BASE SEQUENCE ANALYSIS METHOD AND GENE ANALYSIS DEVICE

Abstract

A gene analysis instrument is provided that is configured to: acquire electrophoresis data, which are time-series data of signal intensities of a variety of frequencies, acquired by subjecting a sample to electrophoresis; identify a single fluorescence spectrum time, at which a single fluorescence spectrum, which is a spectrum derived from only one base, is present, using the electrophoresis data; compute a spectral shift model using a spectrum obtained from a color conversion matrix at the single fluorescence spectrum time and the single fluorescence spectrum at the single fluorescence spectrum time; correct the electrophoresis data using the spectral shift model; and identify a base sequence of the sample using the corrected electrophoresis data.

Description

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a gene analysis apparatus for analyzing a base sequence of a sample using electrophoresis and a method therefor.

[0002] A capillary electrophoresis device acquires electrophoresis data by irradiating it with excitation light while separating DNA fragments by electrophoresis. The excitation light has a predetermined width, and pull-ups (quasi-peaks) are known to occur due to spectral shift (see, for example, paragraphs 0050 and 0051 of JP 2006-292368 A). The accuracy of base sequence analysis is reduced by these pull-ups. Citation listPatent literature PTL 1: JP 2006-292368 A PTL 2: JP 2014-117222 A PTL 3: JP 2020-41876 A SUMMARY OF THE INVENTION

[0003] JP 2006-292368 A discloses an optical detection unit that reduces pull-ups. However, the spectral shift varies depending on an error in the optical system of each device and environmental differences such as electrophoresis voltage and temperature, making it difficult to accurately model the spectral shift. This invention implements a method for reducing pull-ups through information processing.

[0004] A typical example of the invention disclosed in this application is as follows. Specifically, a base sequence analysis method is provided, which is performed by a genetic analyzer configured to analyze a base sequence of a sample. The base sequence analysis method includes: a first step of acquiring, by the genetic analyzer, electrophoresis data that is time series data of signal intensities of a plurality of frequencies acquired by subjecting the sample to electrophoresis; a second step of identifying, by the genetic analyzer, a single fluorescence spectrum time at which a single fluorescence spectrum, which is a spectrum derived from only one base, exists, using the electrophoresis data;a third step of calculating, by the genetic analyzer, a spectral shift model using a spectrum obtained from a color conversion matrix at the single fluorescence spectrum time and the single fluorescence spectrum at the single fluorescence spectrum time; a fourth step of correcting, by the genetic analyzer, the electrophoresis data using the spectral shift model; and a fifth step of identifying, by the genetic analyzer, a base sequence of the sample using the corrected electrophoresis data.

[0005] According to this invention, it is possible to reduce pull-ups through information processing and improve the accuracy of base sequence analysis by considering a single fluorescence spectrum as a shifted spectrum obtained from a color conversion matrix. Problems, configurations, and effects other than those described above will become apparent from the following description of embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The present invention can be understood from the following description in conjunction with the following figures, in which: Fig. 1 is a diagram illustrating a configuration example of a genetic analysis apparatus 100 of a first embodiment of this invention; Fig. 2 is a diagram illustrating an example of a configuration of the electrophoresis apparatus 110 in the first embodiment; Fig. 3 is a flowchart illustrating an outline of processing executed by the genetic analysis apparatus 100 of the first embodiment; Fig. 4 is a flowchart illustrating electrophoresis processing performed by the electrophoresis apparatus 110 in the first embodiment; Fig. 5 is a flowchart illustrating spectral correction processing executed by the data analysis apparatus 111 in the first embodiment; Fig. 6 is a flowchart illustrating single fluorescence spectrum correction processing executed by the data analysis apparatus 111 in the first embodiment; Fig. 7 is a diagram illustrating images of single fluorescence spectrum correction processing in the first embodiment; Fig. Fig. 8 is a flowchart illustrating non-single fluorescence spectrum correction processing performed by the data analysis apparatus 111 in the first embodiment; and Fig. 9 is a diagram illustrating images of the non-single fluorescence spectrum correction processing in the first embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] At least one embodiment of this invention will now be described with reference to the drawings. It should be noted that this invention should not be construed as limiting the invention to the content described in the following at least one embodiment. One skilled in the art would readily recognize that specific configurations described in the following at least one embodiment can be changed within the scope of the concept and gist of this invention.

[0008] In configurations of the at least one embodiment of this invention described below, the same or similar components or functions are denoted by the same reference numerals, and redundant description thereof is omitted here.

[0009] Notations such as “first,” “second,” and “third” are assigned here to distinguish between components and do not necessarily restrict the number or order of those components.

[0010] The position, size, shape, range, and other details of each component illustrated in the drawings, for example, may not represent the actual position, size, shape, range, and other details to facilitate understanding of this invention. Thus, this invention is not limited to the position, size, shape, range, and other details disclosed in the drawings, for example. [First embodiment]

[0011] Fig. 1 is a diagram illustrating a configuration example of a genetic analysis apparatus 100 of a first embodiment of this invention.

[0012] The genetic analysis device 100 includes an electrophoresis device 110 and a data analysis device 111. The electrophoresis device 110 and the data analysis device 111 are communicatively coupled using a communication cable.

[0013] The data analysis device 111 includes a control device 120, a storage device 121 and a coupling interface 122.

[0014] The control device 120 controls the electrophoresis device 110 and processes data. The control device 120 is, for example, a central processing unit (CPU) or a graphics processing unit (GPU).

[0015] The storage device 121 stores, for example, programs executed by the control device 120, setting information about the electrophoresis device 110, and information used in various processes. The storage device 121 is, for example, a memory.

[0016] The coupling interface 122 is an interface coupled to an input device and an output device, or an interface coupled to an external device via a network. The data analysis device 111 presents information to the user and receives information input by the user via the coupling interface 122.

[0017] The control device 120 functions as a sample information setting module 131, an electrophoresis device control module 132, a fluorescence intensity calculation module 133, a spectral shift correction module 134, and a base call module 135 by executing programs stored in the storage device 121. In the following description, when processing using a functional module is described as the subject of the sentence, it means that the control device 120 executes a program.

[0018] The electrophoresis device 110 performs electrophoresis on a sample (DNA fragment) and acquires electrophoresis data. The electrophoresis data are time-series data of signal intensities (brightness values) at a variety of frequencies.

[0019] The configuration of the electrophoresis apparatus 110 will now be described. Fig. 2 is a diagram illustrating an example of a configuration of the electrophoresis apparatus 110 in the first embodiment.

[0020] The electrophoresis device 110 includes a detection unit 216, a thermostatic bath 218, a conveying device 225, a high-voltage power source 204, a first ammeter 205, an anode electrode 211, a second ammeter 212, a capillary assembly 217, and a pumping mechanism 203.

[0021] The capillary assembly 217 is a replaceable element that includes a plurality of (for example, eight) capillaries 202, and includes a charging head 229, the detection unit 216, and a capillary head 233. If the capillaries 202 become damaged or deteriorate, the capillary assembly 217 can be replaced with a new capillary assembly 217.

[0022] Each capillary 202 is made of a glass tube having an inner diameter of several tens to several hundred micrometers and an outer diameter of several hundred micrometers, and the surface of the capillary 202 is coated with polyimide to improve strength. However, a light irradiation portion onto which laser light is irradiated has a structure in which the polyimide coating is removed, so that internally emitted light easily escapes to the outside. The interior of the capillaries 202 is filled with a separation medium to provide a difference in migration speed during electrophoresis. The separation medium can be any of a fluid medium and a non-fluid medium, but in the first embodiment, a fluid polymer is used.

[0023] The high-voltage power source 204 applies a high voltage to the capillaries 202. The first ammeter 205 detects the current emitted by the high-voltage power source 204. The second ammeter 212 detects the current flowing through the anode electrode 211.

[0024] An optical detection unit that detects information light obtained from the sample includes a light source 214 that irradiates excitation light to the detection unit 216, an optical detector 215 that detects light emitted in the detection unit 216, and a diffraction grating 232. The detection unit 216 is an element that acquires sample-dependent information.

[0025] When the sample is to be detected in a capillary 202 separated by electrophoresis, excitation light from the light source 214 is irradiated onto the detection unit 216, generating fluorescence with a wavelength that depends on the sample as information light. Further, the diffraction grating 232 scatters the information light in the wavelength direction, and the optical detector 215 detects the scattered information light and analyzes the sample.

[0026] Each capillary cathode end 227 is secured by a hollow metal electrode 226, and the tip of the capillary 202 protrudes approximately 0.5 millimeters from the hollow electrodes 226. Furthermore, all hollow electrodes 226 arranged for the capillaries 202 are attached to the charging head 229 as a unit. Additionally, all hollow electrodes 226 are electrically connected to the high-voltage power source 204 mounted on the main body of the device and function as cathode electrodes when a voltage needs to be applied, such as during electrophoresis and sample introduction.

[0027] The capillary ends (other ends) opposite the capillary cathode ends 227 are bundled together by the capillary head 233. The capillary head 233 can be pressure-tightly connected to a block 207. The high voltage from the high-voltage power source 204 is applied between the charging head 229 and the capillary head 233. New polymer is then filled into the capillaries 202 from the other end using a syringe 206. Refilling the polymer into the capillaries 202 is performed after each measurement to improve measurement performance.

[0028] The pump mechanism 203 includes the syringe 206 and a mechanism system for pressurizing the syringe 206 and injecting the polymer into the capillaries 202.

[0029] The block 207 is a connecting part so that the syringe 206, the capillary assembly 217, an anode buffer container 210 and a polymer container 209 can communicate with each other.

[0030] The thermostat bath 218 is covered with a heat-insulating material to keep the capillaries 202 in the thermostat bath 218 at a constant temperature, and the temperature is controlled by a heating / cooling mechanism 220. Furthermore, a fan 219 circulates and mixes the air in the thermostat bath 218 to keep the temperature of the capillary array 217 positionally uniform and constant.

[0031] The conveying device 225 conveys various containers to the capillary cathode ends 227. The conveying device 225 includes three electric motors and a linear actuator and is movable in three axial directions: up and down, left and right, and depth. Furthermore, at least one or more containers can be placed on a movable table 230 of the conveying device 225. The movable table 230 has an electric handle 231 that can grip and release each container. Therefore, a buffer container 221, a cleaning container 222, a waste liquid container 223, and a sample plate 224 can be conveyed to the capillary cathode ends 227 as needed. Unneeded containers are stored in a predetermined storage space in the electrophoresis device 110.

[0032] The user can use the data analysis device 111 to control various functions of the electrophoresis device 110 and to acquire electrophoresis data detected by the optical detection unit.

[0033] The electrophoresis device 110 may include sensors for detecting information related to an observation environment that affects electrophoresis (observation environment information). The electrophoresis device 110 of Fig. 2 includes internal sensors 240, polymer sensors 241 and a buffer sensor 242.

[0034] The internal sensors 240 are sensors for detecting information related to the internal environment of the electrophoresis device 110 and perform measurements using, for example, a temperature sensor, a humidity sensor, and an atmospheric pressure sensor arranged in the electrophoresis device 110.

[0035] The polymer sensors 241 are sensors for detecting information related to the quality of the polymer and are, for example, a pH sensor and an electrical conductivity sensor. Fig. 2, the polymer sensors 241 are arranged in the polymer container 209, but the arrangement position is not limited thereto.

[0036] The buffer sensor 242 is a sensor for detecting information regarding the quality of the buffer and is, for example, a temperature sensor. Fig. 2, the buffer sensor 242 is arranged in the anode buffer tank 210, but the arrangement position is not limited thereto. For example, the buffer sensor 242 may be arranged in the buffer tank 221.

[0037] Fig. 3 is a flowchart illustrating an outline of processing executed by the genetic analysis apparatus 100 of the first embodiment.

[0038] The electrophoresis device 110 of the genetic analysis device 100 performs electrophoresis processing on a sample to be analyzed (step S101). Details of the electrophoresis processing will be described with reference to Fig. 3 described.

[0039] Next, the data analyzer 111 of the genetic analyzer 100 performs spectral correction processing using the electrophoresis data (step S102). Details of the spectral correction processing will be described with reference to Fig. 5 described.

[0040] Next, the data analysis device 111 of the genetic analysis device 100 performs fluorescence intensity calculation processing using the corrected electrophoresis data (step S103). Specifically, the fluorescence intensity calculation module 133 calculates fluorescence intensity time series data of a fluorescent dye from the corrected electrophoresis data and detects the center position, height, and width of, for example, peaks from the fluorescence intensity time series data.

[0041] Next, the data analysis device 111 of the genetic analysis device 100 performs mobility correction processing on the fluorescence intensity time series data (step S104).

[0042] Next, the data analysis device 111 of the genetic analysis device 100 performs base retrieval using the fluorescence intensity time series data corrected based on the results of the mobility correction processing (step S105). Specifically, the base retrieval module 135 identifies the base sequence of the sample using the corrected fluorescence intensity time series data.

[0043] Fig. 4 is a flowchart illustrating electrophoresis processing performed by the electrophoresis apparatus 110 in the first embodiment.

[0044] The user sets samples and reagents to be analyzed, for example, in the electrophoresis device 110 and instructs the start of electrophoresis processing via the coupling interface 122. The samples are set according to the following steps.

[0045] The user fills the buffer container 221 and the anode buffer container 210 with a buffer to form part of a current-carrying path. The buffer is, for example, an electrolyte solution commercially available from various companies for use in electrophoresis. The user dispenses the samples to be analyzed into wells of the sample plate 224. The samples are, for example, a DNA PCR product. The user dispenses a cleaning solution for cleaning the capillary cathode end 227 into the cleaning container 222. The cleaning solution is, for example, pure water. The user injects a migration medium for electrophoreting the sample into the syringe 206. Examples of the migration medium include polyacrylamide separation gels and polymers commercially available from various companies for use in electrophoresis.The user replaces the capillary assembly 217 when deterioration of the capillaries 202 is expected or changes the length of the capillaries 202.

[0046] At this time, examples of the samples to be set on the sample plate 224 include, in addition to the actual DNA sample to be analyzed, a positive control, a negative control, and an allelic ladder, and each is electrophoreted in a different capillary 202.

[0047] For example, the positive control is a PCR product containing known DNA and is a sample for a control experiment to confirm that the DNA is correctly amplified by PCR. The negative control is a PCR product that does not contain DNA and is a sample for a control experiment to confirm that the PCR amplification product is free of contamination, such as user DNA or dust. The allelic ladder is an artificial sample containing many bases that may commonly be found in DNA markers and is usually provided by a reagent manufacturer as a reagent kit for DNA identification. The allelic ladder is used for the purpose of fine-tuning the correspondence between the DNA fragment lengths of individual DNA markers and alleles.

[0048] Furthermore, a known DNA fragment, called the size standard, labeled with a specific fluorescent dye, is mixed into the entire sample, the positive control, the negative control, and the allelic ladder. The type of fluorescent dye assigned to the size standard varies depending on the reagent kit used.

[0049] For example, the user specifies the type of allelic ladder, the type of size standard, the type of fluorescent reagent, and the type of each sample to be set into the wells on the sample plate 224 corresponding to each capillary 202. In the first embodiment, any of the actual sample, the positive control, the negative control, and the allelic ladder is specified. These information settings are input to the sample information setting module 131 via the coupling interface 122 of the data analyzer 111.

[0050] The above is the description of the sample setting.

[0051] The electrophoresis device control module 132 transmits a signal instructing the start of analysis to the electrophoresis device 110. When the electrophoresis device 110 receives the signal, the electrophoresis device 110 starts the electrophoresis processing described below.

[0052] First, the electrophoresis device 110 fills new migration medium into the capillaries 202 to form a migration path (step S201). Filling the migration medium can be performed automatically after the start of the analysis or can be performed sequentially based on control signals transmitted from the electrophoresis device control module 132.

[0053] Specifically, the electrophoresis device 110 transports the waste liquid container 223 directly below the loading head 229 using the conveyor 225 and closes an electromagnetic valve 213 to allow the spent migration medium discharged from the capillary cathode end 227 to be received. The electrophoresis device 110 then drives the syringe 206, fills the capillary 202 with new migration medium, and discards the spent migration medium. Finally, the electrophoresis device 110 immerses the capillary cathode end 227 in the cleaning solution in the cleaning container 222 to clean the capillary cathode end 227 contaminated with the migration medium.

[0054] Next, the electrophoresis device 110 applies a predetermined voltage to the migration medium to perform a preprocess to bring the migration medium into a state suitable for electrophoresis (step S202). The preprocess may be performed automatically or may be performed sequentially based on control signals transmitted from the electrophoresis device control module 132.

[0055] Specifically, the electrophoresis device 110 uses the conveying device 225 to immerse the capillary cathode end 227 into the buffer in the buffer tank 221 to form a current-carrying path. Then, the electrophoresis device 110 applies a voltage of approximately several kilovolts to several tens of kilovolts from the high-voltage power source 204 to the migration medium for several minutes to several tens of minutes to bring the migration medium into a state suitable for electrophoresis. Finally, the electrophoresis device 110 immerses the capillary cathode end 227 into the cleaning solution in the cleaning tank 222 to clean the capillary cathode ends 227 contaminated with the buffer.

[0056] Next, the electrophoresis device 110 introduces the sample (step S203). Sample introduction may be performed automatically or may be performed sequentially based on control signals sent from the electrophoresis device control module 132.

[0057] Specifically, the electrophoresis device 110 uses the conveyor device 225 to immerse the capillary cathode end 227 into the sample held in the well of the sample plate 224, and then opens the electromagnetic valve 213. This forms a current-carrying path, allowing the sample components to be introduced into the migration path. The electrophoresis device 110 applies a pulse voltage from the high-voltage power source 204 to the current-carrying path to introduce the sample components into the migration path. Finally, the electrophoresis device 110 immerses the capillary cathode end 227 into the cleaning solution in the cleaning tank 222 to clean the capillary cathode end 227 contaminated with the sample.

[0058] Next, the electrophoresis device 110 performs electrophoresis analysis, separating and analyzing each sample component contained in the sample (step S204). The electrophoresis analysis may be performed automatically or may be performed sequentially based on control signals transmitted from the electrophoresis device control module 132.

[0059] Specifically, the electrophoresis device 110 uses the conveyor device 225 to immerse the capillary cathode end 227 into the buffer in the buffer tank 221 to form a current-carrying path. The electrophoresis device 110 applies a high voltage of approximately 15 kilovolts from the high-voltage power source 204 to the current-carrying path to generate an electric field in the migration path. The generated electric field causes each sample component in the migration path to move toward the detection unit 216 at a speed that depends on the characteristics of each sample component. That is, the sample components are separated based on the differences in the speed at which each sample component moves. Then, the sample components that have reached the detection unit 216 are detected sequentially.For example, if the sample contains a large number of DNA fragments with different base lengths, differences in the movement speed of the DNA fragments arise depending on the base length, and therefore the DNA fragments with a shorter base length reach the detection unit 216 before the DNA fragments with a longer base length. A fluorescent dye is attached to each DNA fragment, which depends on its terminal base sequence. When the detection unit 216 is irradiated with excitation light from the light source 214, fluorescence with a wavelength that depends on the sample is generated and emitted to the outside. The electrophoresis device 110 detects the fluorescence using the optical detector 215. During electrophoresis analysis, the optical detector 215 detects this fluorescence at regular time intervals and transmits image data to the data analysis device 111.Note that, to reduce the amount of information to be transmitted, the luminance of only a portion of the image data may be transmitted instead of the image data. For example, for each capillary 202, a luminance value obtained by sampling only wavelength positions at regular intervals may be transmitted. The data transmitted from the electrophoresis device 110 is time-series data of the luminance value of each capillary 202 (fluorescence intensity time-series data) and is stored in the storage device 121.

[0060] When the electrophoresis device 110 has acquired the expected image data, the electrophoresis device 110 stops applying voltage and terminates the electrophoresis analysis. The above is a description of the electrophoresis processing.

[0061] Fig. 5 is a flowchart illustrating spectral correction processing executed by the data analysis apparatus 111 in the first embodiment. Fig. 6 is a flowchart illustrating single fluorescence spectrum correction processing executed by the data analysis apparatus 111 in the first embodiment. Fig. 7 is a diagram illustrating images of the single fluorescence spectrum correction processing in the first embodiment. Fig. 8 is a flowchart illustrating non-single fluorescence spectrum correction processing executed by the data analysis apparatus 111 in the first embodiment. Fig. 9 is a diagram illustrating images of the non-single fluorescence spectrum correction processing in the first embodiment.

[0062] The spectral shift correction module 134 performs single fluorescence spectrum correction processing on time series data (electrophoresis data) of signal intensities of a plurality of frequencies (step S301) and then performs non-single fluorescence spectrum correction processing (step S302).

[0063] As used herein, “single fluorescence spectrum” means a spectrum derived from only one base, and “non-single fluorescence spectrum” means a spectrum derived from a plurality of bases.

[0064] In single fluorescence spectrum correction processing, the spectral shift correction module 134 uses the electrophoresis data to estimate a single fluorescence spectrum time (step S401). As used herein, "single fluorescence spectrum time" means the time at which a single fluorescence spectrum is detected. Examples of the estimation method may include the following.

[0065] The spectral shift correction module 134 compares the spectrum at time "t" and the spectrum obtained from a color conversion matrix. If the spectrum at time "t" is similar to the spectrum obtained from the color conversion matrix, the spectral shift correction module 134 determines that the spectrum at time "t" is a single fluorescence spectrum and records time "t" as the single fluorescence spectrum time. The color conversion matrix is ​​a matrix in which the elements are coefficients for obtaining individual fluorescence intensities from a spectral waveform. The color conversion matrix may be a color conversion matrix obtained in advance using a reagent called a “matrix standard” in which DNA fragments of different lengths are each labeled with a different fluorescent dye for use during electrophoresis (see, for example, JP 2014-117222 A).In addition, a color conversion matrix can be obtained each time a sample is run (see, for example, JP 2020-41876 A).

[0066] The spectral shift correction module 134 calculates a fluorescence intensity vector by applying a color conversion matrix to the spectrum (n-order vector) at time "t." If a pull-up component is equal to or less than a threshold value, that is, if a value other than the value of a given base component of the fluorescence intensity vector is equal to or less than a threshold value, the spectral shift correction module 134 determines that the spectrum at time "t" is a single fluorescence spectrum and records time "t" as the single fluorescence spectrum time.

[0067] Method 1 and Method 2 may be combined. Furthermore, a method in which estimation is performed using, for example, a difference in impulse response waveforms or an FFT characteristic of a wavelength spectrum may also be used.

[0068] The spectral shift correction module 134 selects a single fluorescence spectrum time from the individual fluorescence spectrum times estimated in step S401 (step S402). It is assumed that the selection is performed in chronological order.

[0069] The spectral shift correction module 134 calculates a spectral shift model at the individual fluorescence spectrum time (step S403). Specifically, the spectral shift correction module 134 calculates a spectral shift model using Expression (1). In Expression 1, ω represents the frequency, F0(ω) represents the Fourier transform of the spectrum obtained from the color conversion matrix, F1(ω) represents the Fourier transform of the individual fluorescence spectrum, and H M (ω) represents the spectral shift model. HM(ω)=F1(ω)F0(ω)

[0070] In this embodiment, the single fluorescence spectrum is regarded as a shifted spectrum obtained from the color conversion matrix, and an impulse response for correcting this shift is calculated as the spectral shift model.

[0071] The spectral shift correction module 134 corrects the spectral shift of the individual fluorescence spectrum using the spectral shift model (step S404). Specifically, the spectral shift correction module 134 corrects the spectral shift of the individual fluorescence spectrum by calculating H M (t) by performing an inverse Fourier transform on H M (ω) and folding of H M (t) with the individual fluorescence spectrum F(t).

[0072] The spectral shift correction module 134 determines whether or not the processing for all estimated individual fluorescence spectrum times is completed (step S405).

[0073] If the processing for all estimated individual fluorescence spectrum times is not completed, the spectral shift correction module 134 returns the process to step S402 and performs the same processing.

[0074] When the processing for all estimated individual fluorescence spectrum times is completed, the spectral shift correction module 134 terminates the individual fluorescence spectrum correction processing.

[0075] The single fluorescence spectrum correction processing used in Fig. 6 is an example, and this invention is not limited thereto. For example, a correction method as shown in Fig. 7 illustrates.

[0076] (Process step 1) The spectral shift correction module 134 calculates a spectral shift model for each individual fluorescence spectrum time.

[0077] (Process Step 2) The spectral shift correction module 134 selects a single fluorescence spectrum time t(n) and acquires a spectral shift model for the single fluorescence spectrum time t(n) and each of the preceding and subsequent single fluorescence spectrum times. It should also be noted that the spectral shift models of a predetermined number of single fluorescence spectrum times (a predetermined time range) before and after the single fluorescence spectrum time "t" can be acquired.

[0078] (Process step 3) The spectral shift correction module 134 calculates a plurality of correction spectra by convolving each spectral shift model with the single fluorescence spectrum F(t(n)) at the single fluorescence spectrum time t(n).

[0079] (Process step 4) The spectral shift correction module 134 outputs the linear sum of the plurality of correction spectra as the final correction result.

[0080] The above is the description of single fluorescence spectrum correction processing.

[0081] In the non-single fluorescence spectrum correction processing, the spectral shift correction module 134 selects a non-single fluorescence spectrum time (step S501). The non-single fluorescence spectrum time is different from the single fluorescence spectrum time estimated in step S401. It is assumed that the selections are performed in chronological order.

[0082] The spectral shift correction module 134 selects a spectral shift model to be used from the spectral shift models calculated in the single fluorescence spectrum correction processing (step S502).

[0083] In particular, as in Fig. As illustrated in Figure 9, the spectral shift correction module 134 acquires the spectral shift model of each individual fluorescence spectrum time t0 and t1 before and after a non-single fluorescence spectrum time t2. It should also be noted that the spectral shift model of a predetermined number of individual fluorescence spectrum times before and after a non-single fluorescence spectrum time may be acquired.

[0084] The spectral shift correction module 134 calculates a plurality of correction spectra by convolving each spectral shift model with the non-single fluorescence spectrum at the non-single fluorescence spectrum time (step S503).

[0085] The spectral shift correction module 134 acquires the linear sum of the plurality of correction spectra as the final correction result (step S504).

[0086] The spectral shift correction module 134 determines whether or not the processing for all non-single fluorescence spectrum times is completed (step S505).

[0087] If the processing for all non-single fluorescence spectrum times is not completed, the spectral shift correction module 134 returns the process to step S501 and performs the same processing.

[0088] When the processing for all non-single fluorescence spectrum times is completed, the spectral shift correction module 134 ends the non-single fluorescence spectrum correction processing.

[0089] Whether the spectral correction processing in this embodiment functions effectively can be confirmed by the following method. Migration data in which non-single fluorescence spectra are present in a wide electrophoresis time band at a fixed mixing ratio and a single fluorescence spectrum is present in a part of the time band are input to the spectral shift correction module 134. If the mixing ratio between the non-single fluorescence spectra near a single fluorescence spectrum and other non-single fluorescence spectra among the non-single fluorescence spectra obtained by the spectral shift correction module 134 differs significantly, this indicates that the spectral correction processing in this embodiment functions effectively.

[0090] According to this embodiment, pull-ups caused by spectral shift can be reduced through information processing. As a result, the accuracy of base sequence analysis can be improved. This embodiment also has a feature of high versatility because it does not depend on the implementation of the electrophoresis device 110.

[0091] The present invention is not limited to the above embodiment and includes various modification examples. In addition, for example, the configurations of the above embodiment will be described in detail to clearly describe the present invention. The present invention is not necessarily limited to the embodiment provided with all of the described configurations. In addition, a part of each configuration of the embodiment may be removed, replaced, or added to other configurations.

[0092] Part or all of each of the above configurations, functions, processing units, processing means, and the like can be realized by hardware such as by designing integrated circuits therefor. In addition, the present invention can be realized by program codes of software that realizes the functions of the embodiment. In this case, a storage medium on which the program codes are recorded is provided to a computer, and a CPU equipped in the computer reads the program codes stored on the storage medium. In this case, the program codes read from the storage medium realize the functions of the above embodiment, and the program codes and the storage medium storing the program codes constitute the present invention.Examples of such a storage medium used to provide program code include a flexible floppy disk, a CD-ROM, a DVD-ROM, a hard disk, a solid-state drive (SSD), an optical disk, a magneto-optical disk, a CD-R, a magnetic tape, a non-volatile memory card, and a ROM.

[0093] The program codes that realize the functions written in the present embodiment can be implemented by a wide range of programming and scripting languages ​​such as assembly, C / C++, Perl, shell scripts, PHP, Python, and Java.

[0094] It may also be possible for the program codes of the software that realizes the functions of the embodiment to be stored on storage means such as a hard disk or memory of the computer or on a storage medium such as a CD-RW or a CD-R by distributing the program codes over a network, and for the CPU equipped in the computer to read and execute the program codes stored on the storage means or on the storage medium.

[0095] In the above embodiment, only control lines and information lines considered necessary for description are shown, and all control lines and information lines of a product are not necessarily shown. All configurations of the embodiment can be connected to each other. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] JP 2006-292368 A [0002, 0003] JP 2014-117222 A [0002, 0065] JP 2020-41876 A [0002, 0065]

Claims

[1] A base sequence analysis method performed by a genetic analyzer configured to analyze a base sequence of a sample, the base sequence analysis method comprising: a first step of acquiring, by the genetic analyzer, electrophoresis data which is time series data of signal intensities of a plurality of frequencies acquired by subjecting the sample to electrophoresis; a second step of identifying, by the genetic analyzer, a single fluorescence spectrum time at which a single fluorescence spectrum, which is a spectrum derived from only one base, is present, using the electrophoresis data; a third step of calculating, by the genetic analyzer, a spectral shift model using a spectrum obtained from a color conversion matrix at the single fluorescence spectrum time and the single fluorescence spectrum at the single fluorescence spectrum time; a fourth step of correcting, by the genetic analyzer, the electrophoresis data using the spectral shift model; and a fifth step of identifying, by the genetic analyzer, a base sequence of the sample using the corrected electrophoresis data. [2] Base sequence analysis method according to claim 1, where the spectral shift model is a function of a frequency space, and wherein the fourth step includes a step of correcting, by the genetic analyzer, a spectrum at a given time by performing a convolution operation using the spectrum at the given time and the spectral shift model. [3] The base sequence analysis method according to claim 2, wherein the fourth step includes: a sixth step of correcting, by the genetic analyzer, the single fluorescence spectrum to the single fluorescence spectrum time using the spectral shift model to the single fluorescence spectrum time; and a seventh step of correcting, by the genetic analyzer, at a time other than the single fluorescence spectrum time, a spectrum at the time using the spectral shift model to at least one single fluorescence spectrum time close to the time. [4] The base sequence analysis method according to claim 3, wherein the sixth step includes the following steps: Selecting, by the genetic analyzer, a first single fluorescence spectrum time and detecting the spectral shift model at the first single fluorescence spectrum time; detecting, by the genetic analysis device, the spectral shift model at another single fluorescence spectrum time included in a predetermined time range including the first single fluorescence spectrum time; and Correcting the single fluorescence spectrum to the first single fluorescence spectrum time using a plurality of the spectral shift models. [5] A genetic analysis device for analyzing a base sequence of a sample, the genetic analysis device comprising: a calculation device; and a storage device coupled to the computing device; wherein the computing device is configured to: to acquire electrophoresis data, which is time series data of signal intensities of a plurality of frequencies acquired by subjecting the sample to electrophoresis; to identify a single fluorescence spectrum time at which a single fluorescence spectrum, which is a spectrum derived from only one base, is present using the electrophoresis data; calculate a spectral shift model using a spectrum obtained from a color conversion matrix at the single fluorescence spectrum time and the single fluorescence spectrum at the single fluorescence spectrum time; correct the electrophoresis data using the spectral shift model; and to identify a base sequence of the sample using the corrected electrophoresis data and to output an analysis result of the base sequence of the sample. [6] Gene analysis device according to claim 5, where the spectral shift model is a function of a frequency space, and wherein the calculation device is configured to correct a spectrum at a given time by performing a convolution operation using the spectrum at the given time and the spectral shift model. [7] A genetic analysis device according to claim 6, wherein the calculation device is configured to perform: a first correction processing of correcting the single fluorescence spectrum at the single fluorescence spectrum time using the spectral shift model at the single fluorescence spectrum time; and a second correction processing of correcting, at a time other than the single fluorescence spectrum time, a spectrum at the time using the spectral shift model to at least one single fluorescence spectrum time near the time. [8] The genetic analysis apparatus according to claim 7, wherein the calculation device is configured to, in the first correction processing: selecting a first single fluorescence spectrum time and acquiring the spectral shift model at the first single fluorescence spectrum time; acquire the spectral shift model at another single fluorescence spectrum time contained within a predetermined time range including the first single fluorescence spectrum time; and to correct the single fluorescence spectrum to the first single fluorescence spectrum time using a plurality of spectral shift models.

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