Electrophoresis data processing device and electrophoresis data processing method

Abstract

In order to support the efficient analysis of electrophoresis results, the present invention comprises: a fluorescence calibration unit (201) that calculates fluorescence spectral data (D020) of a second specimen (D002) on the basis of signal charge data (D012) of the second specimen (D002); a color conversion processing unit (202) that calculates fluorescent color signal data (D033) on the basis of signal charge data (D013) of a third specimen (D003) and the fluorescence spectral data (D020), and calculates fluorescent color signal data (D031) on the basis of signal charge data (D011) of a first specimen (D001) and the fluorescence spectral data (D020); an intensity correction factor determination unit (203) that calculates an intensity correction factor (D040) from the fluorescent color signal data (D033); an intensity adjustment processing unit (204) that calculates color signal data (D050) by using the fluorescent color signal data (D031) and the intensity correction factor (D040); and a display device (301) that outputs the color signal data (D050).

Description

[Technical Field]

[0001] The present invention relates to a technique for processing electrophoretic data and a method for processing electrophoretic data. [Background technology]

[0002] A capillary array electrophoresis apparatus (hereinafter referred to as an electrophoresis apparatus) for analyzing base sequence information of DNA (deoxyribonucleic acid) is known. In an electrophoresis apparatus, a sample with DNA attached with multiple fluorescent labels is electrophoresed inside a capillary. During electrophoresis, excitation light is irradiated onto the detection region of the capillary, and fluorescence emitted by the fluorescent labels is detected as a signal. The fluorescence emitted by the sample is split into wavelength regions and detected as a fluorescent signal by a device that converts optical signals into electrical signals for each wavelength region, such as a CCD (charge coupled device) element or a CMOS element.

[0003] A binning function is known that, when acquiring a fluorescent signal, enlarges or reduces the light-receiving area per pixel by pseudo-combining multiple light-receiving surfaces (corresponding to pixels) of an element and treating them as a single pixel. For example, Patent Document 1 discloses a capillary array electrophoresis apparatus, a fluorescent detection apparatus, and a method for acquiring fluorescent signal intensity, which "includes multiple light-receiving surfaces that generate signal charges when irradiated with a fluorescent signal 405, and acquires the fluorescent signal intensity based on the multiple signal charges generated on the light-receiving surfaces. The fluorescent detection apparatus 400 acquires the fluorescent signal intensity by performing either hardware binning, which acquires the fluorescent signal intensity by converting multiple signal charges collectively, or software binning, which converts the signal charges one by one into fluorescent signal intensity and adds the converted fluorescent signal intensities to acquire the fluorescent signal intensity" (see Abstract). The combination of multiple binning regions used during binning is called a binning pattern. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2015-49179 Summary of the Invention [Problem to be solved by the invention]

[0005] Electrophoresis devices must simultaneously detect and analyze fluorescence from multiple fluorescent labels, and satisfactory analysis results cannot be obtained unless variations in fluorescence sensitivity are suppressed.

[0006] The binning function described in Patent Document 1 can improve the data acquisition speed and S / N ratio. However, adjusting the binning area to improve the S / N ratio requires improvements to prevent variations in fluorescence sensitivity that occur depending on the wavelength characteristics of the fluorescent label.

[0007] Therefore, when a binning function is provided, a binning pattern optimized for a set of fluorescent labels is applied to suppress variations in fluorescent sensitivity.

[0008] However, as fluorescence detection speed improves and the acquisition pixel area shrinks, there are cases where data can be acquired without a binning function. In such cases, the lack of a binning function makes it impossible to suppress the variation in fluorescence sensitivity that was previously achieved by applying a binning pattern. Therefore, a method for suppressing variation in fluorescence sensitivity that does not rely on binning is needed.

[0009] The present invention has been made in view of the above background, and an object of the present invention is to support efficient analysis of electrophoresis results. [Means for solving the problem]

[0010] In order to solve the above-mentioned problems, the present invention provides a fluorescence spectrum calculation unit that calculates fluorescence spectrum data, which is data obtained by normalizing wavelength spectra related to signal charge values ​​of fluorescent labels used in a first reference sample, based on first signal charge data, which is the result of electrophoresis for the first reference sample, which is a sample for calibrating data from the real sample; and a fluorescence spectrum calculation unit that calculates second fluorescence color signal data, which is time-series information on signal intensities of the fluorescent labels, based on second signal charge data, which is the result of electrophoresis for a second reference sample, which is a sample for evaluating or calibrating data from the real sample, and the fluorescence spectrum data. a fluorescence color signal data calculation unit that calculates third fluorescence color signal data, which is time-series information on the signal intensity of each of the fluorescent labels, based on third signal charge data, which is the result of electrophoresis, and the fluorescence spectrum data; an intensity correction coefficient calculation unit that calculates, in the second fluorescence color signal data, an intensity correction coefficient, which is the ratio of the signal intensity of each of the fluorescent labels to the reference signal intensity, using the signal intensity of a predetermined fluorescent label as a reference; a color signal data calculation unit that calculates color signal data by multiplying each of the data of the fluorescent labels in the third fluorescence color signal data by the corresponding intensity correction coefficient; and an output unit that outputs the color signal data. [Effects of the Invention]

[0011] According to the present invention, it is possible to support efficient analysis of electrophoresis results. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram schematically illustrating an example of the configuration of an electrophoresis system according to a first embodiment. [Figure 2A] FIG. 1 is a diagram illustrating an example of the configuration of an electrophoresis apparatus. [Figure 2B] FIG. 1 is an enlarged view of the capillary cathode end. [Figure 3A] FIG. 2 is a diagram illustrating an example of the configuration of a detection unit of an electrophoresis apparatus. [Figure 3B]FIG. 2 is a schematic cross-sectional view of a part of the detection unit taken along a plane perpendicular to the capillary. [Figure 4] 4 is a flowchart showing the procedure of a process performed by the electrophoresis device of the first embodiment. [Figure 5] FIG. 10 is a diagram showing an example of signal charge data. [Figure 6] 5 is a flowchart showing the procedure of processing performed by a fluorescence calibration unit in the first embodiment. [Figure 7] FIG. 10 is a diagram showing an example of fluorescence spectrum data. [Figure 8] 5 is a flowchart showing the procedure of processing performed by a fluorescence calibration unit in the first embodiment. [Figure 9] FIG. 10 is a diagram showing an example of fluorescence color signal data. [Figure 10] 6 is a flowchart showing the procedure of a process performed by an intensity correction coefficient determination unit according to the first embodiment. [Figure 11] FIG. 10 is another diagram showing an example of fluorescence color signal data. [Figure 12] 6 is a flowchart showing the procedure of processing performed by an intensity adjustment processing unit of the first embodiment. [Figure 13] FIG. 2 is a diagram illustrating an example of color signal data. [Figure 14A] 10 is a table showing examples of evaluation values ​​for each capillary and each fluorescent label. [Figure 14B] 10 is a table (part 1) showing an example of an intensity correction coefficient calculated based on an evaluation value. [Figure 14C] 10 is a table (part 2) showing an example of an intensity correction coefficient calculated based on an evaluation value. [Figure 15A] FIG. 10 is a diagram (part 1) showing an example of a menu screen. [Figure 15B] FIG. 10 is a diagram illustrating an example of an alert screen. [Figure 15C] FIG. 10 is a diagram (part 2) showing an example of a menu screen. [Figure 15D] FIG. 10 is a diagram illustrating an example of a color intensity adjustment screen. [Figure 16] FIG. 10 is a diagram illustrating an example of the configuration of an electrophoresis system according to a second embodiment. [Figure 17] FIG. 1 is a diagram illustrating an example of the hardware configuration of an electrophoresis data processing device. DETAILED DESCRIPTION OF THE INVENTION

[0013] Next, a mode for carrying out the present invention (referred to as an "embodiment") will be described in detail with reference to the drawings as appropriate. Note that in each drawing, similar components are given the same reference numerals and their description will be omitted.

[0014] [First embodiment] <System configuration diagram> FIG. 1 is a diagram schematically illustrating an example of the configuration of an electrophoresis system 1 according to the first embodiment. The electrophoresis system 1 includes an electrophoresis device 100, an electrophoresis data processing device 200, and a display device 301. In the first embodiment, a first sample (actual sample) D001, a second sample (first reference sample) D002, and a third sample (second reference sample) D003 are each different from one another, and color signal data D050 of the first sample D001 is finally output. The first sample D001, the second sample D002, and the third sample D003 are each given a plurality of fluorescent labels of the same type. In this embodiment, the second sample D002, the third sample D003, and the first sample D001 are passed through each of the capillaries 102 (see FIG. 2A ) included in the electrophoresis apparatus 100 in this order. The first sample D001, the second sample D002, and the third sample D003 may be collectively referred to as the samples.

[0015] The electrophoresis device 100 electrophoreses the samples and acquires signal charge data D011 to D013 (D010) for each of the first to third samples D001 to D003. The signal charge data D011 to D013 (D010) will be described later. For example, when the first sample D001, which is the sample to be measured (actual sample), is electrophoresed in the electrophoresis device 100, signal charge data (third signal charge data) D011 for the first sample D001 is obtained. In other words, the signal charge data D011 for the first sample D001 is the result of electrophoresis for the first sample D001.

[0016] The same is true for the signal charge data (first signal charge data) D012 of the second sample D002 and the signal charge data (second signal charge data) D013 of the third sample D003. That is, the signal charge data D012 of the second sample D002 is the result of electrophoresis for the second sample D002. And the signal charge data D013 of the third sample D003 is the result of electrophoresis for the third sample D003.

[0017] The first sample D001 to be measured by the electrophoresis apparatus 100 is a real sample of DNA molecules or reagents to which multiple fluorescent labels have been attached. When DNA molecules are used as the first sample D001, the DNA molecules are assumed to have fluorescent labels attached to base information (GATC) or characteristic base sequence structures (for example, locations where the same base sequence loops). In this embodiment, four types of fluorescent labels (first fluorescent label, second fluorescent label, third fluorescent label, and fourth fluorescent label) are assumed to be used. However, the types of fluorescent labels used are not limited to four. Furthermore, when DNA molecules are used as the real sample, they will be referred to as a DNA sample as appropriate.

[0018] The second sample D002 is used (e.g., a matrix standard) to calculate the fluorescence spectrum data D020 described below. The second sample D002 is a sample for calibrating data from the first sample D001, which is a real sample. More specifically, the second sample D002 is electrophoresed in advance prior to measurement using the first sample D001, which is a real sample, and is generally used to calibrate the wavelength of fluorescence in order to suppress false signals. The second sample D002 is also labeled with the same fluorescent labels as the first sample D001 (in this embodiment, the first to fourth fluorescent labels). The electrophoresis results of the second sample D002 have independent peaks that do not overlap for each fluorescent label when observed by electrophoresis.

[0019] The third sample D003 is a sample for calculating the intensity correction coefficient D040 (a sample for determining the intensity correction coefficient), which will be described later. The third sample D003 is a sample for evaluating data from the first sample D001. Specifically, the third sample D003 is a sample in which the difference in brightness between the fluorescent labels is reflected in the fluorescent color signal intensity, and has brightness similarity to that of the first sample D001. For example, an allelic ladder may be used as the third sample D003, or a sample independently specified by the user may be used. The allelic ladder is electrophoresed in advance prior to measurement using the first sample D001, which is the actual sample, and serves as a reference sample for the user to determine the length of the DNA molecules in the first sample D001. The allelic ladder is labeled with the same fluorescent labels as the first sample D001 (first to fourth fluorescent labels in this embodiment), and is characterized in that peaks appear at specific base length intervals for each fluorescent label in the electrophoresis results. Both the first sample D001 and the allelic ladder are electrically induced, and by comparing the first sample D001 with the allelic ladder, the base length of the DNA molecules in the first sample D001 is measured.

[0020] The electrophoresis data processing device 200 performs color intensity adjustment processing to equalize the fluorescent color signal intensities of the signal charge data D011 obtained from the electrophoresis device 100. The electrophoresis data processing device 200 includes a fluorescence calibration unit (fluorescence spectrum calculation unit) 201, a color conversion processing unit (fluorescence color signal data calculation unit) 202, an intensity correction coefficient determination unit (intensity correction coefficient calculation unit) 203, and an intensity adjustment processing unit (color signal data calculation unit) 204. The fluorescence calibration unit 201 calculates fluorescence spectrum data D020 based on the signal charge data D012 of the second sample D002 and passes it to the color conversion processing unit 202. The fluorescence spectrum data D020 will be described later, and is data obtained by normalizing the wavelength spectrum related to the signal charge value of the fluorescent label used in the second sample D002.

[0021] The color conversion processing unit 202 calculates fluorescence color signal data (second fluorescence color signal data) D033 (D030) of the third sample D003 based on the signal charge data D013 of the third sample D003 and the fluorescence spectral matrix. The fluorescence color signal data D033, which will be described later, is time-series information on the signal intensity (fluorescence color signal intensity) of each fluorescent label. Furthermore, the color conversion processing unit 202 calculates fluorescence color signal data (third fluorescence color signal data) D031 (D030) of the first sample D001 based on the signal charge data D011 of the first sample D001 and the fluorescence spectral matrix. The fluorescence color signal data D031, which will be described later, is time-series information on the signal intensity of each fluorescent label. The intensity correction coefficient determination unit 203 calculates an intensity correction coefficient D040, which is the ratio of the signal intensity of each fluorescent label to the reference signal intensity, using the signal intensity of a predetermined fluorescent label as a reference for the fluorescent color signal data D033. The intensity correction coefficient D040 will be described later. The intensity adjustment processing unit 204 calculates color signal data D050 by multiplying each piece of data of the fluorescent label in the fluorescence color signal data D031 by the corresponding intensity correction coefficient D040.

[0022] A display device (output unit) 301 displays (outputs) the color signal data D050 calculated by the intensity adjustment processing unit 204 and the like.

[0023] 1, the units 201 to 204 are shown independently, but at least two of the units 201 to 204 may be integrated into one unit. Also, the units 201 to 204 may be configured to perform their processing using one or more central processing units (CPUs). 1, all of units 201-204 are provided in electrophoresis data processing device 200. However, this is not limiting, and each of units 201-204 may have one or more external components. For example, electrophoresis data processing device 200 and electrophoresis device 100 may be integrated into one device. Alternatively, at least one of units 201-204, such as fluorescence calibration unit 201, may be installed outside electrophoresis data processing device 200.

[0024] (Electrophoresis device 100) FIG. 2A is a diagram showing an example of the configuration of an electrophoresis device 100. As shown in FIG. 2A, the electrophoresis apparatus 100 includes a detection unit 150 for optically detecting samples, a thermostatic bath 118 for maintaining the capillaries 102 at a constant temperature, and a transport device 125 for transporting various containers to the cathode ends of the capillaries 102. The electrophoresis apparatus 100 also includes a high-voltage power supply 104 for applying a high voltage to the capillaries 102, a first ammeter 105 for detecting the current generated by the high-voltage power supply 104, and a second ammeter 112 for detecting the current flowing through the anode electrode. The second ammeter 112 is connected to a GND (Ground) electrode 111 installed in an anode buffer container 110. The electrophoresis apparatus 100 also includes a capillary array 117 consisting of one or more capillaries 102, and a pump unit 103 for injecting a polymer into the capillaries 102.

[0025] The capillary array 117 is a replaceable component including a plurality of capillaries 102 (four in the example shown in FIG. 2A), and includes a load header 129, a detection unit 150, and a capillary head. When a user changes the measurement method, the capillary array 117 is replaced and the lengths of the capillaries 102 are adjusted. When a capillary 102 is damaged or its quality deteriorates, the user replaces it with a new capillary array 117.

[0026] The capillary 102, which serves as an electrophoresis path for electrophoretically separating samples (first sample D001, second sample D002, and third sample D003), is made 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. The capillary 102 has a polyimide coating on its surface to enhance its strength. However, in the detection unit 150, which is irradiated with laser light L (excitation light: dotted arrow in FIG. 2A; see FIGS. 3A and 3B), the polyimide coating of the capillary 102 is removed to allow the internal light to easily leak to the outside. The capillary 102 is filled with a separation medium that imparts a difference in migration speed during electrophoresis. Separation media can be either fluid or non-fluid; in this embodiment, a fluid polymer is used.

[0027] The detection unit 150 acquires information dependent on the sample. During electrophoresis, a laser beam L (see FIGS. 3A and 3B) is irradiated and passes through all of the capillaries 102 in succession. As described above, this laser beam L generates fluorescence having a wavelength dependent on the fluorescent label attached to the sample. The sample is analyzed by detecting this fluorescence.

[0028] As shown in Fig. 2B, the capillary cathode ends 140 are fixed through metal hollow electrodes 141, and the tips 142 of the capillaries 102 extend about 0.5 mm from the hollow electrodes 141. All of the hollow electrodes 141 provided in the capillaries 102 are mounted together on a load header 129 shown in Fig. 2A. All of the hollow electrodes 141 are connected to a high-voltage power supply 104 via the load header 129. The hollow electrodes 141 function as cathode electrodes when voltage application is required for electrophoresis, sample introduction, etc.

[0029] The ends (other ends) of the capillaries 102 opposite the capillary cathode ends 140 are bundled together by a capillary head (not shown). The bundle is a pressure-tight, detachable member. The capillary head can be connected to a block 107 in a pressure-tight manner. Then, new polymer is filled into the capillaries 102 from the other ends using a syringe 106. It is desirable to refill the polymer filled in the capillaries 102 after each measurement to improve measurement performance.

[0030] The pump unit 103 pressurizes the syringe 106. The block 107 is a connection unit for connecting the syringe 106, the capillary array 117, the anode buffer container 110, and the polymer container 109 to each other.

[0031] The light source 114 irradiates the detection unit 150 with laser light L (see FIGS. 3A and 3B). The detection unit 150 will be described later.

[0032] The thermostatic chamber 118 is covered with a heat insulating material to maintain a constant temperature inside, and the temperature is controlled by a heating and cooling mechanism 120. A fan 119 circulates and agitates the air inside the thermostatic chamber, thereby maintaining the temperature of the capillary array 117 uniform and constant across its position.

[0033] The transporter 125 is equipped with three electric motors and linear actuators, and is movable in three axes: up and down, left and right, and depth directions. At least one container can be placed on the moving stage 130 of the transporter 125. The moving stage 130 is further equipped with an electric grip 131, which can grip and release each container. Therefore, the buffer container 121, the washing container 122, the waste container 123, and the sample container 124 can be transported to the capillary cathode end 140 as needed. Unnecessary containers are stored in a designated storage location in the electrophoresis apparatus 100.

[0034] Furthermore, electrophoresis device 100 is used while connected to electrophoresis data processing device 200 via a communication cable. A user can control the functions of electrophoresis device 100 using electrophoresis data processing device 200 and exchange data detected by detection unit 150 of electrophoresis device 100.

[0035] (Detection unit 150) Fig. 3A is a diagram showing an example of the configuration of the detection unit 150 of the electrophoresis apparatus 100. Fig. 3B is a schematic cross-sectional view of a part of the detection unit 150 taken along a plane perpendicular to the capillary 102. Fig. 2 will also be referred to as appropriate. The detection unit 150 includes a planar ceramic substrate 151 , a lid 152 , a shutter 154 , an imaging optical lens 155 , and an optical detector 156 . 2A, the detection unit 150 detects light emitted from the sample by the laser light L emitted from the light source 114. In this way, the detection unit 150 detects the fluorescence emitted by the fluorescent labels in the DNA sample separated by electrophoresis.

[0036] 3A and 3B, unlike FIG. 2A, 16 capillaries 102 are arranged on a capillary support surface, which is the flat surface of a planar ceramic substrate 151, and fixed with adhesive or the like to form a capillary array 117. In this way, the areas of the capillaries 102 near the points where the laser light L is irradiated are arranged and fixed to an optically flat surface with a height accuracy of several microns. Each capillary 102 is a quartz glass tube covered with a thin polymer film, but the polymer film is removed at the location corresponding to the opening 153 in the lid 152, exposing the quartz. The inner and outer diameters of the quartz tube are 50 and 323 μm, respectively, and the outer diameter of each capillary 102, including the thin polymer film, is 363 μm.

[0037] As described above, there are 16 capillaries 102. As shown in FIG. 3B, the laser light L first irradiates the capillary 102 at the right end of the drawing, passes through it, and then irradiates the next capillary 102. In this manner, the laser light L passes through the capillaries 102 one after another and is emitted from the capillary 102 at the opposite end. The capillaries 102 have a cylindrical shape and are filled with a polymer, which provides a focusing function similar to that of a convex lens. This suppresses divergence of the laser light L. In this embodiment, the laser light L is irradiated from one direction. However, by irradiating the capillary array 117 with the laser light L from both the left and right directions, it is possible to irradiate substantially all of the capillaries 102 with laser light L of uniform intensity. Therefore, samples flowing through the 16 capillaries 102 can be simultaneously detected while maintaining high sensitivity.

[0038] The fluorescent labels in the DNA sample emit fluorescence when irradiated with laser light L. The fluorescence emitted from the fluorescent labels passes through opening 153 and is imaged on optical detector 156 by imaging optical lens 155. Optical detector 156 outputs the signal charge data D010 (see FIG. 1) described above.

[0039] Normally, laser light L continues to be output during analysis, but the duration of irradiation of laser light L onto the sample flowing inside capillary 102 is controlled by shutter 154. Electrophoresis data processing device 200 (see FIGS. 1 and 2A) controls the duration of irradiation onto capillary 102 by synchronizing the opening and closing of shutter 154 with the timing of data acquisition by optical detector 156. Electrophoresis data processing device 20 also controls the intensity of laser light L so that the signal value acquired by optical detector 156 does not become saturated.

[0040] The electrophoresis device 100 used in this embodiment does not necessarily have to have the configuration shown in FIGS. 2A to 3B.

[0041] (Processing by electrophoresis device 100) 4 is a flowchart showing the procedure of processing performed by the electrophoresis device 100 of the first embodiment. Figures 1 and 2A are referenced as appropriate. In the electrophoresis device 100, a first sample D001, a second sample D002, and a third sample D003 are electrophoresed. The electrophoresis device 100 outputs signal charge data D010 according to the processing flow shown in FIG. First, the electrophoresis apparatus 100 starts separating the sample into a predetermined base length by applying a voltage to the electrophoresis solvent (S101). At this time, the number of detection scans is initialized. The number of detection scans is the number of scans performed, which is the number of times the shutter 154 in FIG. 3A opens. Since the sample is flowing through the capillary 102, each time the shutter 154 opens, a substance flowing inside the capillary 102 at that time is detected. The number of scans is preset to be the maximum number of times for detection.

[0042] Then, the electrophoresis apparatus 100 determines whether the number of detection scans is equal to or less than the maximum number of detection scans (S102). If the number of detection scans is equal to or less than the maximum number of detection scans (S102→Yes), the light source 114 of the electrophoresis apparatus 100 irradiates the sample with excitation light (laser light L) to generate fluorescence (S103).

[0043] Subsequently, the detection unit 150 of the electrophoresis device 100 wavelength-disperses the generated fluorescence (S104), using a wavelength-dispersing element such as a diffraction grating for wavelength dispersion. Thereafter, the detection unit 150 of the electrophoresis apparatus 100 detects the generated fluorescence as a signal charge (S105). The fluorescence is detected using a CCD (Charge Coupled Device) element or a CMOS (Complementary Metal Oxide Semiconductor) element, etc., provided in the optical detector 156. In this embodiment, fluorescence detection using a CCD element is used. The CCD element performs detection for each dispersed wavelength. For example, signals are detected for wavelength regions of 500-510 nm, 510-520 nm, 690-700 nm using CCD elements corresponding to each wavelength region. Furthermore, the processes of steps S103 to S105 are performed any number of times in chronological order as the electrophoresis time elapses.

[0044] The electrophoresis apparatus 100 adds "1" to the number of detection scans (number of detection scans+1) (S106), and returns the process to step S102. In step S102, if the number of detection scans is greater than the maximum number of detections (S102→No), electrophoresis device 100 outputs the detected signal charges as signal charge data D010 to electrophoresis data processing device 200 (S107).

[0045] (Signal charge data D010) 5 is a diagram showing an example of signal charge data D010, with reference to FIG. Signal charge data D010 obtained by performing multiple detections using electrophoresis device 100 is expressed as a graph such as that shown in FIG. 5, with the vertical axis representing the integrated signal charge value and the horizontal axis representing the elapsed time of electrophoresis (the number of scans performed). FIG. 5 shows, for example, signal charge data D012 of the second sample D002. Signal charge data D011 of the first sample D001 and signal charge data D013 of the third sample D003 are similar to those shown in FIG. 5, although the shapes of the graphs are different.

[0046] Each line on the graph indicates the wavelength region detected by each CCD element. As described above, each CCD element provided in the optical detector 135 shown in Figures 2A and 3A detects fluorescence in a corresponding wavelength region. In other words, each line on the graph shown in Figure 5 corresponds to a respective CCD element. Furthermore, each line shown in Figure 5 is the sequential accumulation of signal charge values ​​for each wavelength region.

[0047] In the signal charge data D010, peaks 401 to 404 corresponding to the respective fluorescent labels are shown with respect to the elapsed time of electrophoresis. For example, peak 401 is derived from the first fluorescent label, and peak 402 is derived from the second fluorescent label. Similarly, peak 403 is derived from the third fluorescent label, and peak 404 is derived from the fourth fluorescent label.

[0048] The signal charge data D010 is expressed by a matrix "F" and is shown in equation (1) where m is the number of wavelength regions and n is the number of scans performed.

[0049]

number

[0050] Each element of the matrix "F" shown in equation (1) is f ij In the above, i indicates the wavelength region (CCD element) and j indicates the elapsed time of electrophoresis. Such signal charge data D010 is output for each of the first sample D001, the second sample D002, and the third sample D003. That is, it is output as signal charge data D011 of the first sample D001, signal charge data D012 of the second sample D002, and signal charge data D013 of the third sample D003 in FIG. Electrophoresis data processing device 200 finally outputs color signal data D050 in which the luminance difference of the fluorescent labels has been corrected, using signal charge data D010 received from electrophoresis device 100. The following describes in detail the processing performed by each of units 201 to 204 of electrophoresis data processing device 200.

[0051] <Specific processing of color intensity adjustment processing> (Processing by the fluorescence calibration unit 201) 6 is a flowchart showing the procedure of the process performed by the fluorescence calibration unit 201 of the first embodiment, with reference to FIG. 1 as appropriate. The fluorescence calibration unit 201 calculates the fluorescence spectrum data D020 according to the flow shown in Fig. 6. Steps S201 to S208 shown in Fig. 6 are fluorescence spectrum calculation steps. First, the fluorescence calibration unit 201 acquires signal charge data D010 from the electrophoresis device 100 (S201). The acquired signal charge data D010 is signal charge data D012 of the second sample D002 adjusted for wavelength calibration.

[0052] Furthermore, in step S201, the number of detected peaks is initialized. For example, the value of the number of detected peaks is set to "0." The number of detected peaks is the number of detected peaks 401 to 404 shown in FIG. 5 (four in the example shown in FIG. 5). When peak 401 shown in FIG. 5 is detected first, the number of detected peaks becomes "1" in step S206, and when peak 402 is detected next, the number of detected peaks becomes "2" in step S206.

[0053] The fluorescence calibration unit 201 determines whether the number of detected peaks in the signal charge data D012 is less than the number of fluorescent labels (S202).

[0054] Peak detection is performed by detecting whether a predetermined threshold value is exceeded in the signal charge data D012, or by detecting the point at which the signal charge value changes from increasing to decreasing. The number of fluorescent labels is the number of fluorescent labels used in the first sample D001, the second sample D002, and the third sample D003. In this embodiment, the first to fourth fluorescent labels are used, so the number of fluorescent labels is "4." Note that the signal charge data D010 handled by the fluorescence calibration unit 201 is the signal charge data D010 of the second sample D002, so the number of peaks 401 to 404 matches the number of fluorescent labels. In the signal charge data D011 of the first sample D001 and the signal charge data D013 of the third sample D003, the number of peaks does not necessarily match the number of fluorescent labels.

[0055] If the number of detected peaks is equal to or less than the number of fluorescent labels (S202 → Yes), the fluorescence calibration unit 201 extracts peak positions specific to the fluorescent labels from the acquired signal charge data D012 (S203). The peak positions are extracted as values ​​of the elapsed time of electrophoresis corresponding to peaks 401 to 404 shown in Figure 5. For example, the peak position of peak 401 in Figure 5 is at an elapsed time of electrophoresis of approximately "23". Thereafter, the fluorescence calibration unit 201 acquires the signal charge values ​​of each wavelength region that constitutes the peak (S204). In other words, the fluorescence calibration unit 201 acquires the values ​​of each CCD element at peaks 401 to 404 in FIG. Next, the fluorescence calibration unit 201 normalizes the acquired signal charge values ​​in each wavelength region (S205). For example, the fluorescence calibration unit 201 may normalize the signal charge values ​​by the maximum signal charge value in each wavelength region, or by the total signal charge value in each wavelength region.

[0056] Then, the fluorescence calibration unit 201 adds "1" to the number of detected peaks (number of detected peaks+1) (S206), and returns the process to step S202.

[0057] In step S202, if the number of detected peaks is greater than the number of fluorescent labels (S202→No), the fluorescence calibration unit 201 converts the signal charge values ​​of each wavelength region at the peak of each fluorescent label into a matrix (S207). In step S207, the fluorescence calibration unit 201 converts each normalized wavelength component into a matrix of the number of fluorescent labels x wavelength information. The fluorescence calibration unit 201 then outputs the converted matrix to the color conversion processing unit 202 as fluorescence spectrum data D020 (S208). "S," which represents the fluorescence spectrum data D020 as a matrix, is expressed by the following equation (2), where "m" is the number of wavelength regions and "n" is the number of fluorescent labels. The meaning of each component of "S" will be described later.

[0058]

number

[0059] (Fluorescence spectrum data D020) FIG. 7 is a diagram showing an example of the fluorescence spectrum data D020. When the fluorescence spectrum data D020 created by the fluorescence calibration unit 201 is represented on a graph with the horizontal axis representing the wavelength range and the vertical axis representing the normalized signal charge value, it is expressed by spectra 411 to 414 as shown in Fig. 7. In Fig. 7, spectrum 411 shown by the solid line is derived from the first fluorescent label, spectrum 412 shown by the dashed line is derived from the second fluorescent label, spectrum 413 shown by the dashed line is derived from the third fluorescent label, and spectrum 414 shown by the dashed line is derived from the fourth fluorescent label.

[0060] By the way, each component of "S" in formula (2) is "S ij " i corresponds to the spectrum 411, spectrum 412, spectrum 413, and spectrum 414 in Fig. 7, respectively. " j corresponds to the wavelength region in Fig. 7.

[0061] Such fluorescence spectrum data D020 indicates wavelength region spectra normalized to "1" for each of peaks 401 to 404 shown in FIG. 5. For example, spectrum 411 shown in FIG. 7 indicates the wavelength region spectrum normalized to "1" for peak 401 in FIG. 5. Spectrum 412 shown in FIG. 7 indicates the wavelength region spectrum normalized to "1" for peak 402 in FIG. 5. Spectrum 413 shown in FIG. 7 indicates the wavelength region spectrum normalized to "1" for peak 403 in FIG. 5. Spectrum 414 shown in FIG. 7 indicates the wavelength region spectrum normalized to "1" for peak 404 in FIG. 5.

[0062] In this way, the fluorescence spectrum data D020 is data obtained by normalizing the wavelength spectrum related to the signal charge value of the fluorescent label used in the second sample D002. The fluorescence calibration unit 201 then calculates the fluorescence spectrum data D020 based on the signal charge data D12 of the second sample D002.

[0063] Furthermore, the fluorescence spectrum data D020 ("S" (Equation (2))) which represents the spectra 411 to 414 shown in Fig. 7 as a matrix can be said to be a matrix of weights indicating the importance of each wavelength region for each fluorescent label. For example, it can be seen from spectrum 412 in Fig. 7 that the wavelength region "540 nm" is important for the second fluorescent label.

[0064] (Processing by color conversion processing unit 202) 8 is a flowchart showing the procedure of the process performed by the fluorescence calibration unit 201 of the first embodiment, with reference to FIG. 1 as appropriate. The color conversion processing unit 202 converts the signal charge data D010 (D011, D013) into fluorescence color signal data D030 according to the flow shown in Fig. 8. Steps S301 to S305 in Fig. 8 are fluorescence spectrum calculation steps. First, the color conversion processor 202 acquires signal charge data D010 from the electrophoretic device 100 (S301). At this time, the acquired signal charge data D010 is signal charge data D011 of the first sample D001 and signal charge data D013 of the third sample D003. That is, the following steps S302 to S305 are performed for each of the signal charge data D011 of the first sample D001 and the signal charge data D013 of the third sample D003.

[0065] Next, the color conversion processing unit 202 acquires the fluorescence spectrum data D020 from the fluorescence calibration unit 201 (S302). Next, the color conversion processing unit 202 calculates the pseudo-inverse matrix of the acquired fluorescence spectrum data D020 (S303). Furthermore, the color conversion processing unit 202 multiplies the signal charge data D010 (each of the signal charge data D011 and D013) acquired in step S601 by the pseudo inverse matrix calculated in step S303 (S304). Finally, the color conversion processing unit 202 outputs the result calculated in step S304 as fluorescence color signal data D030 to the intensity correction coefficient determination unit 203 and the intensity adjustment processing unit 204 (S305). In step S305, the color conversion processing unit 202 outputs the fluorescence color signal data D031 of the first sample D001 to the intensity adjustment processing unit 204. Then, in step S305, the color conversion processing unit 202 outputs the fluorescence color signal data D033 of the third sample D003 to the intensity correction coefficient determination unit 203.

[0066] The fluorescence color signal data D030 is actually output in the form of a matrix. If the number of fluorescent labels is "n" and the number of scans performed is "m", the matrix "C" representing the fluorescence color signal data D030 is expressed by the following formula (3).

[0067]

number

[0068] When calculating the fluorescence color signal data D030, the color conversion processing unit 202 calculates the pseudo-inverse matrix "S" of the signal charge data D010 "F" and the fluorescence spectrum data D020. -1 " and the matrix "C" of the fluorescence color signal data D030 are used to express the following equations (11) to (13). Note that the process performed in step S304 of FIG. 8 is the calculation shown in equation (13).

[0069] F = CS (11) FS -1 =CSS -1 ··· (12) FS -1 = C (13)

[0070] That is, the color conversion processing unit 202 calculates the fluorescence color signal data D030 as matrix "C" using equation (13). In this way, the color conversion processing unit 202 calculates the fluorescence color signal data D033 of the third sample D003 by multiplying the signal charge data D013 of the third sample D003 by the pseudo-inverse matrix of the fluorescence spectrum data D020. Similarly, the color conversion processing unit 202 calculates the fluorescence color signal data D031 of the first sample D001 by multiplying the signal charge data D011 of the first sample D001 by the pseudo-inverse matrix of the fluorescence spectrum data D020.

[0071] (Fluorescent color signal data D030) Fig. 9 shows an example of fluorescence color signal data D030. Fig. 9 shows fluorescence color signal data D033 generated based on signal charge data D013 of the third sample D003. The fluorescence color signal data D033 of the first sample D001 is similar to that shown in Fig. 9, although the graph shape is different. The matrix "C" of the fluorescent color signal data D030 calculated by the color conversion processing unit 202 can be represented by graphs 421 to 424 shown in FIG. 9, with the horizontal axis representing the elapsed time of electrophoresis (number of scans performed) and the vertical axis representing the fluorescent color signal intensity. In FIG. 9, graph 421, shown by a solid line, is derived from the first fluorescent label, and graph 422, shown by a dashed line, is derived from the second fluorescent label. Furthermore, graph 423, shown by a dashed line, is derived from the third fluorescent label, and graph 424, shown by a two-dot chain line, is derived from the fourth fluorescent label.

[0072] The component "C" of the fluorescent color signal data D030 "C" shown in equation (3) ij 9, i corresponds to graph 421, graph 422, graph 423, and graph 424. And j corresponds to the horizontal axis of FIG. 9 (elapsed time of electrophoresis).

[0073] As shown in FIG. 9, the fluorescence color signal data D030 is obtained by removing information about the wavelength range from the graphs 421 to 424 shown in FIG. 5, and instead represents information about the signal intensity (fluorescence color signal intensity) of each fluorescent label. The fluorescence color signal data D030 calculated in this manner represents time-series information about the signal intensity of each fluorescent label. The color conversion processing unit 202 then calculates the fluorescence color signal data (second fluorescence color signal data) D033 (D030) of the third sample D003 based on the signal charge data D013 of the third sample D003 and the fluorescence spectral matrix. The fluorescence color signal data D033, which will be described later, represents time-series information about the signal intensity of each fluorescent label. Furthermore, the color conversion processing unit 202 calculates the fluorescence color signal data (third fluorescence color signal data) D031 (D030) of the first sample D001 based on the signal charge data D011 of the first sample D001 and the fluorescence spectral matrix.

[0074] (Processing by the intensity correction coefficient determination unit 203) 10 is a flowchart showing the procedure of the processing performed by the intensity correction coefficient determination unit 203 of the first embodiment, with reference to FIG. The intensity correction coefficient determination unit 203 calculates the intensity correction coefficient D040 according to the flow shown in Fig. 10. Note that steps S401 to S408 in Fig. 10 are intensity correction coefficient calculation steps. First, the intensity correction coefficient determination unit 203 acquires fluorescence color signal data D030 from the color conversion processing unit 202 (S401). The fluorescence color signal data D030 used at this time is fluorescence color signal data D033 obtained by color-converting signal charge data D013 of a third sample D003, which is a sample for determining intensity correction coefficients, in the color conversion processing unit 202. At this time, the number of evaluation values ​​is initialized to, for example, "0."

[0075] Next, the intensity correction coefficient determination unit 203 determines whether the number of evaluation values ​​is less than the number of fluorescent markers (S402). The number of evaluation values ​​corresponds to the number of rows of the matrix "C" that represents the fluorescent color signal data D030 expressed by equation (3). For example, if the first row of the matrix "C" has been processed, the number of evaluation values ​​will be "1" in step S406. Similarly, if the second row of the matrix "C" has been processed, the number of evaluation values ​​will be "2" in step S406. In this way, the intensity correction coefficient determination unit 203 performs the processes of steps S403 to S406 for each row of the matrix "C" expressed by equation (3). As described above, the number of fluorescent markers is the number of fluorescent markers used, and in the example shown in this embodiment, it is "4."

[0076] If the number of evaluation values ​​is less than the number of fluorescent markers (S402→Yes), the intensity correction coefficient determination unit 203 uses the acquired fluorescent color signal data D033 to extract peak positions for the fluorescent marker being processed (S403). Next, the intensity correction coefficient determination unit 203 acquires the fluorescent color signal intensity of the peak (peak position) extracted in step S403 (S404). The peak position is the elapsed time corresponding to the highest fluorescent color signal intensity in each of graphs 421 to 424 in the example of Fig. 9. Then, the intensity correction coefficient determination unit 203 calculates an evaluation value from the information on the fluorescent color signal intensity acquired in step S404 (S405). The evaluation value will be described later.

[0077] Furthermore, the intensity correction coefficient determination unit 203 adds "1" to the number of evaluation values ​​(number of evaluation values+1) (S406), and returns the process to step S402.

[0078] In step S402, if the number of evaluation values ​​is equal to or greater than the number of fluorescent labels (S402→No), the intensity correction coefficient determination unit 203 calculates the relative ratio of the evaluation values ​​of each fluorescent label (S407). The relative ratio will be described later. Then, the intensity correction coefficient determination unit 203 outputs the relative ratio calculated in step S407 to the intensity adjustment processing unit 204 as the intensity correction coefficient D040 (S408).

[0079] The evaluation value calculated in step S405 is the average value of the fluorescent color signal intensity of each peak acquired in step S404. That is, the intensity adjustment processing unit 204 calculates the evaluation value for each fluorescent label by calculating the average value of the peaks originating from each fluorescent label in the fluorescent color signal data D033 of the third sample D003.

[0080] FIG. 11 is another diagram showing an example of the fluorescence color signal data D030. While Fig. 9 shows an example in which one peak is detected for each fluorescent label, Fig. 11 shows an example in which two peaks are detected for each fluorescent label. That is, peaks 421a and 421b correspond to the first fluorescent label, and peaks 422a and 422b correspond to the second fluorescent label. Similarly, peaks 423a and 423b correspond to the third fluorescent label, and peaks 424a and 424b correspond to the fourth fluorescent label. For example, when obtaining an evaluation value from the fluorescent color signal shown in FIG. 11, the evaluation value "C1" of the first fluorescent label is the average value of the fluorescent color signal intensities of peaks 421a and 421b. Furthermore, if X peaks are obtained from the first fluorescent label, the average value of these X peaks becomes the evaluation value "C1." Regarding the evaluation values ​​of the second to fourth fluorescent labels, the intensity correction coefficient determination unit 203 calculates evaluation values ​​"C2" to "C4" in the same manner as "C1."

[0081] The relative ratio calculated in step S407 is the ratio of the evaluation value of each fluorescent label when the evaluation value of a certain fluorescent label is used as a reference. For example, when evaluation values ​​"C1" to "C4" are used, if the reference is "C1" (signal intensity based on the reference; reference evaluation value), the relative ratio of the first fluorescent label is expressed as "C1 / C1" and the relative ratio of the second fluorescent label is expressed as "C1 / C2" (ratio of each fluorescent label to the signal intensity based on the reference). Specifically, the intensity correction determination unit 203 sets an arbitrary evaluation value from among the calculated evaluation values ​​as a reference evaluation value and calculates an intensity correction coefficient D040 for each fluorescent label by dividing the reference evaluation value by the respective evaluation value.

[0082] In this way, the intensity correction coefficient D040 is the ratio of the signal intensity of each fluorescent label to the reference signal intensity (fluorescent color signal intensity) of a predetermined fluorescent label in the fluorescent color signal data D031 of the third sample D003.

[0083] (Processing by the intensity adjustment processing unit 204) FIG. 12 is a flowchart showing the procedure of the processing performed by the intensity adjustment processing unit 204 in the first embodiment. The intensity adjustment processing unit 204 adjusts the fluorescence color signal data D031 to color signal data D050, which is data appropriate for secondary analysis, according to the flow shown in Fig. 12. Note that steps S501 to S503 in Fig. 12 are color signal data calculation steps, and step S504 is an output step. First, the intensity adjustment processing unit 204 acquires the fluorescence color signal data D031 of the first sample D001 from the color conversion processing unit 202 (S501). Next, the intensity adjustment processing unit 204 acquires the intensity correction coefficient D040 from the intensity correction coefficient determination unit 203 (S502). Next, the intensity adjustment processing unit 204 multiplies each fluorescence label in the acquired fluorescence color signal data D031 by an intensity correction coefficient D040 (S503). The intensity adjustment processing unit 204 multiplies each piece of fluorescence label data in the fluorescence color signal data D031 by the corresponding intensity correction coefficient D040. As a result, the intensity adjustment processing unit 204 calculates color signal data D050. Finally, the intensity adjustment processing unit 204 outputs the result of the multiplication in step S503 to the display device 301 as color signal data D050 (S504).

[0084] Fig. 13 is a diagram showing an example of color signal data D050. Fig. 13 shows an example in which the intensity correction coefficient D040 is applied to the fluorescence color signal data D030 of the first sample D001. The color signal data D050 output by the intensity adjustment processing unit 204 is represented by graphs 431 to 434 as shown in FIG. 13, where the horizontal axis represents the elapsed time of electrophoresis (detection scan number) and the vertical axis represents the corrected fluorescent color signal intensity. Note that, like FIG. 9, FIG. 13 shows an example in which one peak is detected for one fluorescent label. Graph 431, shown by a solid line, is derived from the first fluorescent label, and graph 432, shown by a dashed line, is derived from the second fluorescent label. Similarly, graph 433, shown by a dashed line, is derived from the third fluorescent label, and graph 434, shown by a two-dot chain line, is derived from the fourth fluorescent label.

[0085] As shown in FIG. 13, the fluorescent color signal intensities of the fluorescent labels are adjusted to be uniform.

[0086] <Independence of determination of strength correction factor D040> In the electrophoresis apparatus 100, it is conceivable that fluorescence detection is performed using the same light source 114 (see FIG. 2A) for multiple capillaries 102. In this case, the processes by the intensity correction coefficient determination unit 203 and the intensity adjustment processing unit 204 according to the first embodiment are performed independently for each capillary 102. Fig. 14A is a table showing examples of evaluation values ​​for each capillary 102 and each fluorescent label. Fig. 14B and Fig. 14C are tables showing examples of intensity correction coefficients D040 calculated based on the evaluation values ​​shown in Fig. 14A. "Capillary A" and "Capillary B" shown in FIGS. 14A to 14C are different capillaries 102.

[0087] A case will be described in which the evaluation values ​​for each fluorescent marker in the fluorescent color signal data D033 of the third sample D003 for each capillary 102 are the values ​​shown in FIG. 14A. When the third fluorescent label is used as a reference for all capillaries 102, the intensity correction coefficient D040 is expressed as shown in FIG. 14B. In FIG. 14B, the intensity correction coefficient D040 is calculated for both "capillary A" and "capillary B" using the third fluorescent label as a reference. However, as shown in FIG. 14B, the reference fluorescent label does not need to be fixed for all capillaries 102. For example, when evaluation values ​​such as those shown in FIG. 14A are obtained, the intensity correction coefficient D040 may be determined using the first fluorescent label as a reference for "capillary A" and the third fluorescent label as a reference for "capillary B." In this case, the intensity correction coefficient D040 will have a value as shown in FIG. 14C.

[0088] In this way, it is possible to arbitrarily determine which fluorescent label is used as the reference when calculating the intensity correction coefficient D040. Furthermore, the calculation of the color signal data D050 using the intensity correction coefficient D004 is also performed for each capillary 102.

[0089] In this way, the processes by the intensity correction coefficient determination unit 203 and the intensity adjustment processing unit 204 are performed independently in each capillary 102. In this way, even if the second sample D002 and the third sample D003 are different samples or use different fluorescent labels, they do not affect each other's results.

[0090] <Screen example> Next, an example of screen transitions in this embodiment will be described with reference to FIGS. 15A to 15D. Fig. 15A is a diagram showing an example of a menu screen 510, and Fig. 15B is a diagram showing an example of an alert screen 520. Fig. 15C is a diagram showing an example of the menu screen 510, and Fig. 15D is a diagram showing an example of a color intensity adjustment screen 530. In addition to the analysis execution function, the menu screen 510 also includes a button for performing an intensity adjustment function. Specifically, as shown in FIG. 15A, an analysis execution button 511, an analysis sample setting button 512, a color intensity adjustment button 513, and a maintenance button 514 are displayed. When the user selects and inputs the analysis execution button 511, analysis of base sequence information of DNA molecules and the like is performed. That is, analysis is performed on the signal charge data D011 of the first sample D001, which is the actual sample. Note that the selection and input are performed via the input device 302 shown in FIG. 17. When the user selects and inputs the analysis sample setting button 512, a setting screen for the first sample D001, in which multiple fluorescent labels are attached to DNA molecules, and the like, is displayed. When the user selects and inputs the color intensity adjustment button 513, a color intensity adjustment screen 530 (see FIG. 15D) for performing the color intensity adjustment process described in this embodiment is displayed. Then, when the user selects and inputs the maintenance button 514, a maintenance execution screen (not shown) for the electrophoresis apparatus 100 is displayed.

[0091] If the user selects and inputs the analysis execution button 511 when the color intensity adjustment process is incomplete, fluorescent color signal data D030 in which the intensities of the fluorescent labels vary, as shown in FIG. 9, will be acquired. Therefore, in this embodiment, if the analysis execution button 511 is selected and input when the color intensity adjustment process is incomplete, an alert screen 520 such as that shown in FIG. 15B is displayed before the actual analysis is performed. The alert screen 520 displays a dialog (warning information) that warns the user that color intensity adjustment has not been performed. In other words, when an analysis of the signal charge data D011 is attempted when the intensity correction coefficient D040 has not been calculated, warning information indicating that the intensity correction coefficient D040 has not been calculated is output to the display device 301.

[0092] The display of alert screen 520 allows the user to recognize the existence of color intensity adjustment processing. Also, as shown in Fig. 15B, alert screen 520 displays YES button 521 and NO button 522. If the user selects and inputs NO button 522, analysis is executed without performing color intensity adjustment processing. Also, if the user selects and inputs YES button 521, the screen transitions to menu screen 510 as shown in Fig. 15C.

[0093] As shown in FIG. 15C, when the user selects and inputs the color intensity adjustment button 513, the screen transitions to a color intensity adjustment screen 530 shown in FIG. 15D. Adjustment sample buttons 531a to 531d are displayed on the color intensity adjustment screen 530. Each of the adjustment sample buttons 531a to 531d corresponds to a capillary 102. In other words, the example shown in FIG. 15D illustrates an example in which the electrophoresis apparatus 100 has four capillaries 102. When the user selects and inputs one of the adjustment sample buttons 531a to 531d, information regarding the adjustment sample to be set in each capillary 102 can be set. The adjustment sample is a sample for determining the intensity correction coefficient D040, and in the first embodiment, it is the third sample D003. Note that in the example shown in FIG. 15C, the adjustment sample buttons 531c and 531d are blank. This indicates that adjustment samples are set in the capillaries 102 corresponding to the adjustment sample buttons 531a and 531b, and that adjustment samples are not set in the capillaries 102 corresponding to the adjustment sample buttons 531c and 531d. Also, a start button 533 is displayed on the color intensity adjustment screen 530. After setting adjustment samples in each of the capillaries 102 to be used, when the user selects and inputs the start button 533, the color intensity adjustment process starts. In this case, the color intensity adjustment process is performed collectively for all of the capillaries 102 in which adjustment samples are set.

[0094] Furthermore, the color intensity adjustment screen 530 displays individual start buttons 532 corresponding to the respective adjustment sample buttons 531. When the user selects and inputs an individual start button 532, the color intensity adjustment process is started for the corresponding capillary 102. For example, when the user selects and inputs an individual start button 532a, the color intensity adjustment process is started for the capillary 102 corresponding to the adjustment sample button 531a, and the color intensity adjustment process is not executed for the other capillaries 102.

[0095] After setting the adjustment sample in the electrophoresis apparatus 100 in this way, the start button 533 is pressed to calculate the intensity correction coefficient D040.

[0096] In this embodiment, the alert screen 520 is used to make the user aware of the presence of the color intensity adjustment process, but the presence or absence of the color intensity adjustment process may also be clearly stated in a document such as an instruction manual. It is desirable that the screens 510, 520, and 530 shown in FIGS. 15A to 15D are displayed separately from the DNA analysis screen.

[0097] Data acquisition of a DNA sample (first sample D001) obtained by electrophoresis must ensure that all colors are consistent. Previously, binning parameters had to be adjusted before measurement, which was one of the factors that made it difficult to use new reagents in the electrophoresis apparatus 100. According to this embodiment, by using an existing third sample D003, such as an allelic ladder, variations in the fluorescent color signal intensity of the first sample D001 can be suppressed without relying on binning. Furthermore, the fluorescent color signal intensity between fluorescent labels can be uniformed so as not to exceed the maximum allowable intensity of the CCD element of the optical detector 156. Thus, the first embodiment provides a technology that accurately and as quickly as possible calculates an effective intensity correction coefficient D040 for a set of fluorescent labels used and applies it to the analysis target, thereby suppressing variations in sensitivity among the fluorescent labels. This supports efficient analysis of electrophoresis results.

[0098] Furthermore, by using an allelic ladder as the third sample D003, the allelic ladder for evaluating the DNA molecular length of the first sample D001 can be used to calculate the intensity correction coefficient D040.

[0099] [Second embodiment] FIG. 16 is a diagram showing an example of the configuration of an electrophoresis system 1a according to the second embodiment. In the second embodiment, the intensity correction coefficient D040 is calculated based on the second sample D002 instead of the third sample D003 in the first embodiment. In the electrophoresis system 1a, a first sample D001 and a second sample (a first reference sample and a second reference sample) D002, which are different from each other, are used to finally output color signal data D050 of the first sample D001. It is also assumed that the first sample D001 and the second sample D002 are labeled with the same type of fluorescent label. In the configuration of the second embodiment, signal charge data D012 (D010) obtained from the second sample D002 used to calculate the fluorescence spectrum data D020 is input as second signal data to the color conversion processing unit 202, and fluorescence color signal data D032 (D030) of the second sample D002 is output. As described above, a matrix standard is used as the second sample D002. Then, based on the output fluorescence color signal data D032 of the second sample D002, the intensity correction coefficient determination unit 203 calculates an intensity correction coefficient D040.

[0100] In addition to adjusting the intensity of the fluorescent color signals, it was necessary to use a matrix standard, which is a fluorescent calibration reagent intended to suppress the generation of false fluorescent signals. This matrix standard was originally used only for the purpose of suppressing false signals. However, the inventors discovered that, when the intensity ratio was examined, the intensity ratio between the signal obtained from the DNA sample and the signal obtained from the matrix standard was the same. In the second embodiment, this calibration reagent is also used for intensity correction, thereby adjusting each color to be consistent.

[0101] According to the second embodiment, the intensity correction coefficient D040 is calculated from the second sample D002 such as a matrix standard without using the third sample D003, so that the number of samples used can be reduced compared to the first embodiment.

[0102] Until now, matrix standards have been used to adjust wavelength and frequency directions, as described in, for example, International Publication No. 2014 / 188887, but have not been used to adjust fluorescent color signal intensity. Similarly, the allelic ladder used as the third sample D003 in the first embodiment is a reference sample that allows the user to recognize the length of the DNA molecules in the first sample D001, and has not been used to correct fluorescent color signal intensity. This embodiment is characterized in that it uses such an allelic ladder, matrix standard, etc. to adjust for variations in fluorescent color signal intensity.

[0103] In this embodiment, when calculating the intensity correction coefficient D040, fluorescent color signal data D032 and D033 calculated from a second sample D002 such as a matrix standard and a third sample D003 such as an allelic ladder are used. However, the present invention is not limited to this, and any reference sample may be used as long as it is used to evaluate and calibrate data from the first sample D001, which is an actual sample.

[0104] Specifically, the reference sample may have the following characteristics (A1) and (A2). (A1) The fluorescent label used for electrophoresis of the DNA sample and the fluorescent label used for calibration are the same type. (A2) The fluorescence intensity ratios between fluorescent labels for the fluorescent color signal intensities obtained by color-converting the fluorescence from the fluorescent labels have a similar correlation (e.g., correlation coefficient R>0.8).

[0105] [Hardware configuration] FIG. 17 is a diagram showing an example of the hardware configuration of the electrophoresis data processing device 200. As shown in FIG. Electrophoresis data processing device 200 includes memory 211 such as RAM (Random Access Memory), arithmetic unit 212, storage device 213, and communication device 214. Arithmetic unit 212 is configured with a CPU (Central Processing Unit), a GPU (Graphics Processing Unit), etc. Storage device 213 is configured with an HDD (Hard Disk Drive), an SDD (Solid State Drive), etc. Communication device 214 transmits and receives data to and from electrophoresis device 100 (see FIGS. 1 and 16). Electrophoresis data processing device 200 is also connected to display device 301 such as a display and input device 302 such as a keyboard and a mouse.

[0106] A program stored in storage device 213 is loaded into memory 211, and the loaded program is executed by calculation device 212. This embodies the fluorescence calibration unit 201, color conversion processing unit 202, intensity correction coefficient determination unit 203, and intensity adjustment processing unit 204 shown in Fig. 1 and Fig. 17.

[0107] It should be noted that the intensity correction coefficient determination unit 203 is executed independently as a function separate from the function of executing DNA analysis.

[0108] The present invention is not limited to the above-described embodiments and includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present invention, and the present invention is not necessarily limited to those having all of the described configurations. Furthermore, it is possible to replace part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. Furthermore, it is possible to add, delete, or replace part of the configuration of each embodiment with other configurations.

[0109] Furthermore, the above-described configurations, functions, units 201 to 204, storage device 213, etc. may be partly or entirely implemented in hardware by, for example, designing them as integrated circuits. Furthermore, as shown in Fig. 17, the above-described configurations, functions, etc. may be implemented in software by an arithmetic unit 212 such as a CPU interpreting and executing a program that implements each function. Information such as the programs, tables, and files that implement each function can be stored in a HD as shown in Fig. 17, or in a storage device such as memory 211 or an SSD, or in a storage medium such as an IC (Integrated Circuit) card, an SD (Secure Digital) card, or a DVD (Digital Versatile Disc).

[0110] In addition, in each embodiment, the control lines and information lines shown are those that are considered necessary for explanation, and not all control lines and information lines in the product are necessarily shown. In reality, it can be considered that almost all components are interconnected. [Explanation of symbols]

[0111] 1. Electrophoresis system 1a Electrophoresis system 100 Electrophoresis apparatus 102 Capillary 150 Detector 200 Electrophoresis data processing device 201 Fluorescence calibration unit (fluorescence spectrum calculation unit) 202 Color conversion processing unit (fluorescence color signal data calculation unit) 203 Strength correction coefficient determination unit (strength correction coefficient calculation unit) 204 Intensity adjustment processing unit (color signal data calculation unit) 301 Display device (output section) 510 Menu screen 511 Analysis execution button 512 Analysis sample setting button 513 Color intensity adjustment button 520 Alert screen (displays warning information) 530 Color intensity adjustment screen 531 Adjustment sample button 531a Adjustment sample button 531b Adjustment sample button 531c Adjustment sample button 531d Adjustment sample button D001 First sample (actual sample, fluorescent label included) D002 Second sample (first reference sample, containing fluorescent label) D003 Third sample (second reference sample, containing fluorescent label) D010 Signal charge data D011 Signal charge data (third signal charge data) D012 Signal charge data (first signal charge data, second signal charge data) D013 Signal charge data (second signal charge data) D020 Fluorescence spectrum data D030 Fluorescence color signal data D031 Fluorescent color signal data (third fluorescent color signal data) D033 Fluorescent color signal data (second fluorescent color signal data) D040 Strength correction factor D050 Color signal data S201~S208 Fluorescence spectrum calculation steps S301~S305 Fluorescence spectrum calculation steps S401~S408 Strength correction coefficient calculation steps S501 to S503 Color signal data calculation steps S504 Output Step

Claims

1. a fluorescence spectrum calculation unit that calculates fluorescence spectrum data, which is data obtained by normalizing a wavelength spectrum related to a signal charge value of a fluorescent label used in a first reference sample, based on first signal charge data, which is a result of electrophoresis of the first reference sample, which is a sample for calibrating data based on an actual sample; a fluorescence color signal data calculation unit that calculates second fluorescence color signal data, which is time-series information on the signal intensities of the fluorescent labels, based on second signal charge data, which is the result of the electrophoresis of a second reference sample that is a sample for evaluating or calibrating data from the real sample, and the fluorescence spectrum data, and that calculates third fluorescence color signal data, which is time-series information on the signal intensities of the fluorescent labels, based on third signal charge data, which is the result of the electrophoresis of the real sample, and the fluorescence spectrum data; an intensity correction coefficient calculation unit that calculates an intensity correction coefficient, which is a ratio of the signal intensity of each of the fluorescent labels to the reference signal intensity, in the second fluorescent color signal data, using the signal intensity of a predetermined fluorescent label as a reference; a color signal data calculation unit that calculates color signal data by multiplying each of the pieces of data of the fluorescent labels in the third fluorescent color signal data by the corresponding intensity correction coefficient; an output unit that outputs the color signal data; Electrophoresis data processing device comprising:

2. The fluorescence color signal data calculation unit calculating second fluorescent color signal data by multiplying second signal charge data, which is the result of electrophoresis of the second reference sample, by a pseudo-inverse matrix of the fluorescent spectrum data; The third signal charge data is multiplied by the pseudo-inverse matrix of the fluorescence spectrum data to calculate third fluorescence color signal data.

2. The electrophoresis data processing apparatus according to claim 1.

3. The intensity correction coefficient calculation unit In the second fluorescent color signal data, an evaluation value is calculated for each of the fluorescent labels by calculating an average value of peaks derived from each of the fluorescent labels, and an arbitrary evaluation value among the calculated evaluation values ​​is set as a reference evaluation value. The intensity correction coefficient is calculated for each of the fluorescent labels by dividing the reference evaluation value by each of the evaluation values.

2. The electrophoresis data processing apparatus according to claim 1.

4. The first reference sample is a matrix standard and the second reference sample is an allelic ladder.

2. The electrophoresis data processing apparatus according to claim 1.

5. The first reference sample and the second reference sample are matrix standards.

2. The electrophoresis data processing apparatus according to claim 1.

6. an electrophoresis apparatus for performing the electrophoresis is provided with a plurality of capillaries through which the first reference sample, the second reference sample, and the actual sample flow; The processes performed by the fluorescence spectrum calculation unit, the fluorescence color signal data calculation unit, the intensity correction coefficient calculation unit, and the color signal data calculation unit are performed independently for each capillary.

2. The electrophoresis data processing apparatus according to claim 1.

7. When an analysis of the third signal charge data is attempted in a state in which the intensity correction coefficient has not been calculated, warning information indicating that the intensity correction coefficient has not been calculated is output to the output unit.

2. The electrophoresis data processing apparatus according to claim 1.

8. An electrophoresis data processing device, a fluorescence spectrum calculation step of calculating fluorescence spectrum data, which is data obtained by normalizing a wavelength spectrum related to a signal charge value of a fluorescent label used in a first reference sample, based on first signal charge data, which is a result of electrophoresis of the first reference sample, which is a sample for calibrating data based on an actual sample; a fluorescence color signal data calculation step of calculating second fluorescence color signal data, which is time-series information on the signal intensities of the respective fluorescent labels, based on second signal charge data, which is the result of the electrophoresis, for a second reference sample, which is a sample for evaluating or calibrating data from the real sample, and the fluorescence spectrum data, and calculating third fluorescence color signal data, which is time-series information on the signal intensities of the respective fluorescent labels, based on third signal charge data, which is the result of the electrophoresis, for the real sample, and the fluorescence spectrum data; an intensity correction coefficient calculation step of calculating an intensity correction coefficient, which is a ratio of the signal intensity of each of the fluorescent labels to the reference signal intensity, in the second fluorescent color signal data, using the signal intensity of a predetermined fluorescent label as a reference; a color signal data calculation step of calculating color signal data by multiplying each of the data of the fluorescent labels of the third fluorescent color signal data by the corresponding intensity correction coefficient; an output step of outputting the color signal data; Electrophoresis data processing method, characterized by carrying out the steps of:

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