Method for analyzing base sequences and genetic analysis device

Abstract

A gene analyzer that: obtains phoretic data which is time-series data of the signal intensity of multiple frequencies obtained by electrophoresing a sample; by using the phoretic data, identifies a single fluorescence spectral time at which a single fluorescence spectrum, a spectrum derived from only one base, is present; calculates a spectral shift model by using a spectrum obtained from a color transformation matrix at the single fluorescence spectral time and the single fluorescence spectrum at the single fluorescence spectral time; corrects the phoretic data by using the spectral shift model; and identifies the base sequence of the sample by using the corrected phoretic data.

Description

[Technical Field]

[0001] The present invention relates to a genetic analysis device and method for analyzing the base sequence of a sample using electrophoresis. [Background technology]

[0002] A capillary electrophoresis apparatus obtains electrophoresis data by irradiating excitation light while separating DNA fragments through electrophoresis. Because the excitation light has a certain width, it is known that pull-up (false peaks) due to spectral shifts occur (see, for example, paragraphs 0050 and 0051 of Patent Document 1). Pull-up reduces the accuracy of base sequence analysis. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-292368 [Patent Document 2] Japanese Patent Application Laid-Open No. 2014-117222 [Patent Document 3] Japanese Patent Publication No. 2020-41876 Summary of the Invention [Problem to be solved by the invention]

[0004] Patent Document 1 discloses a detection optical unit that reduces pull-up. However, because the spectral shift depends on the error of the optical system of each device and on environmental differences such as migration voltage and temperature, it is difficult to accurately model it. In this invention, a method for reducing pull-up is realized by information processing. [Means for solving the problem]

[0005] A representative example of the invention disclosed in the present application is as follows: That is, a base sequence analysis method executed by a genetic analyzer that analyzes the base sequence of a sample, the base sequence analysis method including: a first step in which the genetic analyzer acquires electrophoresis data, which is time-series data of signal intensities at multiple frequencies obtained by electrophoresis of a sample; a second step in which the genetic analyzer uses the electrophoresis data to identify a single fluorescence spectrum time at which a single fluorescence spectrum, which is a spectrum derived from only one base, is present; a third step in which the genetic analyzer calculates a spectral shift model using a spectrum obtained from a color transformation matrix at the single fluorescence spectrum time and the single fluorescence spectrum at the single fluorescence spectrum time; a fourth step in which the genetic analyzer corrects the electrophoresis data using the spectral shift model; and a fifth step in which the genetic analyzer identifies the base sequence of the sample using the corrected electrophoresis data. [Effects of the Invention]

[0006] According to the present invention, by regarding a single fluorescence spectrum as a shifted spectrum obtained from a color conversion matrix, pull-up can be reduced by information processing, and the accuracy of base sequence analysis can be improved. Other problems, configurations, and advantages will become clearer in the following examples. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a diagram showing an example of the configuration of a gene analysis device according to a first embodiment. [Figure 2] FIG. 1 is a diagram illustrating an example of the configuration of an electrophoresis apparatus according to a first embodiment. [Figure 3] 1 is a flowchart illustrating an outline of a process executed by a gene analysis device according to a first embodiment. [Figure 4] 4 is a flowchart illustrating an electrophoresis process performed by the electrophoresis apparatus of the first embodiment. [Figure 5]4 is a flowchart illustrating a spectrum correction process executed by the data analysis device according to the first embodiment. [Figure 6] 10 is a flowchart illustrating a correction process for a single fluorescence spectrum executed by the data analysis device of the first embodiment. [Figure 7] FIG. 1 is a diagram showing an image of the correction process for a single fluorescence spectrum in Example 1. [Figure 8] 10 is a flowchart illustrating a correction process for a non-single fluorescence spectrum executed by the data analysis device of the first embodiment. [Figure 9] FIG. 1 is a diagram showing an image of the correction process for non-single fluorescence spectrum in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0008] Hereinafter, examples of the present invention will be described with reference to the drawings. However, the present invention should not be construed as being limited to the description of the embodiments shown below. Those skilled in the art will readily understand that the specific configuration can be changed within the scope of the idea or purpose of the present invention.

[0009] In the configuration of the invention described below, the same or similar configurations or functions are denoted by the same reference numerals, and redundant explanations will be omitted.

[0010] In this specification, the terms "first," "second," "third," etc. are used to identify components and do not necessarily limit the number or order.

[0011] To facilitate understanding of the invention, the position, size, shape, range, etc. of each component shown in the drawings etc. may not represent the actual position, size, shape, range, etc. Therefore, the present invention is not limited to the position, size, shape, range, etc. disclosed in the drawings etc. [Example]

[0012] FIG. 1 is a diagram showing an example of the configuration of a gene analysis device 100 according to the first embodiment.

[0013] 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 communicably connected using a communication cable.

[0014] The data analysis device 111 includes a control device 120 , a storage device 121 , and a connection interface 122 .

[0015] The control device 120 controls and processes data of the electrophoretic device 110. The control device 120 is, for example, a central processing unit (CPU) and a graphics processing unit (GPU).

[0016] The storage device 121 stores programs executed by the control device 120, setting information for the electrophoresis device 110, information used for various processes, etc. The storage device 121 is, for example, a memory.

[0017] The connection interface 122 is an interface for connecting to an input device and an output device, or an interface for connecting to an external device via a network. The data analysis device 111 presents information to a user via the connection interface 122, and also accepts information input by the user.

[0018] The control device 120 executes a program stored in the storage device 121 to operate as a sample information setting unit 131, an electrophoresis apparatus control unit 132, a fluorescence intensity calculation unit 133, a spectral shift correction unit 134, and a base call unit 135. In the following description, when a process is described using a functional unit as the subject, it means that the control device 120 is executing the program.

[0019] The electrophoresis device 110 electrophoreses the sample (DNA fragments) and acquires electrophoresis data, which is time-series data of signal intensities (brightness values) at multiple frequencies.

[0020] Here, a description will be given of the configuration of the electrophoresis device 110. Fig. 2 is a diagram showing an example of the configuration of the electrophoresis device 110 of the first embodiment.

[0021] The electrophoresis apparatus 110 includes a detection unit 216 , a thermostatic bath 218 , a transporter 225 , a high-voltage power supply 204 , a first ammeter 205 , an anode electrode 211 , a second ammeter 212 , a capillary array 217 , and a pump mechanism 203 .

[0022] The capillary array 217 is a replaceable component including a plurality of (e.g., eight) capillaries 202, and includes a load header 229, a detection unit 216, and a capillary head 233. When a capillary 202 is damaged or its quality deteriorates, the capillary array 217 can be replaced with a new one.

[0023] The capillary 202 is composed of a glass tube with an inner diameter of several tens to several hundreds of microns and an outer diameter of several hundred microns, and its surface is coated with polyimide to improve its strength. However, the light irradiation section where the laser light is irradiated has a structure in which the polyimide coating has been removed to allow the internal light to easily leak to the outside. The inside of the capillary 202 is filled with a separation medium that creates a difference in migration speed during electrophoresis. Separation media are available in both fluid and non-fluid forms, but in Example 1, a fluid polymer is used.

[0024] The high-voltage power supply 204 applies a high voltage to the capillary 202. The first ammeter 205 detects the current generated from the high-voltage power supply 204. The second ammeter 212 detects the current flowing through the anode electrode 211.

[0025] The optical detection unit that detects the information light obtained from the sample is composed of a light source 214 that irradiates the detection unit 216 with excitation light, an optical detector 215 that detects the luminescence in the detection unit 216, and a diffraction grating 232. The detection unit 216 is a component that acquires information that depends on the sample.

[0026] When detecting a sample in the capillary 202 separated by electrophoresis, the detection unit 216 is irradiated with excitation light from the light source 214, which generates fluorescence having a wavelength dependent on the sample as information light. Furthermore, the diffraction grating 232 separates the information light in the wavelength direction, and the optical detector 215 detects the dispersed information light to analyze the sample.

[0027] The capillary cathode ends 227 are fixed through respective metal hollow electrodes 226, and the tips of the capillaries 202 protrude about 0.5 mm from the hollow electrodes 226. All of the hollow electrodes 226 provided on each capillary 202 are mounted integrally on a load header 229. All of the hollow electrodes 226 are electrically connected to a high-voltage power supply 204 mounted on the main body of the apparatus, and function as cathode electrodes when voltage application is required for electrophoresis, sample introduction, etc.

[0028] The capillary cathode end 227 and the opposite end (other end) of the capillary are bundled together by a capillary head 233. The capillary head 233 can be connected to the block 207 in a pressure-tight sealed manner. A high voltage is applied between the load header 229 and the capillary head 233 by a high-voltage power supply 204. Then, a new polymer is filled into the capillary 202 from the other end by a syringe 206. The polymer in the capillary 202 is refilled for each measurement to improve measurement performance.

[0029] The pump mechanism 203 is composed of a syringe 206 and a mechanism for pressurizing the syringe 206 , and injects the polymer into the capillary 202 .

[0030] The block 207 is a connection portion for communicating the syringe 206, the capillary array 217, the anode buffer container 210, and the polymer container 209, respectively.

[0031] The thermostatic bath 218 is covered with a heat insulating material to keep the capillaries 202 in the thermostatic bath 218 at a constant temperature, and the temperature is controlled by a heating and cooling mechanism 220. A fan 219 circulates and agitates the air in the thermostatic bath 218, keeping the temperature of the capillary array 217 uniform and constant across its position.

[0032] The transporter 225 transports various containers to the capillary cathode end 227. The transporter 225 is equipped with three electric motors and linear actuators, and is movable in three axial directions: up and down, left and right, and depth. At least one container can be placed on the moving stage 230 of the transporter 225. The moving stage 230 is further equipped with an electric grip 231, which can grasp and release each container. Therefore, the buffer container 221, the washing container 222, the waste container 223, and the sample plate 224 can be transported to the capillary cathode end 227 as needed. Unnecessary containers are stored in a designated storage location within the electrophoresis apparatus 110.

[0033] A user can use the data analysis device 111 to control various functions of the electrophoresis device 110 and obtain electrophoresis data detected by the optical detection unit.

[0034] The electrophoresis device 110 may include sensors for acquiring information about the observation environment that affects electrophoresis (observation environment information). The electrophoresis device 110 in FIG. 2 includes an in-device sensor 240, a polymer sensor 241, and a buffer solution sensor 242.

[0035] The internal sensor 240 is a sensor for acquiring information about the internal environment of the electrophoresis device 110, and measures, for example, a temperature sensor, a humidity sensor, and an air pressure sensor within the electrophoresis device 110.

[0036] The polymer sensor 241 is a sensor for acquiring information about the quality of the polymer, such as a pH sensor, an electrical conductivity sensor, etc. Although the polymer sensor 241 is installed in the polymer container 209 in FIG. 2, the installation location is not limited thereto.

[0037] The buffer solution sensor 242 is a sensor for obtaining information about the quality of the buffer solution, and may be, for example, a temperature sensor. Although the buffer solution sensor 242 is shown installed in the anode buffer container 210 in FIG. 2, the installation location is not limited thereto. For example, the buffer solution sensor 242 may be installed in the buffer container 221.

[0038] FIG. 3 is a flowchart outlining the process executed by the genetic analysis device 100 of the first embodiment.

[0039] The electrophoresis device 110 of the genetic analysis device 100 performs electrophoresis processing on the sample to be analyzed (step S101). Figure 4 This will be explained using:

[0040] Next, the data analysis device 111 of the genetic analysis device 100 executes a spectrum correction process using the electrophoresis data (step S102). Details of the spectrum correction process will be explained using FIG.

[0041] Next, the data analysis device 111 of the genetic analysis device 100 executes a fluorescence intensity calculation process using the corrected electrophoresis data (step S103). Specifically, the fluorescence intensity calculation unit 133 calculates time-series data of the fluorescence intensity of the fluorescent dye from the corrected electrophoresis data, and detects the center position, height, width, etc. of the peak from the time-series data of the fluorescence intensity.

[0042] Next, the data analysis device 111 of the genetic analysis device 100 executes a mobility correction process on the time-series data of the fluorescence intensity (step S104).

[0043] Next, the data analyzer 111 of the genetic analyzer 100 executes base calling using the time-series data of the fluorescence intensity corrected based on the result of the mobility correction process (step S105). Specifically, the base calling unit 135 identifies the base sequence of the sample using the time-series data of the corrected fluorescence intensity.

[0044] FIG. 4 is a flowchart illustrating the electrophoresis process executed by the electrophoresis device 110 of the first embodiment.

[0045] The user sets a sample to be analyzed, a reagent, and the like in the electrophoresis device 110, and issues an instruction to start the electrophoresis process via the connection interface 122. The sample is set in the following procedure.

[0046] The user fills the buffer reservoir 221 and the anode buffer reservoir 210 with a buffer solution that forms part of the current path. The buffer solution is, for example, an electrolyte solution commercially available from various companies for electrophoresis. The user dispenses a sample to be analyzed into the wells of the sample plate 224. The sample is, for example, a DNA PCR product. The user dispenses a cleaning solution for cleaning the capillary cathode end 227 into the cleaning reservoir 222. The cleaning solution is, for example, pure water. The user injects a migration medium for electrophoresis of the sample into the syringe 206. The migration medium is, for example, a polyacrylamide-based separation gel or polymer commercially available from various companies for electrophoresis. The user replaces the capillary array 217 when deterioration of the capillaries 202 is expected or when the length of the capillaries 202 is to be changed.

[0047] At this time, the samples set on the sample plate 224 include the actual sample of DNA to be analyzed, as well as a positive control, a negative control, an allelic ladder, etc., each of which is electrophoresed in a different capillary 202 .

[0048] A positive control is, for example, a PCR product containing known DNA, and is a control sample used to verify that the PCR amplification is correct. A negative control is a PCR product containing no DNA, and is a control sample used to verify that the PCR amplification product is free of contamination from the user's DNA or dust. An allelic ladder is an artificial sample containing many bases that are likely to be commonly found in DNA markers, and is usually provided by reagent manufacturers as part of a DNA testing kit. Allelic ladders are used to fine-tune the correspondence between the DNA fragment length of each DNA marker and its allele.

[0049] In addition, all of the actual samples, positive controls, negative controls, and allelic ladders are mixed with known DNA fragments labeled with specific fluorescent dyes, called size standards. The type of fluorescent dye assigned to the size standard varies depending on the reagent kit used.

[0050] The user specifies the type of allelic ladder, the type of size standard, the type of fluorescent reagent, and the type of sample set in the well on the sample plate 224 corresponding to each capillary 202. In Example 1, the type is specified as either a real sample, a positive control, a negative control, or an allelic ladder. The settings of this information are input to the sample information setting unit 131 via the connection interface 122 of the data analysis device 111.

[0051] This concludes the description of the sample set.

[0052] The electrophoresis apparatus control unit 132 transmits a signal instructing the start of analysis to the electrophoresis apparatus 110. Upon receiving the signal, the electrophoresis apparatus 110 starts the electrophoresis process described below.

[0053] The electrophoresis device 110 first fills the capillary 202 with a new migration medium to form a migration path (step S201). The filling of the migration medium may be performed automatically after the start of analysis, or may be performed sequentially based on a control signal transmitted from the electrophoresis device control unit 132.

[0054] Specifically, the electrophoresis apparatus 110 uses the transport device 225 to transport the waste liquid container 223 to directly below the load header 229, closes the solenoid valve 213, and allows the used migration medium discharged from the capillary cathode end 227 to be received. Then, the electrophoresis apparatus 110 drives the syringe 206 to fill the capillary 202 with new migration medium and discards the used migration medium. Finally, the electrophoresis apparatus 110 immerses the capillary cathode end 227 in a cleaning solution in the cleaning container 222 to clean the capillary cathode end 227 contaminated with the migration medium.

[0055] Next, the electrophoresis device 110 applies a predetermined voltage to the migration medium to perform a preliminary migration to prepare the migration medium for electrophoresis (step S202). The preliminary migration may be performed automatically or sequentially based on a control signal transmitted from the electrophoresis device control unit 132.

[0056] Specifically, the electrophoresis apparatus 110 uses the conveyor 225 to immerse the capillary cathode end 227 in the buffer solution in the buffer container 221 to form a current path. Then, the electrophoresis apparatus 110 uses the high-voltage power supply 204 to apply a voltage of several to several tens of kilovolts to the migration medium for several to several tens of minutes to make the migration medium suitable for electrophoresis. Finally, the electrophoresis apparatus 110 immerses the capillary cathode end 227 in a cleaning solution in the cleaning container 222 to clean the capillary cathode end 227 contaminated by the buffer solution.

[0057] Next, the electrophoresis apparatus 110 introduces the sample (step S203). The introduction of the sample may be performed automatically, or may be performed sequentially based on a control signal transmitted from the electrophoresis apparatus control unit 132.

[0058] Specifically, the electrophoresis device 110 uses the transporter 225 to immerse the capillary cathode end 227 in the sample held in the well of the sample plate 224, and then opens the solenoid valve 213. This forms a current path, and the sample components are transported to the migration path. Ready to be introduced The electrophoresis device 110 applies a pulse voltage to the current path using the high-voltage power supply 204, thereby introducing the sample components into the migration path. Finally, the electrophoresis device 110 immerses the capillary cathode end 227 in a washing solution in the washing container 222, and washes the capillary cathode end 227 contaminated with the sample.

[0059] Next, the electrophoresis device 110 performs electrophoresis analysis to separate and analyze each sample component contained in the sample (step S204). The electrophoresis analysis may be performed automatically or sequentially based on control signals transmitted from the electrophoresis device control unit 132.

[0060] Specifically, the electrophoresis device 110 uses the transporter 225 to immerse the capillary cathode end 227 in the buffer solution in the buffer container 221, forming a current path. The electrophoresis device 110 uses the high-voltage power supply 204 to apply a high voltage of approximately 15 kV to the current path, generating an electric field in the migration path. The generated electric field causes each sample component in the migration path to move to the detection unit 216 at a speed dependent on the properties of each sample component. In other words, the sample components are separated based on the difference in their migration speed. The sample components are then detected in order, starting with the sample components that reach the detection unit 216. For example, if a sample contains multiple DNAs with different base lengths, differences in migration speed will occur depending on the base lengths, and the DNAs with the shortest base lengths will reach the detection unit 216 in order. Each DNA is attached with a fluorescent dye that 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 dependent on the sample is generated and emitted to the outside. The electrophoresis device 110 detects 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, in order to reduce the amount of information to be transmitted, it is also possible to transmit the luminance of only a portion of the image data, rather than the image data. For example, it is also possible to transmit luminance values ​​sampled only at wavelength positions at regular intervals for each capillary 202. The data transmitted from the electrophoresis device 110 is time-series data of the luminance values ​​of each capillary 202 (fluorescence intensity time-series data), and is stored in the storage device 121.

[0061] When the expected image data has been acquired, the electrophoresis device 110 stops applying voltage and ends the electrophoresis analysis. This concludes the description of the electrophoresis process.

[0062] FIG. 5 is a flowchart illustrating the spectrum correction process executed by the data analysis device 111 of the first embodiment. FIG. 6 is a flowchart illustrating the correction process for a single fluorescence spectrum executed by the data analysis device 111 of the first embodiment. FIG. 7 is a diagram illustrating an image of the correction process for a single fluorescence spectrum of the first embodiment. FIG. 8 is a flowchart illustrating the correction process for a non-single fluorescence spectrum executed by the data analysis device 111 of the first embodiment. FIG. 9 is a diagram illustrating an image of the correction process for a non-single fluorescence spectrum of the first embodiment.

[0063] The spectral shift correction unit 134 performs correction processing for a single fluorescence spectrum on time-series data (electrophoresis data) of signal intensities at a plurality of frequencies (step S301), and then performs correction processing for non-single fluorescence spectra (step S302).

[0064] Here, a single fluorescence spectrum refers to a spectrum derived from only one base, whereas a non-single fluorescence spectrum refers to a spectrum derived from multiple bases.

[0065] In the correction process for a single fluorescence spectrum, the spectral shift correction unit 134 estimates a single fluorescence spectrum time using electrophoresis data (step S401). Here, the single fluorescence spectrum time means the time when a single fluorescence spectrum is detected. The following estimation methods are possible:

[0066] (Method 1) The spectral shift correction unit 134 compares the spectrum at time t with the spectrum obtained from the color transformation matrix. If the spectrum at time t is similar to the spectrum obtained from the color transformation matrix, the spectral shift correction unit 134 determines that the spectrum at time t is a single fluorescence spectrum and records the time t as the time of the single fluorescence spectrum. Here, the color transformation matrix is ​​a matrix whose elements are coefficients for obtaining individual fluorescence intensities from the spectral waveform. The color transformation matrix may be calculated in advance using reagents called matrix standards, in which DNA fragments of different lengths are labeled with respective fluorescent dyes, and this may be used during electrophoresis (see, for example, Patent Document 2). Alternatively, the color transformation matrix may be calculated each time a sample is electrophoresed (see, for example, Patent Document 3).

[0067] (Method 2) The spectral shift correction unit 134 calculates a fluorescence intensity vector by applying a color transformation matrix to the spectrum (n-th order vector) at time t. If the pull-up component is equal to or less than the threshold, that is, if the values ​​of the fluorescence intensity vector other than the components of any base are equal to or less than the threshold, the spectral shift correction unit 134 determines that the spectrum at time t is a single fluorescence spectrum and records that time t as the time of the single fluorescence spectrum.

[0068] It is also possible to combine Method 1 and Method 2. In addition, a method of performing estimation using differences in impulse response waveforms, FFT characteristics of wavelength spectra, etc. may also be considered.

[0069] The spectral shift correction unit 134 selects one single fluorescence spectrum time from the single fluorescence spectrum times estimated in step S401 (step S402). Here, it is assumed that the selection is made in chronological order.

[0070] The spectral shift correction unit 134 calculates the spectral shift model at the single fluorescence spectrum time (step S403). Specifically, the spectral shift correction unit 134 calculates the spectral shift model using equation (1). Here, ω represents frequency, F0(ω) represents the Fourier transform of the spectrum obtained from the color transformation matrix, F1(ω) represents the Fourier transform of the single fluorescence spectrum, and H M (ω) represents the spectral shift model.

[0071]

number

[0072] In this example, the single fluorescence spectrum is considered to be a spectrum obtained by shifting the spectrum obtained from the color conversion matrix, and an impulse response for correcting this shift is calculated as a spectrum shift model.

[0073] The spectral shift correction unit 134 corrects the spectral shift of the single fluorescence spectrum using the spectral shift model (step S404). M (ω) is inverse Fourier transformed to H M (t) and calculate the single fluorescence spectrum F(t) using H M Correction for spectral shifts is performed on a single fluorescence spectrum by convolving (t).

[0074] The spectral shift correction unit 134 determines whether or not the processing has been completed for all the estimated single fluorescence spectrum times (step S405).

[0075] If the processing has not been completed for all estimated single fluorescence spectrum times, the spectral shift correcting unit 134 returns to step S402 and executes the same processing.

[0076] When the processing is completed for all estimated single fluorescence spectrum times, the spectral shift correction unit 134 ends the correction processing for the single fluorescence spectrum.

[0077] The correction process for the single fluorescence spectrum shown in Fig. 6 is an example and is not limiting. For example, a correction method such as that shown in Fig. 7 is also possible.

[0078] (Step 1) The spectral shift correction unit 134 calculates a spectral shift model for each single fluorescence spectrum time.

[0079] (Step 2) The spectral shift correction unit 134 selects a single fluorescence spectrum time t(n) and obtains spectral shift models for the single fluorescence spectrum time t(n) and each of the single fluorescence spectrum times before and after it. Note that spectral shift models for a predetermined number of single fluorescence spectrum times before and after (a predetermined time range) centered on the single fluorescence spectrum time t may also be obtained.

[0080] (Step 3) The spectral shift correction unit 134 calculates a plurality of corrected spectra by convolving each spectral shift model with the single fluorescence spectrum F(t(n)) at the single fluorescence spectrum time t(n).

[0081] (Step 4) The spectrum shift correction unit 134 outputs the linear sum of the multiple corrected spectra as the final correction result.

[0082] The above is the explanation of the correction process for a single fluorescence spectrum.

[0083] In the correction process for a non-single fluorescence spectrum, the spectral shift correction unit 134 selects a non-single fluorescence spectrum time (step S501). The non-single fluorescence spectrum time is a time other than the single fluorescence spectrum time estimated in step S401. Here, the selection is made in chronological order.

[0084] The spectral shift correction unit 134 selects a spectral shift model to be used from among the spectral shift models calculated in the correction process for the single fluorescence spectrum (step S502).

[0085] 9, the spectral shift correction unit 134 acquires spectral shift models for the single-mode fluorescence spectrum times t0 and t1 before and after the non-single-mode fluorescence spectrum time t2. Note that spectral shift models for a predetermined number of single-mode fluorescence spectrum times before and after the non-single-mode fluorescence spectrum time may also be acquired.

[0086] The spectral shift correction unit 134 calculates a plurality of corrected spectra by convolving each spectral shift model with the non-single fluorescence spectrum at the non-single fluorescence spectrum time (step S503).

[0087] The spectral shift corrector 134 obtains the linear sum of the multiple corrected spectra as the final correction result (step S504).

[0088] The spectral shift correction unit 134 determines whether or not the process has been completed for all non-single fluorescence spectrum times (step S505).

[0089] If the processing has not been completed for all non-single fluorescence spectrum times, the spectral shift corrector 134 returns to step S501 and executes the same processing.

[0090] When the processing is completed for all non-single fluorescence spectrum times, the spectral shift correction unit 134 ends the correction processing for the non-single fluorescence spectrum.

[0091] Whether the spectral correction process of this embodiment is functioning effectively can be confirmed by the following method: Electrophoresis data in which non-single-stranded fluorescence spectra are present at a certain mixing ratio over a wide electrophoresis time period, and in which single-stranded fluorescence spectra are also present in some areas, is input to the spectral shift correction unit 134. If the non-single-stranded fluorescence spectrum obtained from the spectral shift correction unit 134 has a significantly different mixing ratio between the non-single-stranded fluorescence spectra near the single-stranded fluorescence spectrum and other non-single-stranded fluorescence spectra, this indicates that the spectral correction process of this embodiment is functioning effectively.

[0092] According to this embodiment, pull-up caused by spectral shift can be reduced by information processing. This improves the accuracy of base sequence analysis. This embodiment is not dependent on the implementation of the electrophoresis device 110, and therefore has the advantage of being highly versatile.

[0093] The present invention is not limited to the above-described embodiments, but includes various modifications. For example, the above-described embodiments are provided to explain the present invention in detail, and the present invention is not necessarily limited to those including all of the described configurations. Furthermore, some of the configurations of each embodiment can be added to, deleted from, or replaced with other configurations.

[0094] Furthermore, the above-described configurations, functions, processing units, processing means, etc. may be partially or entirely implemented in hardware, for example, by designing them as integrated circuits. The present invention can also be realized by software program code that implements the functions of the embodiments. In this case, a storage medium on which the program code is recorded is provided to a computer, and a processor included in the computer reads the program code stored in the storage medium. In this case, the program code itself read from the storage medium implements the functions of the above-described embodiments, and the program code itself and the storage medium on which it is stored constitute the present invention. Examples of storage media for providing such program code include flexible disks, CD-ROMs, DVD-ROMs, hard disks, solid-state drives (SSDs), optical disks, magneto-optical disks, CD-Rs, magnetic tapes, non-volatile memory cards, and ROMs.

[0095] Furthermore, the program code that realizes the functions described in this embodiment can be implemented in a wide range of program or script languages, such as assembler, C / C++, perl, Shell, PHP, Python, and Java.

[0096] Furthermore, the program code of the software that realizes the functions of the embodiments may be distributed via a network and stored in a storage means such as a computer's hard disk or memory, or in a storage medium such as a CD-RW or CD-R, and the processor of the computer may read and execute the program code stored in the storage means or storage medium.

[0097] In the above-described embodiment, the control lines and information lines are those that are considered necessary for the explanation, and not all control lines and information lines are necessarily shown in the product. All components may be interconnected.

Claims

1. A method for analyzing a base sequence performed by a genetic analyzer that analyzes the base sequence of a sample, comprising: The method for analyzing a base sequence comprises: a first step in which the genetic analyzer acquires electrophoresis data, which is time-series data of signal intensities at a plurality of frequencies acquired by electrophoresis of a sample; a second step in which the genetic analysis device uses the electrophoresis data to identify a single fluorescence spectrum time at which a single fluorescence spectrum, which is a spectrum derived from only one base, is present; a third step in which the genetic analysis device calculates a spectral shift model using a spectrum obtained from a color transformation matrix at the single fluorescence spectrum time and the single fluorescence spectrum at the single fluorescence spectrum time; a fourth step in which the genetic analysis device corrects the electrophoresis data using the spectral shift model; a fifth step in which the genetic analyzer identifies the base sequence of the sample using the corrected electrophoresis data; A method for analyzing a base sequence, comprising:

2. The method for analyzing a base sequence according to claim 1, The spectral shift model is a function of frequency space, The fourth step of the method for analyzing a base sequence comprises a step in which the genetic analysis device corrects the spectrum at the arbitrary time by performing a convolution operation using the spectrum at the arbitrary time and the spectral shift model.

3. The method for analyzing a base sequence according to claim 2, The fourth step includes: a sixth step in which the genetic analysis device corrects the single fluorescence spectrum at the single fluorescence spectrum time using the spectral shift model at the single fluorescence spectrum time; a seventh step in which the genetic analysis device corrects the spectrum at a time other than the single fluorescence spectrum time by using the spectral shift model for at least one single fluorescence spectrum time near the time; A method for analyzing a base sequence, comprising:

4. The method for analyzing a base sequence according to claim 3, The sixth step includes: the genetic analysis device selecting a first single fluorescence spectrum time and acquiring the spectral shift model at the first single fluorescence spectrum time; a step in which the genetic analysis device acquires 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 at 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, a computing device and a storage device connected to the computing device, The computing device acquiring electrophoresis data, which is time-series data of signal intensities at a plurality of frequencies obtained by electrophoresis of the sample; using the electrophoresis data, identifying a single fluorescence spectrum time at which a single fluorescence spectrum, which is a spectrum derived from only one base, exists; calculating a spectral shift model using a spectrum obtained from a color transformation matrix at the single fluorescence spectrum time and the single fluorescence spectrum at the single fluorescence spectrum time; correcting the electrophoretic data using the spectral shift model; A genetic analysis device that identifies the base sequence of the sample using the corrected electrophoresis data and outputs an analysis result of the base sequence of the sample.

6. The genetic analysis device according to claim 5, The spectral shift model is a function of frequency space, The genetic analysis device is characterized in that the calculation device corrects the spectrum at any time by performing a convolution calculation using the spectrum at the any time and the spectral shift model.

7. The genetic analysis device according to claim 6, The computing device a first correction process 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 process for correcting the spectrum at a time other than the single fluorescence spectrum time using the spectral shift model for at least one single fluorescence spectrum time near the time.

8. The genetic analysis device according to claim 7, In the first correction process, the arithmetic device selecting a first single fluorescence spectral time; and obtaining the spectral shift model at the first single fluorescence spectral time; acquiring the spectral shift model at another single fluorescence spectrum time included in a predetermined time range including the first single fluorescence spectrum time; A genetic analysis device, characterized in that the single fluorescence spectrum at the first single fluorescence spectrum time is corrected using a plurality of the spectral shift models.

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