and the hybridization reaction specifically includes: before hybridization, the micro-pools are treated with a pre-hybridization buffer to reduce non-specific binding, the hybridization temperature is 30-50°C, and the hybridization time is 0.5-2h.

[0091] b. Wash the hybridized micro-pools to remove unbound fluorescent probes, wherein the washing includes using a washing solution containing SSC buffer and SDS, the washing temperature is 30-50°C, and the washing number is 1-5 times.

[0092] c.Using multi-channel fluorescence detection technology, the signals of multiple fluorescent probes in the micro-pool are detected simultaneously, the fluorescence crosstalk is corrected by spectral separation algorithm, the fluorescence signal intensity of each probe is generated, and the data set is formed.

[0093] The following provides specific examples, which can make those skilled in the art more fully understand the present application, but in no way limit the present application.

[0094] Examples

[0095] Taking the English text information "Where there is a will, there is a way." as an example, the color threshold of four fluorescent probes in each micro-pool on the 96-well plate is calculated, and the threshold obtained by different fluorescent information is different. The test results using fluorescent signal values are shown in Tables 1-4. For an 8x12 data storage matrix, the fluorescent signal values in 96 data storage micro-pools are tested, wherein the numbers 1 / 0 represent the algorithm judgment of yes / no, i.e. the actual writing of yes / no. In practical application, the algorithm can be continuously optimized according to experimental data, and gradually form a judgment method based on statistical principles and with universality, which can adapt to different operators and various experimental conditions.

[0096] Table 1: Fluorescence detection information decoding example 1

[0097]

[0098] Table 2: Fluorescence detection information decoding example 2

[0099]

[0100] Table 3: Fluorescence detection information decoding example 3

[0101]

[0102] Table 4: Fluorescence detection information decoding example 4

[0103]

[0104] When converting the detection data of fluorescent probes into a string, the detection results of fluorescent probes in a single micro-well can be listed as 1 or 0, and then four tables are selected in turn according to the progressive order of adjacent micro-pool data to form a machine code, and then the machine code is converted into letters, symbols, Chinese characters or other information.

[0105] As shown in Figure 3 , it is the software interface for applying automatic threshold calculation. It can be seen that for the color threshold calculation of four fluorescent probes in each micro-pool, the threshold values calculated by different color values are different.

[0106] AsFigure 4 As shown, the intermediate interface of the software applying automatic threshold calculation restores the text, clearly showing the information stored in each microwell.

[0107] The fluorescence signal threshold discrimination algorithm is based on a statistical method, which dynamically sets a reasonable threshold by calculating the overall mean (M1), strong signal mean (M2) and weak signal mean (M3) of the fluorescence detection data, to ensure the accuracy of information reading. This method overcomes the limitations of the fixed threshold method, making the fluorescence signal discrimination more scientific, stable, and able to adapt to different experimental conditions. Experimental results show that this algorithm can maintain a high discrimination accuracy in various environments, achieving efficient decoding of DNA short-chain hybrid storage data and improving the practicality and reliability of information storage technology.

[0108] The principles and implementation modes of the present application are described in this paper by applying specific examples, and the above example descriptions are only used to help understand the method of the present application and its core idea. The above is only a preferred embodiment of the present application, but the protection scope of the present application is not limited thereto, and any skilled person in the art can make equivalent substitutions or changes within the technical scope disclosed by the present application according to the technical solution and inventive concept of the present application, which should be within the protection scope of the present application.

Claims

1. A method for calculating and distinguishing the threshold value of DNA short chain hybridization fluorescence signal, characterized in that: The steps include: Acquire fluorescence signal intensity data of DNA coding chains in multiple micro-pools to form a data set; Calculating at least three statistics based on the data set, the statistics including a strong signal mean and a weak signal mean; calculating a dynamic threshold based on the statistic; Comparing the fluorescence signal intensity data with the dynamic threshold value to determine the presence status of the DNA coding chain in each micropool; Binary information is generated according to the presence state combination.

2. The method for calculating and distinguishing the threshold value of DNA short-chain hybridization fluorescence signal according to claim 1, characterized in that: The calculating of at least three statistics specifically includes: Calculate the mean of all fluorescence signals where x i is the fluorescence signal intensity of the i-th micropool, and n is the total number of micropools; Calculate the mean of strong signals Where S2={x i |x i >M1}; Calculate the mean of weak signals Where S3={x i |x i ≤M1}; The calculating of the dynamic threshold specifically includes: calculating the threshold according to the formula T=a×M2+b×M3, wherein a and b are predetermined weights, satisfying a+b=1 and 0.2≤a≤0.

4.

3. The method for calculating and distinguishing the threshold value of DNA short-chain hybridization fluorescence signal according to claim 2, characterized in that: The predetermined weights a=0.382, b=0.

618.

4. The method for calculating and distinguishing the threshold value of DNA short chain hybridization fluorescence signal according to claim 1, characterized in that: The calculating of at least three statistics specifically includes: Calculate the mean of all fluorescence signals where x i is the fluorescence signal intensity of the i-th micropool, and n is the total number of micropools; Calculate the mean of strong signals Where S2={x i |x i >M1}; Calculate the mean of weak signals Where S3={x i |x i ≤M1}; The calculation of the dynamic threshold specifically includes: according to the variance σ of the data set total and resolution index Optimize the weights, select a that maximizes S(a) as the adaptive weight, b=1-a, and calculate the threshold T=a×M2+b×M3.

5. The method for calculating and distinguishing the threshold value of DNA short chain hybridization fluorescence signal according to claim 4, characterized in that: The weight optimization specifically includes: traversing a in the range of [0.2, 0.4] with a step size of 0.001, calculating the corresponding S(a) value, and selecting a=arg max S(a).

6. The method for calculating and distinguishing a DNA short-chain hybridization fluorescence signal threshold according to any one of claims 2 to 5, wherein: The determination of the presence of the DNA coding chain in each micro-pool specifically includes: if the fluorescence signal intensity x i >T, the DNA coding chain in the corresponding micropool is determined to exist, otherwise it is determined to be absent; Generating binary information specifically includes: combining the existence states in a predetermined order, wherein the existence states are derived from detection results of fluorescent probes of one or more colors, and decoding them into text, pictures, audio or video data through a predetermined coding table.

7. The method for calculating and distinguishing the threshold value of a DNA short-chain hybridization fluorescence signal according to any one of claims 1 to 5, characterized in that: The method is applied to an n-well plate, and m fluorescent probes of different colors can be added to the micropool. The fluorescent signal intensity data corresponds to the detection results of the m fluorescent probes, where n is an integer multiple of 4 and m is a natural number greater than 1.

8. The method for calculating and distinguishing the threshold value of DNA short-chain hybridization fluorescence signal according to claim 6, characterized in that: Combining the existence states in a predetermined order specifically includes: if it is determined that the DNA coding chain exists in the micropool, recording it as a binary value 1, otherwise recording it as a binary value 0; and arranging the binary values ​​of each micropool in sequence according to the predetermined order of the micropools to form a binary information sequence.

9. The method for calculating and distinguishing the threshold value of a DNA short-chain hybridization fluorescence signal according to claim 6, wherein: The data set is obtained by hybridizing a fluorescent probe to a DNA coding strand followed by washing and detection; The predetermined coding table includes machine codes or ASCII codes.

10. The method for calculating and distinguishing the threshold value of DNA short chain hybridization fluorescence signal according to any one of claims 1 to 5, characterized in that: The step of obtaining the fluorescence signal intensity data of the DNA coding chains in the plurality of micro-pools specifically comprises the following steps: a) adding multiple sets of fluorescent probes to the microcell in stages, allowing each set of fluorescent probes to undergo a hybridization reaction with a corresponding DNA coding strand, wherein each set of fluorescent probes corresponds to one coding unit, the hybridization reaction specifically comprising: treating the microcell with a prehybridization buffer before hybridization to reduce nonspecific binding, the hybridization temperature being 30 to 50° C., and the hybridization time being 0.5 to 2 hours; b) washing the hybridized micropool to remove unbound fluorescent probes, wherein the washing comprises using a washing solution containing SSC buffer and SDS, the washing temperature is 30 to 50° C., and the number of washes is 1 to 5; c) using a multi-channel fluorescence detection technology to simultaneously detect the signals of multiple fluorescent probes in the microcell, correcting fluorescence interference through a spectral separation algorithm, generating the fluorescence signal intensity of each probe, and forming the data set.

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