Method for analyzing base sequence and genetic analysis device

Abstract

The gene analyzer performs analysis of the base sequence of a sample, and associates and manages the measurement environment and mobility correction amount data for correcting the position in the time direction of time series data for the signal strength for a plurality of bases that is acquired by performing electrophoresis on the sample. Upon receiving time series data for the signal strength for a plurality of bases that has been acquired by performing electrophoresis on the sample in a first measurement environment, the gene analyzer produces default mobility correction amount data by scaling mobility correction amount data that corresponds to a measurement environment different from the first measurement environment. Using a mobility correction amount optimization algorithm and the default mobility correction amount data, the gene analyzer corrects the position in the time direction of the time series data for the signal strength for a plurality of bases and specifies the base sequence of the sample using the corrected time series data for the signal strength for a plurality of bases.

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] In the analysis of DNA base sequences using a capillary electrophoresis device, a mobility correction process is performed to correct differences in the electrophoretic speed of bases (see, for example, Non-Patent Document 1). For mobility correction processes, techniques described in, for example, Patent Documents 1 and 2 are known.

[0003] Patent Document 1 describes the following: "Detection data for four types of bases is obtained (S1), the detection data of the reference base is used as the reference detection data to move the other detection data (S2), the total area of ​​the apex waveform is calculated (S3), the mobility correction function is linearly shifted to determine the shift amount of the other detection data that maximizes the total area of ​​the apex waveform (S4), and the position information of the detection data is corrected (S5). A tentative base sequence is determined (S6), the detection data of the reference base is used as the reference detection data, a target peak that exists before and after the reference peak of the reference detection data is selected from the other detection data (S7), the peak intervals between the target peak and the reference peaks before and after it are respectively determined (S8), the shift amount of the target peak is calculated so that both peak intervals are equal (S9), and the position information of the detection data is corrected as the shift amount of the detection data to which the target peak belongs (S10)."

[0004] Patent Document 2 describes that "a method for determining the base sequence of a nucleic acid includes the following steps (A) to (C) in this order: (A) a base peak extraction step of extracting a base peak from electrophoretic data containing peaks of four base types obtained by electrophoretically separating a sample nucleic acid; (B) a condition setting step of setting a search starting base peak and a peak interval reference value at which a search will begin in time series data composed of the extracted base peaks; and (C) a base sequence is determined by sequentially scanning between adjacent base peaks in the forward and backward directions of the time series starting from the search starting base peak in the time series data, comparing the interval between base peaks with the peak interval reference value, and adding an interpolated peak to a peak missing section." [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2002-228633 A [Patent Document 2] International Publication No. 2008 / 050426 [Non-patent literature]

[0006] [Non-Patent Document 1] Michael C. Giddings, Jessica Severin, Michael Westphall, Jiazhen Wu, and Lloyd M. Smith, "A Software System for Data Analysis in Automated DNA?Sequencing", Genome Res. 1998 Jun;8(6):644-665. Summary of the Invention [Problem to be solved by the invention]

[0007] In recent years, there has been an increasing need to miniaturize electrophoretic devices. However, when an electrophoretic device is miniaturized, there is a problem in that the accuracy of signal resolution decreases, making it difficult to perform correction using the signal waveform.

[0008] In addition, when the accuracy of the signal resolution decreases, the conventional mobility correction method does not function properly. do not have For example, in the case of a sample that contains a long mixed base sequence, or in the case of a signal that contains a large amount of noise, the signal waveforms are corrected to reduce waveform overlap, and the correct base sequence may not be identified.

[0009] An object of the present invention is to provide a mobility correction technique that solves the above problems. [Means for solving the problem]

[0010] A representative example of the invention disclosed in the present application is as follows: That is, a method for analyzing a base sequence executed by a genetic analyzer that analyzes a base sequence of a sample using time-series data of signal intensities of a plurality of bases obtained by electrophoresis of the sample, the genetic analyzer manages an observation environment and mobility correction amount data for correcting a position in a time direction of the time-series data of signal intensities of the plurality of bases in association with each other, the method for analyzing a base sequence includes a first step of generating default mobility correction amount data by scaling the mobility correction amount data corresponding to an observation environment different from the first observation environment when the genetic analyzer receives time-series data of signal intensities of a plurality of bases obtained by electrophoresis of the sample under a first observation environment, a second step of the genetic analyzer correcting the position in a time direction of the time-series data of signal intensities of the plurality of bases using an optimization algorithm for the mobility correction amount and the default mobility correction amount data, and a third step of identifying the base sequence of the sample using the corrected time-series data of signal intensities of the plurality of bases. Effect of the Invention

[0011] According to the present invention, it is possible to realize mobility correction with high analytical accuracy for the base sequence of a sample. Problems, configurations and effects other than those described above will become apparent from the following description of the embodiments. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram showing an example of the configuration of a gene analysis device according to a first embodiment. [Diagram 2] FIG. 1 is a diagram illustrating an example of the configuration of an electrophoresis apparatus according to a first embodiment. [Diagram 3] FIG. 4 is a diagram showing an example of electrophoretic characteristic information stored in a storage device of the first embodiment. [Figure 4A] FIG. 11 is a diagram illustrating an example of mobility correction amount information stored in the storage device of the first embodiment. [Figure 4B] FIG. 11 is a diagram illustrating an example of mobility correction amount information stored in the storage device of the first embodiment. [Figure 4C] FIG. 11 is a diagram illustrating an example of mobility correction amount information stored in the storage device of the first embodiment. [Diagram 5] 2 is a flowchart for explaining an overview of a process executed by the gene analysis device of the first embodiment. [Figure 6] 4 is a flowchart illustrating an electrophoresis process executed by the electrophoresis device of the first embodiment. [Figure 7] 4 is a flowchart illustrating a mobility correction process executed by the data analysis device according to the first embodiment. [Figure 8] FIG. 2 is a diagram showing an image of a method for calculating a scale in the first embodiment. [Figure 9] FIG. 13 is a diagram showing an image of scaling of mobility correction amount data in the first embodiment. [Figure 10A] FIG. 4 is a diagram showing an example of a method for correcting automatic mobility correction amount data in the first embodiment. [Figure 10B] FIG. 4 is a diagram showing an example of a method for correcting automatic mobility correction amount data in the first embodiment. [Figure 11] FIG. 13 is a diagram showing a configuration example of a gene analysis device according to a second embodiment. [Figure 12]11 is a flowchart illustrating a data generation process executed by a gene analysis device according to a second embodiment. [Figure 13] 11 is a flowchart illustrating a mobility-corrected amount data generating process executed by a gene analyzer according to a second embodiment. [Figure 14] FIG. 13 is a diagram showing an example of electrophoretic characteristic data and mobility correction amount data stored in a storage device according to a third embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Hereinafter, examples of the present invention will be described with reference to the drawings. However, the present invention should not be interpreted as being limited to the description of the embodiments shown below. It will be easily understood by those skilled in the art that the specific configuration can be changed without departing from the concept or purpose of the present invention.

[0014] In the configurations of the invention described below, the same or similar configurations or functions are given the same reference numerals, and duplicated explanations are omitted.

[0015] In this specification, the terms "first," "second," "third," and the like are used to identify components and do not necessarily limit the number or order.

[0016] In order 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. EXAMPLES

[0017] FIG. 1 is a diagram showing an example of the configuration of a gene analysis device 100 according to a first embodiment.

[0018] 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 connected using a communication cable.

[0019] The data analysis apparatus 111 includes a control device 120 , a storage device 121 , and a connection interface 122 .

[0020] The control device 120 executes control and data processing of the electrophoretic device 110. The control device 120 is, for example, a central processing unit (CPU) and a graphics processing unit (GPU).

[0021] The storage device 121 stores programs executed by the control device 120, setting information for the electrophoretic device 110, information used for various processes, etc. The storage device 121 is, for example, a memory.

[0022] 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.

[0023] 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 mobility correction unit 134, and a base call unit 135. In the following explanation, when a process is explained using a functional unit as the subject, it indicates that the control device 120 is executing the program.

[0024] The electrophoresis device 110 electrophoreses a sample (DNA fragments) and obtains electrophoresis data, which is time-series data of the luminance values ​​of DNA fragments labeled with fluorescent dyes.

[0025] Here, a description will be given of the configuration of the electrophoretic device 110. Fig. 2 is a diagram showing an example of the configuration of the electrophoretic device 110 of the first embodiment.

[0026] The electrophoresis apparatus 110 includes a detection unit 216 , a thermostatic bath 218 , a transport machine 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 .

[0027] The capillary array 217 is a replaceable member 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 the capillary 202 is damaged or its quality deteriorates, it can be replaced with a new capillary array 217.

[0028] 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 hundreds of microns, and its surface is coated with polyimide to improve its strength. However, the light irradiating section where the laser light is irradiated has a structure where the polyimide coating has been removed so that the internal light emission can easily leak to the outside. The inside of the capillary 202 is filled with a separation medium that creates a migration speed difference during electrophoresis. There are both fluid and non-fluid separation media, but in Example 1, a fluid polymer is used.

[0029] The high voltage power supply 204 applies a high voltage to the capillary 202. The first ammeter 205 detects a current generated from the high voltage power supply 204. The second ammeter 212 detects a current flowing through the anode electrode 211.

[0030] 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 member that acquires information that depends on the sample.

[0031] 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 to generate 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 separated information light to analyze the sample.

[0032] The capillary cathode ends 227 are fixed through respective metallic 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 the capillaries 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.

[0033] The capillary cathode end 227 and the opposite capillary end (the other end) are bound together by a capillary head 233. The capillary head 233 can be connected to the block 207 in a pressure-tight 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 in order to improve the measurement performance.

[0034] The pump mechanism 203 is composed of a syringe 206 and a mechanism for pressurizing the syringe 206 , and injects a polymer into the capillary 202 .

[0035] 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.

[0036] The thermostatic chamber 218 is covered with a heat insulating material in order to keep the capillaries 202 in the thermostatic chamber 218 at a constant temperature, and the temperature is controlled by a heating and cooling mechanism 220. A fan 219 circulates and stirs the air in the thermostatic chamber 218, thereby keeping the temperature of the capillary array 217 uniform and constant in each position.

[0037] The transporter 225 transports various containers to the capillary cathode end 227. The transporter 225 is equipped with three electric motors and a linear actuator, and can move in three axial directions, namely up and down, left and right, and depth. At least one container can be placed on the moving stage 230 of the transporter 225. Furthermore, the moving stage 230 is equipped with an electric grip 231, which can grip and release each container. Therefore, the buffer container 221, the washing container 222, the waste liquid container 223, and the sample plate 224 can be transported to the capillary cathode end 227 as necessary. Unnecessary containers are stored in a predetermined storage location in the electrophoresis device 110.

[0038] A user can use the data analysis device 111 to control various functions of the electrophoresis device 110 and obtain the electrophoresis data detected by the optical detection unit.

[0039] 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.

[0040] The internal sensor 240 is a sensor for acquiring information about the internal environment of the electrophoresis device 110, for example. 、 Temperature sensor, humidity sensor, air pressure sensor, etc. is .

[0041] The polymer sensor 241 is a sensor for acquiring information on the quality of the polymer, and is, for example, a pH sensor, an electrical conductivity sensor, etc. The polymer sensor 241 is installed in the polymer container 209 in FIG. 2, but the installation location is not limited thereto.

[0042] The buffer solution sensor 242 is a sensor for acquiring information regarding the quality of the buffer solution, and may be, for example, a temperature sensor. Although the buffer solution sensor 242 is 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.

[0043] FIG. 3 is a diagram showing an example of the electrophoretic characteristic information stored in the storage device 121 of the first embodiment.

[0044] The storage device 121 stores electrophoretic characteristic information for managing electrophoretic characteristic data representing the relationship between electrophoretic time (t) and the position (p) of the base for each base type. The electrophoretic characteristic information includes the electrophoretic characteristic data for each observation environment in the electrophoretic device 110. Electrophoretic characteristics data is stored.

[0045] The electrophoretic characteristic data is managed as a function Y(p). In this embodiment, it is assumed that parameters representing the function are managed, rather than the function itself. FIG. 3 shows electrophoretic characteristic data with different voltages applied by the high-voltage power supply 204. Y5(p) is electrophoretic characteristic data when the voltage is 5.0 kV, Y8(p) is electrophoretic characteristic data when the voltage is 8.0 kV, and Y 11 (p) shows the migration characteristics data at a voltage of 11.0 kV.

[0046] The electrophoretic characteristic information may be stored in advance in the storage device 121, or may be generated using electrophoretic data obtained by actually electrophoresing a sample with a known length, such as a size standard, in the electrophoretic device 110.

[0047] 4A, 4B, and 4C are diagrams illustrating an example of mobility correction amount information stored in the storage device 121 of the first embodiment.

[0048] The storage device 121 stores mobility correction amount information for managing mobility correction amount data that indicates the relationship between migration time and the correction amount of mobility for each base type. The mobility correction amount information stores default mobility correction amount data for each observation environment in the electrophoresis device 110. In the first embodiment, the mobility of the base C (cytosine), base A (adenine), and base T (thymine) is corrected based on the base G (guanine). Therefore, the storage device 121 stores mobility correction amount information for each of the base C, base A, and base T.

[0049] Fig. 4A shows the mobility correction amount information of the base C, Fig. 4B shows the mobility correction amount information of the base A, and Fig. 4C shows the mobility correction amount information of the base T. The curves show the default mobility correction amount data.

[0050] The mobility correction amount information may be stored in advance in the storage device 121, or may be generated using electrophoretic data obtained by electrophoresing a sample with a known length, such as a size standard, in the electrophoretic device 110.

[0051] FIG. 5 is a flowchart outlining the process executed by the gene analysis device 100 of the first embodiment.

[0052] The electrophoresis device 110 of the genetic analysis device 100 performs an electrophoresis process on the sample to be analyzed (step S101). The details of the electrophoresis process will be described with reference to FIG.

[0053] Next, the data analyzer 111 of the genetic analyzer 100 executes a fluorescence intensity calculation process using the electrophoretic data (step S102). Specifically, the fluorescence intensity calculation unit 133 calculates time series data of the fluorescence intensity of the fluorescent dye from the electrophoretic data, and detects the center position, height, width, etc. of the peak from the time series data of the fluorescence intensity.

[0054] 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 S103). The details of the mobility correction process will be described with reference to Figs. 7 to 10. The genetic analysis device 100 of the first embodiment is characterized by the mobility correction process, as described later.

[0055] 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 S104). Specifically, the base calling unit 135 identifies the base sequence of the sample using the time series data of the corrected fluorescence intensity.

[0056] FIG. 6 is a flowchart illustrating the electrophoresis process executed by the electrophoresis device 110 of the first embodiment.

[0057] A user sets a sample to be analyzed, a reagent, and the like in the electrophoresis device 110, and issues an instruction to start electrophoresis processing via the connection interface 122. Setting of the sample is performed in the following procedure.

[0058] The user fills the buffer container 221 and the anode buffer container 210 with a buffer solution that forms a part of the electric 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 PCR product of DNA. 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 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 degradation of the capillary 202 is expected or when the length of the capillary 202 is changed.

[0059] At this time, the samples set on the sample plate 224 include, in addition to the actual sample of DNA to be analyzed, a positive control, a negative control, an allelic ladder, and the like, each of which is electrophoresed in a different capillary 202 .

[0060] A positive control is, for example, a PCR product containing known DNA, and is a control sample used to confirm that DNA is amplified correctly by PCR. A negative control is a PCR product containing no DNA, and is a control sample used to confirm that the PCR amplified product is not contaminated by the user's DNA or dust. An allelic ladder is an artificial sample that contains many bases that may generally be contained in DNA markers, and is usually provided by reagent manufacturers as part of a reagent kit for DNA identification. Allelic ladders are used to fine-tune the correspondence between the DNA fragment length and alleles of each DNA marker.

[0061] 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.

[0062] The user specifies the type of allelic ladder, the type of size standard, the type of fluorescent reagent, the type of sample set in the well on the sample plate 224 corresponding to each capillary 202, etc. In the first embodiment, any one of the types of real sample, positive control, negative control, and allelic ladder is specified. 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.

[0063] This concludes the description of the sample set.

[0064] 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.

[0065] First, the electrophoresis device 110 fills the capillary 202 with a new migration medium to form a migration path (step S201). The migration medium may be filled automatically after the start of the analysis, or may be filled sequentially based on a control signal transmitted from the electrophoresis device control unit 132.

[0066] Specifically, the electrophoresis apparatus 110 uses the transport device 225 to transport the waste liquid container 223 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, and washes the capillary cathode end 227 contaminated by the migration medium.

[0067] Next, the electrophoretic device 110 applies a predetermined voltage to the electrophoretic medium to perform a preliminary electrophoresis to make the electrophoretic medium suitable for electrophoresis (step S202). The preliminary electrophoresis may be performed automatically or sequentially based on a control signal transmitted from the electrophoretic device control unit 132.

[0068] Specifically, the electrophoresis device 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 device 110 uses the high-voltage power supply 204 to apply a voltage of several to several tens of kilovolts to the electrophoresis medium for several to several tens of minutes to make the electrophoresis medium in a state suitable for electrophoresis. Finally, the electrophoresis device 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.

[0069] 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.

[0070] Specifically, the electrophoresis apparatus 110 uses the conveyor 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 an electric current path, and the sample components can be introduced into the migration path. The electrophoresis apparatus 110 applies a pulse voltage to the electric current path using the high-voltage power supply 204, and introduces the sample components into the migration path. Finally, the electrophoresis apparatus 110 immerses the capillary cathode end 227 in a cleaning solution in the cleaning container 222, and cleans the capillary cathode end 227 contaminated by the sample.

[0071] Next, the electrophoretic device 110 executes electrophoretic analysis in which each sample component contained in the sample is separated and analyzed (step S204). The electrophoretic analysis may be performed automatically or sequentially based on a control signal transmitted from the electrophoretic device control unit 132.

[0072] Specifically, the electrophoresis device 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. The electrophoresis device 110 uses the high-voltage power supply 204 to apply a high voltage of about 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 the detection unit 216 at a speed that depends on the properties of each sample component. In other words, the sample components are separated based on the difference in their migration speed. Then, the sample components that reach the detection unit 216 are detected in order. For example, when the sample contains a large number of DNAs with different base lengths, the migration speed differs depending on the base length, and the DNAs with the shortest base lengths reach the detection unit 216 in order. A fluorescent dye that depends on the terminal base sequence is attached to each DNA. When the detection unit 216 is irradiated with excitation light from the light source 214, fluorescence having a wavelength that depends on the sample is generated and released to the outside. The electrophoresis device 110 detects the fluorescence by the optical detector 215. During the electrophoresis analysis, the optical detector 215 detects the fluorescence at regular time intervals and transmits image data to the data analysis device 111. In order to reduce the amount of information to be transmitted, the luminance of only a part of the image data may be transmitted instead of the image data. For example, luminance values ​​sampled only at wavelength positions at regular intervals may be transmitted for each capillary 202. The data transmitted from the electrophoresis device 110 is time-series data of the luminance values ​​of each capillary 202, and is stored in the storage device 121.

[0073] When the expected image data has been acquired, the electrophoretic device 110 stops applying the voltage and ends the electrophoretic analysis. This concludes the description of the electrophoretic process.

[0074] Fig. 7 is a flowchart for explaining the mobility correction process executed by the data analysis device 111 of the first embodiment. Fig. 8 is a diagram showing an image of a scale calculation method of the first embodiment. Fig. 9 is a diagram showing an image of scaling of mobility correction amount data of the first embodiment. Figs. 10A and 10B are diagrams showing an example of a method of correcting automatic mobility correction amount data of the first embodiment.

[0075] Mobility corrector 134 calculates the scale based on observation environment information of electrophoretic device 110 (step S301). Here, a method of calculating the scale will be described with a focus on the voltage in the observation environment.

[0076] (S301-1) The mobility correction unit 134 refers to the migration characteristic information and searches for migration characteristic data corresponding to the voltage (target voltage) included in the observation environment information. If migration characteristic data corresponding to the target voltage exists, default mobility correction amount data corresponding to the target voltage exists, and the mobility correction unit 134 ends the process of step S301. In this case, the scale is not calculated.

[0077] (S301-2) If there is no migration characteristic data corresponding to the target voltage, the mobility correction unit 134 generates migration characteristic data for the target voltage (target migration characteristic data) using migration characteristic data for a voltage that is less different from the target voltage. In this way, migration characteristic data for an observation environment similar to the actual observation environment is used to estimate the migration characteristic data for the actual observation environment. Here, similarity of the observation environments refers to a similar combination of physical quantities representing the observation environments.

[0078] For example, when the target voltage is 9.0 kV, as shown in FIG. 8, the electrophoretic characteristic data Y8(p) at 8.0 kV and the electrophoretic characteristic data Y 11 (p) is used to generate target electrophoretic characteristic data. When linear interpolation is used, for example, electrophoretic characteristic data of the target voltage can be generated by the formula (1).

[0079]

number

[0080] (S301-3) The mobility correction unit 134 calculates a scale between the electrophoretic characteristic data used to generate the target electrophoretic characteristic data at time t and the target electrophoretic characteristic data.

[0081] For example, when the target voltage is 9.0 kV, the scale S8(t) for the electrophoretic characteristic data Y8(p) at 8.0 kV at time t is given by Equation (2), and the scale S8(t) for the electrophoretic characteristic data Y8(p) at 11.0 kV at time t is given by 11 Scale S for (p) 11 (t) is given by equation (3).

[0082]

number

[0083]

number

[0084] This concludes the explanation of how to calculate the scale.

[0085] Next, the mobility correction unit 134 generates default mobility correction amount data (step S302). Here, a method for calculating the default mobility correction amount data will be described with a focus on the voltage in the observation environment.

[0086] (S302-1) The mobility correction unit 134 refers to the mobility correction amount information of each base and searches for default mobility correction amount data corresponding to the voltage (target voltage) included in the observation environment information. If default mobility correction amount data corresponding to the target voltage exists, the mobility correction unit 134 ends the process of step S302. In this case, the searched default mobility correction amount data is used as is.

[0087] (S302-2) If there is no default mobility correction amount data corresponding to the target voltage, the mobility correction unit 134 identifies the voltage of the electrophoretic characteristic data used to generate the target electrophoretic characteristic data in step S301. The mobility correction unit 134 refers to the mobility correction amount information of each base and acquires the default mobility correction amount data corresponding to the identified voltage.

[0088] (S302-3) As shown in FIG. 9, the mobility correction unit 134 calculates default mobility correction amount data for the target voltage by multiplying each point of the default mobility correction amount data by a scale corresponding to the specified voltage.

[0089] For example, when the target voltage is 9.0 kV, the mobility correction unit 134 multiplies the default mobility correction amount data of 8.0 kV by the scale S8(t) to calculate first default mobility correction amount data, and multiplies the default mobility correction amount data of 11.0 kV by the scale S 11 (t) to calculate second default mobility correction amount data. Furthermore, the mobility correction unit 134 calculates the average of the first default mobility correction amount data and the second default mobility correction amount data as the default mobility correction amount data for 9.0 kV.

[0090] In this way, the default mobility correction amount data for each observation environment in the first embodiment has a scale relationship.

[0091] This concludes the description of the method for calculating the default mobility correction amount data.

[0092] Next, the mobility correction unit 134 optimizes the amount of mobility correction for the time-series data of the fluorescence intensity (step S303).

[0093] The mobility correction amount can be optimized using a known optimization algorithm for the mobility correction amount, and therefore a detailed description will be omitted. For example, the following optimization algorithm can be considered.

[0094] The mobility correction unit 134 sets a block of a predetermined time size, and while moving the block in the time direction with respect to the time series data of the fluorescence intensity, searches for the mobility correction amount of each base so that the overlap of the waveforms of each base in the block is minimized. The result of the search process is plotted, and further, mobility correction amount data (automatic mobility correction amount data) is obtained by smoothly interpolating between the plotted points.

[0095] The default mobility correction amount data may be used as an initial value for optimization.

[0096] Next, the mobility correction unit 134 determines the mobility correction amount data based on the automatic mobility correction amount data and the default mobility correction amount data (step S304). For example, the following processing may be performed.

[0097] (Process 1) When the difference between the automatic mobility correction amount data and the default mobility correction amount data is large, the mobility correction unit 134 adopts the default mobility correction amount data, and when the difference is small, the mobility correction unit 134 adopts the automatic mobility correction amount data. The difference can be evaluated based on the maximum value of the difference between the automatic mobility correction amount data and the default mobility correction amount data, the total value of the differences between the automatic mobility correction amount data and the default mobility correction amount data at each point, and the like. Note that the automatic mobility correction amount data may be adopted for a portion where the difference between the automatic mobility correction amount data and the default mobility correction amount data is small, and the default mobility correction amount data may be adopted for a portion where the difference between the automatic mobility correction amount data and the default mobility correction amount data is large.

[0098] (Process 2) As shown in Fig. 10A, the mobility correction unit 134 identifies a portion (dotted rectangular area) where the automatic mobility correction amount data (solid line graph) differs greatly from the default mobility correction amount data (dotted line graph) and where the fluctuation rate is large, as shown in Fig. 10B, the mobility correction unit 134 corrects the identified portion so that the difference from the default mobility correction amount data becomes small and the fluctuation rate becomes small.

[0099] In the automatic mobility correction process, the mobility correction amount is calculated so as to minimize overlap, so if the mixed base sequence is long, the mobility correction may not be calculated appropriately. In such cases, the difference from the default mobility correction amount data is likely to be large.

[0100] The mobility correction unit 134 of the first embodiment performs automatic correction using the default mobility correction amount data as a correction constraint, thereby enabling accurate mobility correction even for a sample containing a long mixed base sequence or electrophoretic data containing large noise.

[0101] The mobility correction amount data actually used is adjusted based on the default mobility correction amount data, and therefore part or all of the mobility correction amount data has characteristics similar to those of the default mobility correction amount data.

[0102] In the first embodiment, the default mobility correction amount data for an arbitrary observation environment is generated by scaling the default mobility correction amount data for a different observation environment, thereby making it possible to deal with various observation environments. EXAMPLES

[0103] In the second embodiment, the data analysis device 111 generates migration characteristic data and default mobility correction amount data for each observation environment. The second embodiment will be described below, focusing on the differences from the first embodiment.

[0104] FIG. 11 is a diagram showing an example of the configuration of a gene analysis device 100 according to the second embodiment.

[0105] The configuration of the electrophoresis apparatus 110 of the second embodiment is the same as that of the first embodiment. The hardware configuration of the data analysis apparatus 111 of the second embodiment is the same as that of the first embodiment.

[0106] The data analysis device 111 of the second embodiment has a partially different software configuration. Specifically, the data analysis device 111 of the second embodiment differs from that of the first embodiment in that it includes a migration characteristic data generating unit 136. The migration characteristic data generating unit 136 generates migration characteristic data and default mobility correction amount data.

[0107] The process performed by the genetic analysis device 100 of the second embodiment is the same as that of the first embodiment. The electrophoresis process performed by the electrophoresis device 110 of the second embodiment is the same as that of the first embodiment. Data of the second embodiment Analysis device 111The mobility correction process executed by is the same as that in the first embodiment.

[0108] It is assumed that reference electrophoretic characteristic data, mobility correction amount data, and base sequence of any sample in any observation environment are stored in the storage device 121. The sample is preferably a sample with a single base sequence that is easy to base call.

[0109] FIG. 12 is a flowchart illustrating the data generation process executed by the gene analysis device 100 of the second embodiment.

[0110] When receiving an instruction from a user, the gene analysis device 100 executes a data generation process.

[0111] The genetic analysis device 100 executes a mobility-corrected amount data generation process (step S401). In the mobility-corrected amount data generation process, mobility-corrected amount data is generated for a plurality of observation environments, and base calling of the base sequence is performed. The details of the process will be described with reference to FIG. 12.

[0112] Next, the gene analysis device 100 performs mapping between the reference base sequence and the base sequence of an arbitrary observation environment (step S402).

[0113] Specifically, the electrophoretic characteristic data generating unit 136 of the data analyzing device 111 calculates the base position of the base G in the base sequence of each observation environment by the above mapping.

[0114] Next, the genetic analyzer 100 executes a process for generating electrophoretic characteristic data (step S403). Specifically, the following process is executed.

[0115] (S403-1) The electrophoretic characteristic data generating unit 136 of the data analyzing device 111 plots points in a space with the base positions and electrophoretic time as axes, based on the base sequence in a certain observation environment.

[0116] (S403-2) The electrophoretic characteristic data generating unit 136 may, if necessary, interpolate between the points plotted in S403-1.

[0117] (S403-3) The migration characteristic data generating unit 136 obtains an approximation equation from the plotted points. The migration characteristic data generating unit 136 stores the parameters of the approximation equation and the like in the storage device 121 as migration characteristic data in association with the observation environment.

[0118] This concludes the description of the process in step S403.

[0119] FIG. 13 is a flowchart illustrating the mobility-corrected amount data generation process executed by the gene analysis apparatus 100 of the second embodiment.

[0120] The electrophoresis device 110 of the genetic analysis device 100 executes electrophoresis processing of the sample for a plurality of observation environments (step S501). The electrophoresis processing of the second embodiment is the same as that of the first embodiment.

[0121] It is assumed that the observation environment is specified by the user, and that for one observation environment, electrophoresis processing is performed multiple times to obtain multiple pieces of electrophoretic data.

[0122] Next, the data analysis device 111 of the gene analysis device 100 starts a loop process of the observation environment (step S502). Specifically, the data analysis device 111 selects one observation environment.

[0123] Next, the data analysis device 111 of the genetic analysis device 100 executes a fluorescence intensity calculation process for the plurality of electrophoretic data (step S503). The fluorescence intensity calculation process of the second embodiment is the same as that of the first embodiment.

[0124] Next, the data analysis device 111 of the genetic analysis device 100 optimizes the amount of mobility correction for each piece of time-series data of the fluorescence intensity (step S504). The optimization of the amount of mobility correction in the second embodiment uses a known technique.

[0125] Next, the data analysis device 111 of the genetic analysis device 100 executes base calling using the corrected time-series data of the fluorescence intensity (step S505).

[0126] Next, the data analysis device 111 of the gene analysis device 100 generates mobility correction amount data for the observation environment (step S506).

[0127] Specifically, the electrophoretic characteristic data generating unit 136 calculates an average of the multiple mobility correction amount data calculated in step S504. The electrophoretic characteristic data generating unit 136 stores the calculated mobility correction amount data in the storage device 121 in association with the observation environment. Note that mobility correction amount data for which the base call of the base sequence is determined to be incorrect is excluded.

[0128] Next, the data analysis device 11 of the gene analysis device 100 1 Then, it is determined whether the processing has been completed for all the observation environments (step S507).

[0129] If the process has not been completed for all observation environments, the data analyzer 111 of the genetic analyzer 100 returns to step S502 and executes the same process. If the process has been completed for all observation environments, the genetic analyzer 100 ends the mobility correction amount data generation process.

[0130] According to the second embodiment, by preparing migration characteristic data for a plurality of observation environments, it is possible to reduce the cost of calculating the scale and the mobility correction amount data. EXAMPLES

[0131] In the third embodiment, the data analysis device 111 adds the electrophoretic characteristic data and the mobility correction amount calculated in the actual analysis. Hereinafter, the third embodiment will be described, focusing on the differences from the first embodiment.

[0132] The configuration of the genetic analysis device 100 of the third embodiment is the same as that of the first embodiment. The configurations of the electrophoresis device 110 and the data analysis device 111 of the third embodiment are the same as those of the first embodiment.

[0133] In the third embodiment, the process executed by the genetic analyzer 100 is partially different. Specifically, after the data analyzer 111 performs base calling (step S104), the data analyzer 111 stores the electrophoretic characteristic data and the mobility correction amount data calculated in the series of processes in the storage device 121 in association with the observation environment based on the user's instruction.

[0134] FIG. 14 is a diagram showing an image of the electrophoretic characteristic data and the mobility correction amount data stored in the storage device 121 of the third embodiment.

[0135] FIG. 14 shows the distribution of data in a space representing an observation environment consisting of temperature and voltage. In the initial storage device 121, the data of the observation environment corresponding to the black circles are Electrophoretic characteristics data and mobility correction data are stored. Electrophoretic characteristics data It can be seen that mobility correction amount data has been added.

[0136] The electrophoresis process and the mobility correction process in the embodiment 3 are similar to those in the embodiment 1. Furthermore, the gene analysis apparatus 100 in the embodiment 3 may execute a data generation process.

[0137] According to the third embodiment, by adding electrophoretic characteristic data and mobility correction amount data in accordance with the aging of the gene analysis device 100 and the environment in which it is actually used, it is possible to improve the accuracy of base calling and reduce costs.

[0138] The present invention is not limited to the above-mentioned embodiment, but includes various modified examples. For example, the above-mentioned embodiment describes the configuration in detail to easily explain the present invention, and the present invention is not necessarily limited to the configuration including all the described configurations. Also, it is possible to add, delete, or replace a part of the configuration of each embodiment with another configuration.

[0139] In addition, each of the above configurations, functions, processing units, processing means, etc. may be realized in part or in whole by hardware, for example, by designing them as integrated circuits. The present invention can also be realized by software program code that realizes 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 realizes the functions of the above-mentioned embodiments, and the program code itself and the storage medium storing it constitute the present invention. Examples of storage media for supplying such program code include flexible disks, CD-ROMs, DVD-ROMs, hard disks, SSDs (Solid State Drives), optical disks, magneto-optical disks, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, etc.

[0140] Furthermore, the program code for realizing 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, Java, etc.

[0141] Furthermore, the program code of the software that realizes the functions of the embodiments may be distributed over 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 out and execute the program code stored in the storage means or storage medium.

[0142] In the above-mentioned embodiment, the control lines and information lines are shown as those considered necessary for the explanation, and not all the control lines and information lines are shown in the product. All the components may be connected to each other.

Claims

1. A method for analyzing a base sequence performed by a genetic analyzer that analyzes a base sequence of a sample using time-series data of signal intensities of a plurality of bases obtained by electrophoresis of the sample, comprising: the genetic analysis device manages an observation environment in association with mobility correction amount data for correcting a position in a time direction of the time series data of the signal intensities of the plurality of bases; The method for analyzing a base sequence comprises: a first step of generating default mobility correction amount data by scaling the mobility correction amount data corresponding to the observation environment different from the first observation environment when the genetic analysis device receives time series data of signal intensities of a plurality of bases obtained by electrophoresis of the sample under a first observation environment; a second step in which the genetic analysis device corrects the time-direction position of the time-series data of the signal intensities of the plurality of bases by using an optimization algorithm for the mobility correction amount and the default mobility correction amount data; and a third step of identifying the base sequence of the sample using the corrected time series data of signal intensities of the multiple bases.

2. The method for analyzing a base sequence according to claim 1, the genetic analysis device manages an observation environment in association with electrophoretic characteristic data that indicates a relationship between a position of the base by electrophoresis and an electrophoretic time; The first step includes: a step of calculating a scale by the genetic analysis device using the electrophoretic characteristic data corresponding to the first observation environment and the electrophoretic characteristic data corresponding to the observation environment different from the first observation environment; and a step of scaling the mobility correction amount data corresponding to the observation environment different from the first observation environment based on the scale by the genetic analysis device.

3. The method for analyzing a base sequence according to claim 1, The second step includes: a fourth step in which the genetic analyzer generates automatic mobility correction amount data based on an optimization algorithm for the mobility correction amount; and a fifth step in which the genetic analysis device corrects the automatic mobility correction amount data based on a difference between the automatic mobility correction amount data and the default mobility correction amount data.

4. The method for analyzing a base sequence according to claim 3, The fifth step of the method for analyzing base sequences is characterized in that it includes a step in which, when the overall difference between the automatic mobility correction amount data and the default mobility correction amount data is large, the genetic analysis device replaces the automatic mobility correction amount data with the default mobility correction amount data.

5. The method for analyzing a base sequence according to claim 3, The fifth step includes: a step of the genetic analysis device identifying a portion where a difference between the automatic mobility-corrected amount data and the default mobility-corrected amount data is large; A method for analyzing a base sequence, comprising a step in which the genetic analysis apparatus replaces the identified portion with the default mobility-corrected amount data.

6. The method for analyzing a base sequence according to claim 3, The fifth step includes: a step of the genetic analysis device identifying a portion where a difference between the automatic mobility-corrected amount data and the default mobility-corrected amount data is large and where a rate of variation is large; and correcting the identified portion by the genetic analysis device so that a difference between the identified portion and the default mobility-corrected amount data becomes small.

7. The method for analyzing a base sequence according to claim 1, The method for analyzing a base sequence, wherein the third step includes a step in which the genetic analysis device stores the first observation environment and the mobility correction amount data used in association with each other.

8. The method for analyzing a base sequence according to claim 2, generating, by the genetic analysis device, the electrophoretic characteristic data of the plurality of bases in the second observation environment, using mobility correction amount data of time series data of signal intensity of the plurality of bases obtained by electrophoresis of the sample in a second observation environment, and the base sequence of the sample identified using the corrected time series data of signal intensity of the plurality of bases; and storing the second observation environment and the generated electrophoretic characteristic data in association with each other.

9. A genetic analysis device that analyzes a base sequence of a sample using time-series data of signal intensities of a plurality of bases obtained by electrophoresis of the sample, comprising: managing the observation environment in association with mobility correction amount data for correcting the time-direction position of the time-series data of the signal intensities of the plurality of bases; a first process for generating default mobility correction amount data by scaling the mobility correction amount data corresponding to the observation environment different from the first observation environment when time-series data of signal intensities of a plurality of bases obtained by electrophoresis of the sample under a first observation environment is received; a second process of correcting a time direction position of the time series data of the signal intensities of the plurality of bases by using an optimization algorithm for the mobility correction amount and the default mobility correction amount data; and a third process of identifying a base sequence of the sample using the corrected time series data of signal intensities of the multiple bases.

10. The genetic analysis device according to claim 9, managing the observation environment in association with electrophoretic characteristic data that indicates a relationship between the position of the base in electrophoresis and the electrophoretic time; In the first process, Calculating a scale using the electrophoretic characteristic data corresponding to the first observation environment and the electrophoretic characteristic data corresponding to the observation environment different from the first observation environment; A genetic analysis apparatus comprising: a step of scaling the mobility correction amount data corresponding to the observation environment different from the first observation environment based on the scale.

11. The genetic analysis device according to claim 9, In the second process, a fourth process of generating automatic mobility correction amount data based on an optimization algorithm for the mobility correction amount; and a fifth process of correcting the automatic mobility-corrected amount data based on a difference between the automatic mobility-corrected amount data and the default mobility-corrected amount data.

12. The genetic analysis device according to claim 11, In the fifth process, when the overall difference between the automatic mobility correction amount data and the default mobility correction amount data is large, the automatic mobility correction amount data is replaced with the default mobility correction amount data.

13. The genetic analysis device according to claim 11, In the fifth process, Identifying a portion where a difference between the automatic mobility correction amount data and the default mobility correction amount data is large; A gene analysis apparatus comprising: a step of replacing the specified portion with the default mobility correction amount data.

14. The genetic analysis device according to claim 11, In the fifth process, Identifying a portion where the difference between the automatic mobility correction amount data and the default mobility correction amount data is large and where the fluctuation rate is large; A gene analysis device comprising: a step of correcting the specified portion so that a difference between the specified portion and the default mobility correction amount data becomes small.

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