JP7050122B2 - Capillary electrophoresis device - Google Patents

Description

The present invention relates to an analysis system and an analysis method.

Multiple types of components contained in biological samples such as DNA, proteins, cells, etc. are labeled with multiple types of phosphors (however, not necessarily one-to-one correspondence), and from multiple types of phosphors. Analytical methods that analyze multiple types of components by detecting while identifying the emission fluorescence of the protein are widely used. Such analytical methods include, for example, chromatography, DNA sequencer, DNA fragment analysis, flow cytometer, PCR, HPLC, Western Northern Southern blot, microscopic observation and the like.

In general, the fluorescence spectra of a plurality of types of phosphors used have overlaps with each other (hereinafter, referred to as spectrum overlap). Furthermore, the fluorescence emission of a plurality of types of phosphors overlaps each other temporally or spatially (hereinafter, referred to as spatiotemporal overlap). In the above-mentioned situation with overlap, a technique for detecting emission fluorescence from a plurality of types of phosphors while distinguishing them is required.

Next, this technique will be described using a DNA sequencer using electrophoresis as an example. DNA sequencers using electrophoresis started with slab electrophoresis in the 1980s and have changed to methods using capillary electrophoresis since the 1990s, but the technology to solve the above problems is basically unchanged. FIG. 3 of Non-Patent Document 1 is a method in slab electrophoresis. This technique is also used in capillary electrophoresis. The basic process of the present technology is composed of the following (1) to (4) on condition that M ≦ N.
(1) A sample containing M types of DNA fragments labeled with M types of phosphors is separated by electrophoresis and irradiated with a laser beam to emit light, and N colors are detected in N types of wavelength bands. Acquire time-series data of N-color fluorescence intensity.
(2) Time-series data Color conversion is performed for each time of (1), and time-series data of the concentration of M-type phosphors, that is, M-type DNA fragments is acquired.
(3) Each time of the time series data (2) is corrected based on the difference in mobility of M types of phosphors (hereinafter referred to as mobility correction), and M types of phosphors, that is, M types of phosphors are corrected. The mobility-corrected time-series data of the concentration of the DNA fragment is acquired.
(4) Make a base call based on the time series data (3).

Based on the above (1) to (4), FIG. 3 of Non-Patent Document 1 will be described in detail. Here, M = 4 and N = 4. In the following, the case of analyzing one sample using one electrophoresis path will be described. When analyzing multiple samples using multiple electrophoresis paths, do the following in parallel:

First, (1) will be described. When making copies of DNA fragments of various lengths to template DNA by the Sanger reaction, the DNA fragment is divided into four types of fluorophore (below) depending on the base species C, A, G, and T at the ends. , For simplicity, these phosphors are labeled with C, A, G, and T, respectively). The DNA fragments labeled with the fluorophore are separated in length by electrophoresis and sequentially irradiated with a laser beam to cause the fluorophore to emit light. The emitted fluorescence is detected in four types of wavelength bands b, g, y, and r (each wavelength band should be matched to the maximum emission wavelengths of C, A, G, and T) (hereinafter, four color detection). Called). As a result, time-series data of the fluorescence intensities I (b), I (g), I (y), and I (r) of those four colors are acquired. FIG. 3A of Non-Patent Document 1 is these time-series data (sometimes called raw data), and I (b), I (g), I (y), and I (r) are blue, respectively. Shown in green, black, and red.

ここで，４行４列の行列Ｗの要素ｗ（ＸＹ）は，スペクトルオーバーラップによって，蛍光体種Ｙ（Ｃ，Ａ，Ｇ，またはＴ）が波長帯Ｘ（ｂ，ｇ，ｙ，またはｒ）で検出される強度比率を表す。ｗ（ＸＹ）は，蛍光体種Ｙ（Ｃ，Ａ，Ｇ，またはＴ）および波長帯Ｘ（ｂ，ｇ，ｙ，またはｒ）の特性のみで決まる固定値であり，電気泳動中に変化しない。 Next, (2) will be described. The fluorescence intensities of the four colors at each time are the concentrations D (C), D (A), D (G) of the four types of phosphors C, A, G, and T at that time, as shown in equation (1). , And D (T).

Here, in the element w (XY) of the matrix W of 4 rows and 4 columns, the phosphor type Y (C, A, G, or T) has the wavelength band X (b, g, y, or r) due to the spectral overlap. ) Represents the detected intensity ratio. w (XY) is a fixed value determined only by the characteristics of the fluorophore type Y (C, A, G, or T) and the wavelength band X (b, g, y, or r), and does not change during electrophoresis. ..

このように，逆行列Ｗ^－１を，４色の蛍光強度に乗じることによってスペクトルオーバーラップを解消する（以下では，この工程を色変換と呼ぶ）。これにより，Ｃ，Ａ，Ｇ，およびＴの４種類の蛍光体濃度，すなわち末端が４種類の塩基のＤＮＡ断片の濃度の時系列データが取得される。 Therefore, the concentrations of the four types of phosphors at each time can be obtained from the fluorescence intensities of the four colors at that time, as shown in equation (2).

In this way, the spectral overlap is eliminated by multiplying the inverse matrix W ^-1 by the fluorescence intensities of the four colors (hereinafter, this step is referred to as color conversion). As a result, time-series data of the concentrations of the four types of phosphors C, A, G, and T, that is, the concentrations of the DNA fragments of the bases having four types at the ends are acquired.

FIG. 3B of Non-Patent Document 1 is this time series data. Color conversion can be performed with or without spatiotemporal overlap. As can be seen in FIG. 3B, the presence of multiple types of phosphor concentrations at the same time indicates the presence of spatiotemporal overlap.

FIG. 3C of Non-Patent Document 1 is a process that does not correspond to (1) to (4) and is not necessarily necessary. A signal representing the concentration of a single length DNA fragment labeled with the time series data of FIG. 3B by deconvolution with individual peaks, one of the four fluorophore C, A, G, and T. By separating into, the spatiotemporal overlap is eliminated.

Finally, (3) and (4) will be described. Generally, by electrophoresis, DNA fragments of various lengths, which differ by one base length, are separated at approximately equal intervals. However, the mobility (electrophoretic rate) may change due to the influence of the phosphor labeled on the DNA fragment, and the above equal intervals may be disrupted. Therefore, the magnitude relationship of the mobility depending on the type of the labeled phosphor is investigated in advance, and the time series data of FIG. 3B or FIG. 3C of Non-Patent Document 1 is corrected based on the information. As a result, time-series data in which DNA fragments of various lengths different by one base length are arranged at substantially equal intervals is acquired. FIG. 3D of Non-Patent Document 1 is this time series data. The blue, green, black, and red peaks each represent the concentration of a single length DNA fragment with terminal base species C, A, G, and T, each of which is one base different in length. They are lined up in order. Therefore, as shown in FIG. 3D, the result of the base call can be obtained by listing the terminal base species in order.

In addition, in each of the above steps, or before and after each step, processing such as smoothing, noise filtering, and baseline removal for time-series data may be performed as appropriate.

In the color conversion step (2), as represented by the equation (1), the four types of phosphor concentrations D (C) and D (A) of C, A, G, and T are used for each time. , D (G), and D (T) unknowns, four types of fluorescence detection intensities I (b), I (g), I (y), and I (r), b, g, y, and r. It is obtained by solving a quaternary simultaneous equation composed of the known numbers of. In general, the condition of M ≦ N is required as described above in order to correspond to solving M kinds of unknowns by N-element simultaneous equations. If M> N, the solution cannot be uniquely obtained (that is, a plurality of solutions can exist), so that the color conversion cannot be performed as in Eq. (2).

このとき，行列Ｗは３行４列となり，その逆行列は存在しないため，式（２）のように，４種類の蛍光体の濃度を一意に求めることができない。上述の通り，一般には，Ｍ＞Ｎの条件下で解を求めることができないが，以下のように，前提条件を加えることによって解を求めることができるようになる。 However, Non-Patent Document 2 deals with the DNA sequence by electrophoresis under the condition of M> N of M = 4, N = 3. When emission fluorescence is detected in three types of wavelength bands b, g, and r (hereinafter referred to as three-color detection), the four-color fluorescence intensity at each time of the formula (1) is 3 at each time of the formula (3). Replaced by color fluorescence intensity.

At this time, since the matrix W has 3 rows and 4 columns and its inverse matrix does not exist, the concentrations of the four types of phosphors cannot be uniquely obtained as in the equation (2). As described above, in general, the solution cannot be obtained under the condition of M> N, but the solution can be obtained by adding the preconditions as follows.

First, in the first step, it is assumed that there is no spatiotemporal overlap of multiple types of phosphors, that is, only one type of phosphor emits light at a time. At this time, at each time, the ratio of the fluorescence intensities (I (b) I (g) I (r)) ^T of the three colors and each of the four columns of the matrix W (w (bY) w (gY) w (rY). )) Y (C, A, G, or T) with the closest ratio of ^T can be selected. In other words, in equation (3), one type of phosphor is selected and the concentration of the remaining three types of phosphors is set to zero, that is, (D (C) D (A) D (G) D. (T)) ^T , (D (C) 0 0 0) ^T , (0 D (A) 0 0) ^T , (0 0 D (G) 0) ^T , or (0 0 0 D (T)) When ^T is used, D (Y) (Y is C, A, G, or T) at which the difference between the left side and the right side is the smallest is obtained. At that time, Y (C, A, G, or T) having the smallest difference between the left side and the right side can be selected. Here, if the difference between the left side and the right side is not sufficiently small in either case, the process proceeds to the next second step.

In the second step, it is assumed that only two types of fluorophore emit light at a time. This time, in the formula (3), two types of phosphors are selected and the concentrations of the remaining two types of phosphors are set to zero, that is, (D (C) D (A) D (G) D (T)). ^T , (D (C) D (A) 0 0) ^T , (D (C) 0 D (G) 0) ^T , (D (C) 0 0 D (T)) ^T , (0 D (A) ) D (G) 0) ^T , (0 D (A) 0 D (T)) ^T , or (0 0 D (G) D (T)) ^T , the difference between the left side and the right side is the smallest. Two types of D (Y) (Y is C, A, G, or T) are obtained. At that time, two types of Y (C, A, G, or T) having the smallest difference between the left side and the right side can be selected.

In this way, similar to the step (2) in Non-Patent Document 1, time-series data of the concentrations of the four types of phosphors, that is, the four types of DNA fragments are acquired. After that, the same steps as the steps (3) and (4) in Non-Patent Document 1 can be performed, and the result of the base call can be obtained.

The spatiotemporal overlap is small in order for the conditions for carrying out the first and second steps in Non-Patent Document 2, that is, the assumption that only one or two kinds of phosphors emit light at a time are satisfied. That is, the following two must be established.
(A) In the time-series data of the concentrations of the four types of DNA fragments, a plurality of peaks, each derived from a DNA fragment of a single length, are arranged at approximately equal intervals. (B) Of the four types of DNA fragments. In the time series data of concentration, the separation of two adjacent peaks derived from DNA fragments with different base lengths is good.

In Non-Patent Document 2, four different types of phosphors are labeled on each of the four types of primers used in the Sanger reaction (to be exact, the labeling method of the three types of fluorescent substances is changed), and the fluorescent substance is labeled on the DNA fragment. The difference in mobility due to the influence of is sufficiently small. In addition, the conditions are such that the electrophoresis separation performance is sufficiently high. As a result, time-series data of the fluorescence intensities (I (b) I (g) I (r)) ^T of the three colors for which the above (a) and (b) are satisfied are acquired (Non-Patent Document). 2 in the upper part of Fig. 2). In addition, under these conditions, the above first and second steps are carried out. As a result, time-series data on the concentrations of four types of phosphors, that is, four types of DNA fragments, is acquired, and the result of the base call is obtained (the lower part of FIG. 2 of Non-Patent Document 2).

Genome Res. 1998 Jun; 8 (6): 644-65. Electrophoresis. 1998 Jun; 19 (8-9): 1403-14.

The RGB color sensor is a two-dimensional sensor in which three types of pixels that detect the red (R), green (G), and blue (B) wavelength bands, which correspond to the three primary colors that can be identified by the human eye, are arranged. be. RGB color sensors have been used not only in single-lens reflex digital cameras and compact digital cameras, but also in digital cameras installed in smartphones in recent years, and have become explosively popular all over the world. Therefore, its performance is significantly improved and its price is also significantly reduced. Therefore, it is extremely useful to apply the RGB color sensor to an analytical method that detects emission fluorescence from a plurality of types of phosphors while discriminating them and detects a plurality of types of components. However, since the RGB color sensor can detect only three colors, it is difficult to distinguish the emission fluorescence of four or more types of phosphors. As described above, in general, in order to identify the emission fluorescence of M types of phosphors, when the M types of phosphors have spectral overlap and spatiotemporal overlap, the condition of M ≦ N is N. It is necessary to perform N color detection in various wavelength bands.

In Non-Patent Document 2, in the case of a DNA sequencer using electrophoresis, when the emission fluorescence of M = 4 types of phosphors is detected in N = 3 colors (that is, in the case of M> N), there is a spatiotemporal overlap. The above difficulties are solved by setting preconditions. The condition was that only one or two types of phosphors emit fluorescent light at a time, that is, the above (a) and (b) were satisfied.

However, in general, the above conditions (a) and (b) are often not satisfied. Even in the time-series data of the fluorescence intensities of the three colors (upper row) and the time-series data of the concentrations of the four types of phosphors (lower row) in FIG. However, due to a phenomenon called compression, three or more peaks, each derived from a DNA fragment of a single length, are densely packed. As a result, the above condition (a) is broken and the correct base call has not been reached. Further, in the latter stage of the electrophoresis in FIG. 2 of Non-Patent Document 2, the separation performance of the electrophoresis deteriorates. Therefore, the separation of two adjacent peaks derived from DNA fragments having different base lengths is insufficient, the above condition (b) is broken, and the correct base call is not reached.

In Non-Patent Document 2, the condition (a) is realized by using a primer labeling method in which four different types of phosphors are labeled on each of the four types of primers used in the Sanger reaction. However, in recent years, instead of the primer labeling method, a terminator labeling method in which four different types of phosphors are labeled on each of the four types of terminators used in the Sanger reaction is mainly used. Whereas the primer labeling method required the Sanger reaction using four types of primers to be performed separately, that is, in four different sample tubes, the terminator labeling method required the Sanger reaction using four types of terminators together. That is, it can be done in one sample tube. Therefore, the terminator label method can greatly simplify the Sanger reaction.

However, in the primer labeling method, the difference in mobility of DNA fragments labeled with four types of phosphors is small, whereas in the terminator labeling method, the difference in mobility of DNA fragments labeled with four types of phosphors is large. Therefore, the condition (a) is inevitably not satisfied. That is, not only one or two types of phosphors emit fluorescent light, but three or more types of phosphors may emit fluorescent light at one time. Therefore, at least when the terminator label method is used, it is difficult to perform DNA sequencing by the method of Non-Patent Document 2.

Therefore, in the present invention, in a state where the emission fluorescence from the M types of phosphors has spectral overlap and spatiotemporal overlap, M types are detected by N color detection in the wavelength band of N types (M> N). Provides an analysis technique for detecting the components of.

For example, in order to solve the above problem, the configuration described in the claims is adopted. The present application includes a plurality of means for solving the above-mentioned problems, and to give an example thereof, a sample containing a plurality of components labeled with any of the M types of phosphors is separated by chromatography, and the plurality of the above-mentioned plurality. An analyzer that acquires the first time-series data of the fluorescence signal detected in the N types (M> N) wavelength band in a state where at least a part of the components is not completely separated, and the plurality of components. By comparing the storage unit that stores the second time-series data of each model fluorescence signal with the first time-series data and the second time-series data, each of the plurality of components is included in the M types. An analytical system is provided that includes a computer to determine which fluorophore is labeled.

Further, according to another example, a sample containing a plurality of components labeled with any of the M types of phosphors was separated by chromatography, and at least a part of the plurality of components was not completely separated. In the state, the step of acquiring the first time-series data of the fluorescence signal detected in the wavelength band of N types (M> N), the first time-series data, and the individual model fluorescence signals of the plurality of components. An analysis method is provided that includes, by comparing with the second time series data, a step of determining which of the M types each of the plurality of components is labeled with a fluorophore. Fluorescent.

According to the present invention, M types of components can be detected even if M> N in a state where the emission fluorescence from the M types of phosphor has spectral overlap and spatiotemporal overlap. Further features related to the present invention will be clarified from the description of the present specification and the accompanying drawings. In addition, problems, configurations, and effects other than those described above will be clarified by the following description of the examples.

It is a figure which shows the DNA sequencing method of Non-Patent Document 1 using the model data. It is a figure which shows the DNA sequencing method of Non-Patent Document 2 using the model data. It is a figure which shows an example of the DNA sequencing method using the model data which concerns on Example 1. FIG. It is a figure which shows another example of the DNA sequencing method using the model data which concerns on Example 1. FIG. It is a figure which shows the processing process and system block diagram which concerns on Example 1. FIG. It is a figure which shows the processing process and system block diagram which concerns on Example 1. FIG. It is a figure which shows the processing process and the system block diagram which concerns on Example 2. FIG. It is a figure which shows the processing process and the system block diagram which concerns on Example 3 (M = 4, N = 3). It is a figure which shows the processing process and the system block diagram which concerns on Example 4 (M = 4, N = 2). It is a block diagram of the capillary electrophoresis apparatus. It is a block diagram of the multicolor detection device of a multi-capillary electrophoresis device. It is a block diagram of a computer. Example 5 in which the DNA sequence was performed using the processing steps and system configurations shown in FIGS. 9 to 12 is shown. It is a figure which shows the time-series data of the two-color fluorescence intensity of the model peak of the DNA fragment of a single length labeled with four kinds of phosphors which concerns on Example 5. FIG. It is a figure explaining the process of the DNA sequence which concerns on Example 5. FIG. It is a figure explaining the process of the DNA sequence which concerns on Example 5. FIG. It is the figure which summarized the terminal base type, fitting accuracy, and QV (quality value) in order of the electrophoresis time for each model peak of FIG. 15 (5). It is the figure which summarized the terminal base type, fitting accuracy, and QV in the order of the corrected electrophoresis time for each model peak of FIG. 15 (6).

Hereinafter, examples of the present invention will be described with reference to the accompanying drawings. The accompanying drawings show specific examples in accordance with the principles of the present invention, but these are for the purpose of understanding the present invention and are never used for a limited interpretation of the present invention. is not.

The following examples relate to an apparatus for analyzing each component by detecting a sample containing a plurality of components labeled with a plurality of phosphors while distinguishing each fluorescence. The following examples are applicable, for example, in the fields of chromatography, DNA sequencer, DNA fragment analysis, flow cytometer, PCR, HPLC, Western Northern Southern blot, microscopic observation and the like.

The contents of Non-Patent Documents 1 and 2 will be described in more detail with reference to FIGS. 1 to 4 in the case of performing DNA sequencing by electrophoresis.

FIG. 1 shows the method of Non-Patent Document 1. FIG. 1 (1) shows the four-color fluorescence intensity obtained by detecting the emission fluorescence of the four types of phosphors C, A, G, and T in four colors in the four wavelength bands b, g, y, and r. Time series data of I (b), I (g), I (y), and I (r) are shown. The horizontal axis of the figure is time, and the vertical axis is fluorescence intensity.

FIG. 1 (W) shows the elements w (XY) of the matrix W having 4 rows and 4 columns. For example, the four black bar graphs show w (bC), w (gC), w (yC), and w (rC) in order from the left. Similarly, the horizontal striped bar graph is w (XA) (X is b, g, y, and r), the diagonally striped bar graph is w (XG) (X is b, g, y, and r), and the check bar graph. Indicates w (XT) (where X is b, g, y, and r). Further, w (bY), w (gY), w (yY), and w (rY) are standardized so that w (bY) + w (gY) + w (yY) + w (rY) = 1, respectively. (Y is C, A, G, or T).

Specifically, the matrix W and its inverse matrix W ^-1 are as follows.

By multiplying the inverse matrix W ^-1 of the matrix W by I (b), I (g), I (y), and I (r) at each time in FIG. 1 (1) (ie, by color conversion), As shown in FIG. 1 (2), the concentrations D (C) of the four types of phosphors C, A, G, and T, that is, the four types of DNA fragments whose ends are the base types C, A, G, and T. ), D (A), D (G), and D (T) time series data are acquired.

In FIG. 1 (2), four peaks C, A, G, and T are obtained. The height of the apex of each peak, that is, the concentration (arbitrary unit) is D (C) = 100, D (A) = 80, D (G) = 90, and D (T) = 80. The time (arbitrary unit) of the apex of each peak is 30, 55, 60, and 75. The peak of A and the peak of G have a large spatiotemporal overlap with each other, and even in such a case, the color conversion can correctly obtain the respective densities.

FIG. 1 (3) shows four types of fluorescence obtained by performing mobility correction on the result of FIG. 1 (2) based on the difference in mobility depending on the type of labeled phosphor examined in advance. It is time series data of the concentration D (C), D (A), D (G), and D (T) of a body or a DNA fragment. Specifically, it was found that the mobility of the DNA fragment labeled with the fluorescent substance A is delayed by 10 in the time width (arbitrary unit) with respect to the mobility of the DNA fragment labeled with the other fluorescent substance. Therefore, mobility correction is performed in which the peak detection time (arbitrary unit) of the DNA fragment labeled with the phosphor A is preceded by 10 by 10. That is, the detection time (arbitrary unit) of the peak A in FIG. 1 (2) is corrected from 55 to 45. As a result of this mobility correction, time-series data in which DNA fragments of various lengths different by one base length are arranged at substantially equal intervals are acquired.

FIG. 1 (4) is the result of a base call made based on FIG. 1 (3). The labeled fluorophore species or terminal base species of the DNA fragment to which each peak belongs may be read in chronological order.

FIG. 2 shows the method of Non-Patent Document 2. FIG. 2 (1) shows the three-color fluorescence intensity I (3 color fluorescence intensity I) obtained by detecting the emission fluorescence of the four types of phosphors C, A, G, and T in three colors in the three types of wavelength bands b, g, and r. b), I (g), and I (r) time series data are shown.

FIG. 2 (1) is obtained by removing only the time series data of I (y) from FIG. 1 (1), and the time series data of I (b), I (g), and I (r) are both. It is the same in the figure.

FIG. 2 (W) shows the elements w (XY) of the matrix W in 3 rows and 4 columns. For example, the four black bar graphs show w (bC), w (gC), and w (rC) in order from the left. Similarly, the horizontal striped bar graph is w (XA) (X is b, g, and r), the diagonal striped bar graph is w (XG) (X is b, g, and r), and the check bar graph is w (XT). (X is b, g, and r). Further, w (bY), w (gY), and w (rY) are standardized so that w (bY) + w (gY) + w (rY) = 1 (Y is C, A, respectively). G, or T).

Specifically, the matrix W is as follows.

As a first step, consider the case where only one type of phosphor emits light at a time in FIG. 2 (1) using FIG. 2 (W). In FIG. 2 (1), the two peaks observed on the left side and the right side have a ratio of three-color fluorescence intensity (I (b) I (g) I (r)) ^T , respectively (0.63 0. 31 0.06) Since it is close to the ratio of ^T and (0.13 0.25 0.63) ^T , it can be judged that it is a single peak of C and T, respectively. The heights of the peaks of C and T, that is, the concentrations (arbitrary units) are D (C) = 100 and D (T) = 80, and the time (arbitrary units) of the peaks of each peak is 30. , And 75.

On the other hand, in the peak observed in the center of FIG. 2 (1), the ratio of the three-color fluorescence intensity (I (b) I (g) I (r)) ^T is (w (bY) w (gY) w (rY). )) Not close to any ratio of ^T (Y is C, A, G, or T). Therefore, as a second step, consider the case where only two types of phosphors emit light at a time by using FIG. 2 (W). Here, a solution can be derived in which the peaks of A and G are detected at the same time. Specifically, the height of the peak peak, that is, the concentration (arbitrary unit) is D (A) = 80 and D (G) = 90, and the time (arbitrary unit) of the peak of each peak is 55. , And 55, the difference between the left side and the right side of Eq. (3) becomes small, and the peak observed in the center of FIG. 2 (1) can be explained. From the above results, the concentration D of the four types of phosphors C, A, G, and T in FIG. 2 (2), that is, the four types of DNA fragments whose ends are the base types C, A, G, and T ( Time-series data of C), D (A), D (G), and D (T) are acquired.

2 (3) shows the concentrations D (C), D (A), D (G), of the four types of phosphors or DNA fragments obtained by performing mobility correction, as in FIG. 1 (3). And D (T) time series data. The peak detection time (arbitrary unit) of A is advanced by 10 and corrected from 55 to 45. Further, in order to make the peak intervals uniform, the peak detection time (arbitrary unit) of G is set back by 5 and corrected from 55 to 60. As a result, FIG. 2 (3) and FIG. 1 (3) are the same. FIG. 2 (4) is the result of the base call made in the same manner as in FIG. 1, and is the same as in FIG. 1 (4).

On the other hand, FIG. 2 shows that the steps from FIG. 2 (1) to FIG. 2 (2) cannot be determined in a single step. The first step is the same as above, but in the second step another solution is derived and FIG. 2 (2)'is obtained instead of FIG. 2 (2). That is, a solution can be derived in which the peaks of A and T are detected at the same time. Specifically, the height of the peak peak, that is, the concentration (arbitrary unit) is D (A) = 107, and D (G) = 34, and the time (arbitrary unit) of each peak peak is 55, and When it is set to 55, the difference between the left side and the right side of the equation (3) becomes small, and the peak observed in the center of FIG. 2 (1) can be explained. From the above results, the concentrations D of the four types of phosphors C, A, G, and T in FIG. 2 (2)', that is, the four types of DNA fragments whose ends are the base types C, A, G, and T. (C), D (A), D (G), and D (T) time series data are acquired.

However, in FIG. 2 (2)', G is not detected and D (G) = 0. 2 (3)'shows the concentrations D (C), D (A), D (G) of the four types of phosphors or DNA fragments obtained by performing mobility correction, as in FIG. 1 (3). , And D (T) time series data. The peak detection time (arbitrary unit) of A is preceded by 10 and corrected from 55 to 45. FIG. 2 (4)'is the result of a base call made based on FIG. 2 (3)'. Despite using the same FIGS. 2 (1) and 2 (W), FIGS. 2 (2), 2 (3), and 2 (4), 2 (2)', 2 (3)'and FIG. 2 (4)' are completely different, leading to different base call results. In the model data used here, since the base call result shown in FIG. 1 (4) is correct, FIG. 2 (4) is a correct base call result, but FIG. 2 (4)'is incorrect. This is the result of the base call.

Therefore, in the method of Non-Patent Document 2, a plurality of solutions may be derived from the same measurement result, and there is a risk of producing an incorrect base call result, generally an incorrect analysis result.

[Example 1]
FIG. 3 is a diagram showing a DNA sequencing method using the model data according to the first embodiment. FIG. 3 (1) shows the three-color fluorescence intensity I (3 color fluorescence intensity I) obtained by detecting the emission fluorescence of the four types of phosphors C, A, G, and T in three colors in the three types of wavelength bands b, g, and r. b), I (g), and I (r) time-series data, which is the same as FIG. 2 (1).

FIG. 3 (5) shows the three-color fluorescence intensities I (b), I (g), and model peaks of the model peaks when a single-length DNA fragment labeled with phosphor C was detected in three colors. It is time series data of I (r). The vertical axis of FIG. 3 (5) is the fluorescence intensity, and the horizontal axis is time. The data in FIG. 3 (5) show the fluorescence intensity ratio between the model peaks of three-color fluorescence (b, g, and r) when a single length DNA fragment labeled with phosphor C was detected. It is the data which shows the time change of. Here, the three-color fluorescence intensity ratio at each time is (w (bC) w (gC) w (rC)) ^T = (0.63 0.31 0.06) ^T in the matrix W of the equation (6). It has become. Similarly, FIGS. 3 (6), (7), and (8) are models when single length DNA fragments labeled with the fluorophores A, G, and T were detected in three colors. It is the time series data of the three-color fluorescence intensity I (b), I (g), and I (r) of the peak. The three-color fluorescence intensity ratios in FIGS. 3 (6), (7), and (8) are (0.33 0.56 0.11) ^T and (0.27 0.40 0.33) ^T , respectively. (0.13 0.25 0.63) It is ^T.

The shape of each model peak is here Gaussian, and its variance is consistent with that of the single length DNA fragment observed in the experiment. The shape of each model peak is not limited to this example, and other configurations may be used. Here, using FIGS. 3 (5), (6), (7), and (8), only the height and time of the apex of the model peak are changed, and the fitting to the time series data of FIG. 3 (1) is performed. Processing is done. An example of the fitting process will be described with reference to FIG. For example, the fitting process is executed from the left end of the data in FIG. 3 (1). For example, when the height (fluorescence intensity) and the median value of the Gaussian distribution (electrophoresis time) in FIG. 3 (5) are varied and the error from the data in FIG. 3 (1) becomes smaller than a predetermined error. ， It is judged that it fits. That is, here, in the data of FIG. 3 (1), the place where the fluorescence intensity and the electrophoresis time most match is searched for. The fitting may be performed while changing the width of the Gaussian distribution. If it does not fit in FIG. 3 (5), it is determined whether it fits with other data (FIGS. 3 (6), (7), and (8)) or in combination with other data. In this way, fitting to the data of FIG. 3 (1) is performed by any one of FIGS. 3 (5), (6), (7), and (8), or a combination thereof. The error in the fitting process may be calculated, for example, by the difference between the shape of the peak in FIG. 3 (1) and the shape of the model peak. Various known methods may be applied to calculate the error. From the viewpoint of efficiency, it is preferable that the fitting is performed in order from the end of the data in FIG. 3 (1), for example. This is because the tail of the adjacent peak leaks from the peak of a certain fluorescence, so it is more efficient to perform the fitting from the edge of the data because the fitting can be performed in consideration of the leakage of the adjacent peak. This is because.

FIG. 3 (2) shows the result of performing the fitting process. At this time, the peak shape of C is the time-series data obtained by multiplying the height of the time-series data of I (b) by 1 / w (bC) = 1 / 0.63 = 1.59. The peak shape of A is time-series data obtained by multiplying the height of the time-series data of I (g) by 1 / w (gA) = 1 / 0.56 = 1.79. The peak shape of G is time-series data obtained by multiplying the height of the time-series data of I (g) by 1 / w (gG) = 1 / 0.40 = 2.50. The peak shape of T is time-series data obtained by multiplying the height of the time-series data of I (r) by 1 / w (rT) = 1 / 0.63 = 1.59.

As a result, FIG. 3 (2) became the same as FIG. 1 (2). Subsequent methods for acquiring FIGS. 3 (3) and (4) are the same as the methods for acquiring FIGS. 1 (3) and (4). According to this embodiment, since the process of deriving FIG. 3 (1) to FIG. 3 (2) is uniquely determined, a correct base call result can be obtained as shown in FIG. 3 (4).

In the method of Non-Patent Document 2 of FIG. 2, when deriving FIG. 2 (2) or FIG. 2 (2)'from FIG. 2 (1), the matrix W shown in the equation (6), that is, the emission of each phosphor. Only the three-color fluorescence detection intensity ratio of fluorescence is used. On the other hand, in this embodiment shown in FIG. 3, when deriving FIG. 3 (1) to FIG. 3 (2), the matrix W shown in the equation (6), that is, the three-color fluorescence of the emission fluorescence of each phosphor. In addition to the detection intensity ratio, the peak shape of the emission fluorescence of each phosphor, that is, the temporal change information is used. These differences make a difference in whether the solution can be uniquely derived, that is, whether the correct base call result can be obtained.

FIG. 4 shows an example in which the three-color detection carried out in FIG. 3 is further narrowed down to the two-color detection. FIG. 4 (1) shows the two-color fluorescence intensity I (b) obtained by detecting the emission fluorescence of the four types of phosphors C, A, G, and T in two colors in the two types of wavelength bands b and r. It is the time-series data of I (r), and the time-series data of I (g) is removed from FIG. 3 (1).

FIG. 4 (5) shows the two-color fluorescence intensities I (b) and I (r) of the model peak when a single-length DNA fragment labeled with phosphor C is detected in two colors. It is a series data, and the time series data of I (g) is removed from FIG. 3 (5). Similarly, FIGS. 4 (6), (7), and (8) are models when single length DNA fragments labeled with fluorophores A, G, and T are detected in two colors. It is the time-series data of the two-color fluorescence intensities I (b) and I (r) of the peak, and the time-series data of I (g) is removed from FIGS. 3 (6), (7), and (8), respectively. It is a thing.

Here, using FIGS. 4 (5), (6), (7), and (8), only the height and time of the apex of the model peak are changed, and the fitting to the time series data of FIG. 4 (1) is performed. Processing is done. FIG. 4 (2) shows the result of performing the fitting process. At this time, the peak shape of C is the time-series data obtained by multiplying the height of the time-series data of I (b) by 1 / w (bC) = 1 / 0.63 = 1.59. The peak shape of A is time-series data obtained by multiplying the height of the time-series data of I (b) by 1 / w (bA) = 1 / 0.33 = 3.03. The peak shape of G is time-series data obtained by multiplying the height of the time-series data of I (r) by 1 / w (rG) = 1 / 0.33 = 3.03. The peak of T is the time series data obtained by multiplying the height of the time series data of I (r) by 1 / w (rT) = 1 / 0.63 = 1.59.

As a result, FIG. 4 (2) became the same as FIG. 1 (2). Subsequent methods for acquiring FIGS. 4 (3) and (4) are the same as the methods for acquiring FIGS. 1 (3) and (4). According to this embodiment, since the process of deriving FIG. 4 (1) to FIG. 4 (2) is uniquely determined, a correct base call result can be obtained as shown in FIG. 4 (4).

FIG. 5 shows the processing process and the system configuration of this embodiment. The system according to the first embodiment includes an analyzer 510, a computer 520, and a display device 530. The analyzer 510 is, for example, a liquid chromatography device. The computer 520 may be realized by using, for example, a general-purpose computer. The processing unit of the computer 520 may be realized as a function of a program executed on a computer. A computer includes at least a processor such as a CPU (Central Processing Unit) and a storage unit such as a memory. The processing of the computer 520 may be realized by storing the program code corresponding to each processing in the memory and executing each program code by the processor.

In this configuration, the analyzer 510 chromatographically separates a sample containing a plurality of components labeled with any of the M types of phosphors, and at least a part of the plurality of components is not completely separated. In the state, the first time series data of the fluorescence signal detected in the wavelength band of N types (M> N) is acquired. The first time-series data of the fluorescence signal corresponds to the N-color detection time-series data 513 of the M color-labeled sample described below. The computer 520 includes a storage unit (for example, a memory, an HDD, etc.), and the storage unit stores in advance the second time-series data of the individual model fluorescence signals of the plurality of components. The second time-series data of the individual model fluorescence signals of the plurality of components corresponds to the N-color detection time-series data 541 of a single peak of each M color label described below. By comparing the first time-series data with the second time-series data, the computer 520 determines which of the M types of phosphors each of the plurality of components is labeled with. do. The display device 530 displays the third time-series data of the concentrations of the M types of phosphors that contribute to the fluorescence signal. The third time-series data of the concentration of the phosphor corresponds to the M color-labeled time-series data 523 described below. Hereinafter, the above processing will be described more specifically.

First, the M color-labeled sample 501 containing a plurality of components labeled with M-type phosphors is charged into the analyzer 510. Next, in the analyzer 510, the separation analysis process 511 of the plurality of components contained in the sample 501 is performed. The analyzer 510 detects the emission fluorescence of M types of phosphors in N types (M> N) wavelength bands (N color detection), and N color detection time series data (fluorescence detection time series data) of the M color labeled sample. ) Get 513. Here, not all of the plurality of components are well separated. That is, fluorescence is detected in a state where some of the different components labeled with different phosphors overlap in space and time. The analyzer 510 outputs the N color detection time series data 513 to the computer 520.

Next, the computer 520 uses the N color detection time series data 513 and the N color detection time series data of a single component labeled with any of the M types of phosphors, that is, a single peak of each M color label. N color detection time series data 541 and the above are acquired as input information. The N color detection time series data 541 of a single peak of each M color label is, for example, data corresponding to FIGS. 3 (5), (6), (7), and (8). The N color detection time series data 541 for a single peak of each M color indicator is stored in advance in the first database 540.

Next, the computer 520 executes a comparative analysis process 521 of the N color detection time series data 513 and the N color detection time series data 541 of a single peak of each M color label. As a result, the computer 520 acquires the M color-labeled time-series data 523, which is the time-series data of the detected concentrations of the M-type phosphors, that is, the concentrations of the components labeled with the M-type phosphors. The M color indicator time series data 523 is, for example, data corresponding to FIG. 3 (2). Finally, the display device 530 performs the display process 531 of the M color indicator time series data 523.

FIG. 6 is a diagram embodying the comparative analysis in the computer 520 of FIG. As a comparative analysis, the computer 520 performs a fitting analysis process 522 on the N color detection time series data 513 using the N color detection time series data 541 of a single peak of each M color label. As a result, the computer 520 acquires the fitting error data (or fitting accuracy data) 524, which is the difference between the N color detection time series data 541 and the fitting result, together with the M color indicator time series data 523. The display device 530 performs display processing 531 of either or both of the M color indicator time series data 523 and the fitting error data 524.

[Example 2]
FIG. 7 shows a processing process and a system configuration when the present invention is applied to electrophoretic analysis of DNA fragments. The plurality of components to be analyzed may be nucleic acid fragments of different lengths or different compositions, and the chromatography may be electrophoresis.

The analyzer 510 is an electrophoresis device. First, an M color-labeled DNA sample 502 containing a plurality of types of DNA fragments labeled with M types of phosphors is charged into the analyzer 510. Next, in the analyzer 510, the electrophoretic separation analysis process 512 of a plurality of types of DNA fragments contained in the DNA sample is performed. The analyzer 510 detects the emission fluorescence of M types of phosphors in the wavelength band of N types (M> N) (N color detection), and acquires N color detection time series data 513. Here, not all of the multiple types of DNA fragments are well separated. That is, a part of DNA fragments of different types labeled with different fluorescent substances is detected by fluorescence in a state of spatiotemporal overlap. The analyzer 510 outputs the N color detection time series data 513 to the computer 520.

Next, the computer 520 is a single M color label, which is N color detection time series data 513 and N color detection time series data of a single type of DNA fragment labeled with any of M types of phosphors. The N color detection time series data 541 of one peak is acquired as input information. Next, the computer 520 performs a comparative analysis between the N color detection time series data 513 and the N color detection time series data 541 of a single peak of each M color label. Specifically, the computer 520 performs fitting analysis processing 522 on the N color detection time series data 513 using the N color detection time series data 541 of a single peak of each M color label. As a result, the computer 520 acquires the M color-labeled time-series data 523, which is the time-series data of the detected concentrations of the M-type phosphors, that is, the concentrations of the DNA fragments labeled with the M-type phosphors. At the same time, the computer 520 acquires the fitting error data (or fitting accuracy data) 524.

Here, the mobility of the DNA fragment by electrophoresis is affected by the labeled M types of phosphors. Therefore, in order to reduce the influence thereof, the computer 520 performs a process using the mobility difference data 551 of each M color label showing the difference in mobility between the labeled M types of phosphors. The mobility difference data 551 of each M color sign is stored in the second database 550 in advance. The computer 520 executes the mobility correction process 525 on the M color indicator time series data 523 using the mobility difference data 551 of each M color indicator, and the mobility correction data of the M color indicator time series data. (Hereinafter referred to as correction data) 526 is acquired. The correction data 526 is, for example, data corresponding to FIG. 3 (3).

Finally, the display device 530 performs a part or all display processing 531 of the M color indicator time series data 523, the fitting error data (or fitting accuracy data) 524, and the correction data 526.

[Example 3]
FIG. 8 shows a processing process and a system configuration when the present invention is applied to a DNA sequence by electrophoresis. In FIG. 8, N = 3 and M = 4. The analyzer 510 is a DNA sequencer.

First, a four-color labeled DNA sequence sample 503 containing four types of DNA fragments labeled with four types of phosphors according to four types of terminal base species prepared by the Sanger method using the target DNA as a template is prepared. .. Then, the four-color labeled DNA sequence sample 503 is put into the analyzer 510. Next, in the analyzer 510, the electrophoretic separation analysis process 512 of the four types of DNA fragments contained in the DNA sequence sample is performed. The analyzer 510 detects the emission fluorescence of four types of phosphors in three types of wavelength bands (three-color detection), and acquires three-color detection time-series data 513. Here, not all of the four types of DNA fragments are well separated. That is, a part of DNA fragments of different lengths labeled with different phosphors are detected by fluorescence in a state of spatiotemporal overlap. The analyzer 510 outputs the three-color detection time-series data 513 to the computer 520.

Next, the computer 520 has three-color detection time-series data 513 and three-color detection time-series data of a single-length DNA fragment labeled with any of the four types of phosphors, each of which is a four-color label. The three-color detection time-series data 541 of a single peak is acquired as input information. The three-color detection time-series data 541 of a single peak of each four-color indicator is stored in advance in the first database 540. The computer 520 performs comparative analysis between the three-color detection time-series data 513 and the three-color detection time-series data 541 with a single peak of each of the four-color labels. Specifically, the computer 520 uses the three-color detection time-series data 541 for a single peak of each four-color label to perform fitting analysis processing 522 for the three-color detection time-series data 513 of the four-color labeled DNA sequence sample. I do. As a result, the computer 520 is a time-series data of the concentrations of the detected four types of phosphors, that is, the concentrations of the four types of DNA fragments labeled with the four types of phosphors and having different terminal base types, four colors. The indicator time series data 523 is acquired. At the same time, the computer 520 acquires the fitting error data (or fitting accuracy data) 524.

Here, the mobility of the DNA fragment by electrophoresis is affected by the four types of fluorescent substances labeled. Therefore, in order to reduce the influence, processing is performed using the mobility difference data 551 of each of the four color labels, which shows the difference in mobility between the four types of labeled phosphors. The mobility difference data 551 of each of the four color signs is stored in advance in the second database 550. The computer 520 executes the mobility correction process 525 on the four-color labeling time-series data 523 using the mobility difference data 551 of each four-color labeling, and corrects the four-color labeling time-series data (hereinafter, correction). (Called data) 526 is acquired.

Further, the computer 520 performs the DNA base sequence determination process 528 using the correction data 526. On the other hand, the computer 520 acquires the base sequence determination error data (or base sequence determination accuracy data) 527 for each base whose DNA base sequence has been determined by using the fitting error data (or fitting accuracy data) 524.

Finally, the display device 530 displays the four-color labeling time series data 523, the fitting error data (or fitting accuracy data) 524, the correction data 526, the DNA base sequence determination result, and the base sequence determination error data (or the base sequence determination accuracy data). ) Perform some or all of the display processing 531 of 527.

[Example 4]
FIG. 9 shows a processing process and a system configuration when the configuration of FIG. 8 is replaced by the conditions of N = 2 and M = 4. Since the processing process and the system configuration are the same as those in FIG. 8, the description thereof will be omitted.

FIG. 10 is a block diagram of a capillary electrophoresis apparatus which is an example of the analyzer 510. The capillary electrophoresis apparatus 100 is used as a DNA sequencer, a DNA fragment analysis apparatus, and the like. The sample injection end 2 and the sample elution end 3 of the capillary 1 filled with the electrophoresis separation medium containing an electrolyte are immersed in the cathode side electrolyte solution 4 and the anode side electrolyte solution 5, respectively. Further, the negative electrode 6 is immersed in the cathode side electrolyte solution 4, and the positive electrode 7 is immersed in the anode side electrolyte solution 5. Electrophoresis is performed by applying a high voltage between the negative electrode 6 and the positive electrode 7 by the high voltage power source 8.

The sample injection into the capillary 1 is carried out by immersing the sample injection end 2 and the negative electrode 6 in the sample solution 9 and applying a high voltage between the negative electrode 6 and the positive electrode 7 for a short time by the high voltage power source 8. The sample solution 9 contains a plurality of types of components labeled with a plurality of types of phosphors. After the sample is injected, the sample injection end 2 and the negative electrode 6 are immersed in the cathode side electrolyte solution 4 again, and a high voltage is applied between the negative electrode 6 and the positive electrode 7 to perform electrophoresis.

The negatively charged component contained in the sample, for example, a DNA fragment, is electrophoresed in the capillary 1 from the sample injection end 2 toward the sample elution end 3 in the electrophoresis direction 10 indicated by the arrow. Due to the difference in mobility due to electrophoresis, a plurality of types of components contained in the sample solution 9 are gradually separated. The laser beam 12 emitted from the laser light source 11 is irradiated to a position (laser beam irradiation position 15) electrophoresed by a certain distance on the capillary 1. It promotes the emission of fluorescence 13 by a plurality of types of phosphors labeled with components that sequentially pass through the laser beam irradiation position 15. The fluorescence 13 that changes with time with electrophoresis is measured by a multicolor detection device 14 that detects light in a plurality of wavelength bands. Although only one capillary 1 is drawn in FIG. 10, a multi-capillary electrophoresis apparatus that performs electrophoresis analysis in parallel using a plurality of capillaries 1 may be used.

FIG. 11 shows an example of a multicolor detection device of a multicapillary electrophoresis device. The laser beam irradiation positions 15 of the plurality of capillaries 1 are arranged on the same plane at equal intervals. The left view of FIG. 11 is a cross-sectional view perpendicular to the long axis of a plurality of capillaries 1, and the right view of FIG. 11 is a cross-sectional view parallel to the long axis of any capillary 1.

The laser beam 12 is irradiated along the arrangement plane of the plurality of capillaries 1. As a result, the laser beam 12 simultaneously irradiates a plurality of capillaries 1. The fluorescence 13 emitted from each capillary 1 is focused in parallel by the individual lenses 16. Each of the collected light fluxes is directly incident on the two-dimensional color sensor 17. The two-dimensional color sensor 17 is an RGB color sensor capable of detecting three colors in three kinds of wavelength bands. Since the fluorescence 13 emitted from each capillary 1 forms spots at different positions on the two-dimensional color sensor 17, each can be independently detected in three colors.

FIG. 12 shows a configuration example of the computer 520. As shown in FIGS. 5 to 9, the computer 520 is connected to the analyzer 510. The computer 520 may control not only the data analysis described with reference to FIGS. 5 to 9 but also the analyzer 510. In FIGS. 5 to 9, the display device 530 and the databases 540 and 550 are drawn outside the computer 520, but these may be included in the computer 520.

The computer 520 includes a CPU (processor) 1201, a memory 1202, a display unit 1203, an HDD 1204, an input unit 1205, and a network interface (NIF) 1206. The display unit 1203 is, for example, a display, and may be used as a display device 530. The input unit 1205 is an input device such as a keyboard or a mouse. The user can set the conditions for data analysis and the control conditions for the analyzer 510 via the input unit 1205. The N color detection time series data 513 output from the analyzer 510 is sequentially stored in the memory 1202.

HDD 1204 may include databases 540,550. Further, the HDD 1204 may include a program for performing fitting analysis processing, mobility correction processing, DNA base sequence determination processing, and the like of the computer 520. The processing of the computer 520 may be realized by storing the program code corresponding to each processing in the memory 1202 and executing each program code by the CPU 1201.

For example, the N color detection time series data 541 of a single peak of each M color indicator stored in the HDD 1204 is stored in the memory 1202, and the CPU 1201 has the N color detection time series data 513 and a single peak of each M color indicator. The comparative analysis process is executed using the N color detection time series data 541 of. The display unit 1203 displays the analysis result. The analysis result may be collated with the information on the network via NIF1206.

[Example 5]
13 to 18 show examples in which DNA sequencing was performed using the processing steps and system configurations shown in FIGS. 9 to 12.

As a 4-color labeled DNA sequence sample, 3500/3500xL Sequencing Standards, BigDye Terminator v3.1 (Thermo Fisher Scientific) dissolved in 300 μL formamide was used. This sample was labeled with the four phosphors dROX (maximum emission wavelength 618 nm), dR6G (568 nm), dR110 (541 nm), and dTAMRA (595 nm), respectively. It contains four types of DNA fragments,, G, and T.

There are four capillaries 1, each of which has an outer diameter of 360 μm, an inner diameter of 50 μm, a total length of 56 cm, and an effective length of 36 cm. As the electrophoresis separation medium, POP-7 (Thermo Fisher Scientific), which is a polymer solution, was used. During electrophoresis, the capillary 1 was adjusted to 60 ° C. and the electric field strength was 182 V / cm. The sample was injected by electric field injection at an electric field strength of 27 V / cm for 8 seconds. The laser beam 12 has a wavelength of 505 nm and an output of 20 mW. A long-pass filter for blocking the laser beam 12 was used between the lens 16 and the two-dimensional color sensor.

FIG. 13 (1) corresponds to FIG. 4 (1), and was obtained by detecting the fluorescence 13 emitted from one of the four capillarys 1 with an RGB color sensor during electrophoresis. Two-color detection time-series data. The RGB color sensor used in this embodiment can detect three colors in three types of wavelength bands r, g, and b corresponding to R (red), G (green), and B (blue). , The emission fluorescence of the above four types of phosphors was hardly detected in the wavelength band of b, and was detected only in the wavelength bands of r and g. Therefore, FIG. 13 (1) shows the time series of the fluorescence intensities I (r) and I (g) of these two colors. The horizontal axis shows the time elapsed from the start of electrophoresis (electrophoresis time) in seconds, and the vertical axis shows the fluorescence intensity in arbitrary units.

FIG. 14 (1) corresponds to FIG. 4 (5), and is a model peak when two colors of DNA fragments of a single length terminal base species C labeled with the fluorescent substance dROX are detected. It is time series data of two-color fluorescence intensity I (g) and I (r). Here, the two-color fluorescence intensity ratio at each time is (w (gC) w (rC)) ^T = (0.02 0.98) ^T. Similarly, FIG. 14 (2) corresponds to FIG. 4 (6), when two colors of DNA fragments of a single length terminal base species A labeled with the fluorescent substance dR6G were detected. It is the time series data of the two-color fluorescence intensity I (g) and I (r) of the model peak. The two-color fluorescence intensity ratio is (w (gA) w (rA)) ^T = (0.50 0.50) ^T. FIG. 14 (3) corresponds to FIG. 4 (7), and is a model peak when two colors of DNA fragments of a single length terminal base species G labeled with the fluorescent substance dR110 are detected. It is time series data of two-color fluorescence intensity I (g) and I (r). The two-color fluorescence intensity ratio is (w (gG) w (rG)) ^T = (0.71 0.29) ^T. FIG. 14 (4) corresponds to FIG. 4 (8), which is a model peak when two colors of DNA fragments of a single length terminal base species T labeled with the fluorescent substance dTAMRA are detected. It is time series data of two-color fluorescence intensity I (g) and I (r). The two-color fluorescence intensity ratio is (w (gT) w (rT)) ^T = (0.16 0.84) ^T. The shape of each model peak was Gaussian, the offset was zero, and the standard deviation was 1 second. This is the result of matching the shape of the peak of a single length DNA fragment measured under this electrophoresis condition.

The two-color detection time-series data of the four types of model peaks shown in FIGS. 14 (1) to 14 (4) were sequentially fitted to the two-color detection time-series data obtained by the electrophoretic analysis of FIG. 13 (1). .. In the fitting, only the median (electrometry time) and height (fluorescence intensity) of the Gaussian distribution are changed while maintaining the two-color fluorescence intensity ratio and standard deviation of the Gaussian distribution, and the error from the two-color detection time series data. The median and height were determined to minimize.

FIG. 13 (2) shows the result of accurately fitting the model peak of G (dR110) of FIG. 14 (3) to the peak of g observed on the leftmost side of FIG. 13 (1). FIG. 13 (3) is a result of accurately fitting the model peak of T (dTAMRA) of FIG. 14 (4) to the peak of r to the right of the peak of g in FIG. 13 (1). .. FIG. 13 (4) is a result of accurately fitting the model peak of C (dROX) of FIG. 14 (4) to the peak of r to the right of the peak of r in FIG. 13 (1). .. 13 (5) shows the superimposed two-color detection time series data of FIGS. 13 (2), (3) and (4), that is, I (g) of FIGS. 13 (2), (3) and (4). It is a time series data obtained by adding I (r) and I (r). FIG. 13 (5) faithfully reproduces the corresponding portion of FIG. 13 (1).

The fitting error and accuracy were evaluated as follows. The fitting error is the difference between the fitted model peak and the measured two-color detection time series data in a 2-second width (twice the standard deviation of the Gaussian distribution) section centered on the time of the peak of the fitted model peak. The standard deviation of was divided by the larger value of the measured two-color detection fluorescence intensity at the time of the peak of the model peak. The fitting accuracy is 1 minus the fitting error. The fitting accuracy becomes 100% when the fitting and the actual measurement completely match, and decreases as the deviation increases, and becomes 0% when the deviation is equal to or more than the larger value of the measured two-color detection fluorescence intensity. I did it. In this embodiment, the fitting error and accuracy are defined as described above, but other definitions may be used.

The single fitting accuracy of the T model peak in FIG. 13 (3) was only 43.41%, but the T model when the G, T, and C model peaks in FIG. 13 (5) were superposed. The peak fitting accuracy reached 95.56%. This means that in the presence of spatiotemporal overlap, it is more important to fit with model peaks before and after coexistence than with a single model peak alone.

Similarly, the two-color detection time-series data of the four types of model peaks shown in FIGS. 14 (1) to 14 (4) were sequentially fitted to all the peaks of g and r in FIG. 13 (1). FIG. 13 (6) is a two-color detection time-series data in which the above-mentioned fitted individual model peaks are superimposed. FIG. 13 (6) can faithfully reproduce the whole of FIG. 13 (1).

FIG. 15 (1) is the sum of I (g) and I (r) of FIG. 13 (2), and shows the time-series data of the concentration of the fluorescent substance dR110, that is, the concentration of the DNA fragment of the terminal base species G. It is shown. FIG. 15 (2) is the sum of I (g) and I (r) in FIG. 13 (3), and shows the time-series data of the concentration of the fluorescent substance dTAMRA, that is, the concentration of the DNA fragment of the terminal base species T. It is shown. FIG. 15 (3) shows the time-series data of the concentration of the fluorescent substance dROX, that is, the concentration of the DNA fragment of the terminal base species C by adding up I (g) and I (r) of FIG. 13 (4). Is. Similarly, FIG. 15 (5) shows time-series data on the concentrations of DNA fragments of terminal base species C, A, G, and T for the individual model peaks fitted in FIG. 13 (6). ..

FIG. 17 summarizes the terminal base species, fitting accuracy, and QV (quality value) for each model peak in FIG. 15 (5) in order of electrophoresis time. Here, QV is obtained by QV = −10 * Log (1-S), where S is the fitting accuracy. During the 200 seconds of the electrophoresis time of 1100 seconds to 1300 seconds, 76 DNA fragments having different lengths of one base were measured, and each terminal base species could be identified. The fitting accuracy is 94.72% on average and the QV is 13.95 on average, and high-precision fitting can be realized. The computer 520 may calculate the data of FIG. 17, and the display device 530 may display the data of FIG.

FIG. 15 (6) shows terminal base types C, A, obtained by performing mobility correction based on the difference in mobility due to the difference in the above four types of phosphors with respect to FIG. 15 (5). It is the correction data of the time series data of the concentration of the DNA fragment of G and T. Specifically, the median electrophoresis time of the model peak of the terminal base species G in FIG. 15 (5) is set back by 1.6 seconds, and the median value of the model peak of the terminal base species T in FIG. 15 (5) is set back. The electrophoresis time was advanced by 1.1 seconds, and the model peaks of terminal base species C and A were not corrected. As a result of the above, the peaks of DNA fragments having different lengths by one base were arranged at almost equal intervals in ascending order. Characteristically, in FIG. 15 (5), the DNA fragments of the terminal base species G and the terminal base species T are reversed (that is, the long DNA fragment is the short DNA fragment) due to the influence of the mobility difference due to the difference in the phosphor. It is measured (overtaking), but in FIG. 15 (6), it is brilliantly corrected.

FIG. 18 summarizes the terminal base species, fitting accuracy, and QV in order of corrected electrophoresis time for each model peak in FIG. 15 (6). FIG. 18 is a rearrangement of FIG. 17, and the numerical values used are the same. The sequence of terminal base species in FIG. 18 gives a DNA sequence result (base call result). The fitting accuracy and QV of each base are indicators that correlate with the accuracy of base sequence determination, but they are not the same. In general, it is expected that the accuracy of base sequence determination will be higher than the accuracy of fitting. In fact, this DNA sequencing result was 100% correct. The computer 520 may calculate the data of FIG. 18, and the display device 530 may display the data of FIG.

FIG. 16 summarizes the steps of the DNA sequence of this example. FIG. 16 (1) is the same as FIG. 15 (1), is two-color detection time-series data obtained by electrophoresis analysis, and corresponds to FIG. 4 (1). FIG. 16 (2) is the same as FIG. 15 (5), and is time-series data of the concentrations of DNA fragments of the terminal base species C, A, G, and T, and corresponds to FIG. 4 (2). FIG. 16 (3) is the same as FIG. 15 (6), and is the correction data obtained by mobility-correcting the time-series data of the concentrations of the DNA fragments of the terminal base species C, A, G, and T. , Corresponds to FIG. 4 (3). Finally, FIG. 16 (4) is the result of making a base call based on the result of FIG. 16 (3), and corresponds to FIG. 4 (4). The base call result in FIG. 16 (4) is consistent with the base sequence of the target DNA.

The means and effects of the above Examples 1 to 5 are summarized. According to the above embodiment, the emission fluorescence from the M types of phosphors has spectral overlap and spatiotemporal overlap, and N color detection in the wavelength band of N types (M> N) is performed. An analytical method for identifying and detecting M types of components can be provided. Hereinafter, following Non-Patent Document 2, a means for detecting emission fluorescence of M = 4 types of phosphors in N = 3 colors will be described for a DNA sequencer using electrophoresis.

The analyzer 510 detects the emission fluorescence of the four types of phosphors C, A, G, and T in three colors in the three types of wavelength bands b, g, and r. It is the same as in Non-Patent Document 2 up to the point where the three-color fluorescence intensity of the formula (3) is obtained for each time. Here, when the HDD (storage unit) 1204 of the computer 520 detects three colors of a single length DNA fragment labeled with any of the four types of phosphors C, A, G, and T. 4 types of time series data, that is, 4 types of model peak data are stored. The three-color fluorescence intensity ratio of the model peak data of the DNA fragment labeled with the phosphor Y (C, A, G, or T) is (w (bY) w (gY) w (rY)) ^T. .. That is, the model peak data contains the same information as the matrix W. In addition, the model peak data contains the peak shape, that is, the time change information.

In Non-Patent Document 2, one or two types of phosphors emitting fluorescent light were selected using the matrix W for each time, and their concentrations were determined. On the other hand, in the above-described embodiment, the computer 520 executes a fitting analysis process using four types of model peak data for the time-series data of the three-color fluorescence intensity represented by the equation (3). Even when the emission fluorescence from the four types of phosphors has spectral overlap and spatiotemporal overlap, the computer 520 can execute the fitting analysis process. For example, three or more types of phosphors may emit fluorescence at one time. The set of model peak data of C, A, G, and T constituting the fitting result is the concentration D (C), D (A), D (G), and D (T) of C, A, G, and T. ) Represents the time series data. That is, even though the color conversion is not performed, the time-series data of the three-color fluorescence intensity is used to obtain the four types of phosphor concentrations corresponding to the formula (2) in Non-Patent Document 1, that is, the four base type concentrations. Time series data can be acquired. Unlike Non-Patent Document 2, the above-mentioned example is characterized in that it utilizes information on the time-varying changes in the concentrations of four types of phosphors. After that, the computer 520 performs the same steps as the steps (3) and (4) in Non-Patent Document 1, and can obtain the result of the base call.

According to the above-mentioned embodiment, it is obtained by N color detection in the wavelength band of N kinds (M> N) in a state where the emission fluorescence from the M kinds of phosphors has spectral overlap and spatiotemporal overlap. By fitting the time-series data of the N-color fluorescence intensity to be obtained using the model peak data of M-type phosphors, the time-series data of the concentrations of M-type phosphors, that is, the M-type components are analyzed. It is possible to do.

In order to perform analysis to identify and detect M types of components by N color detection in N types of wavelength bands in a state where the emission fluorescence from M types of phosphors has spectral overlap and spatiotemporal overlap. Conventionally, it was necessary to set M ≦ N. According to the above-mentioned embodiment, even if M> N, it is possible to analyze by identifying and detecting M kinds of components in the same manner. In other words, it has the effect that the same analysis can be realized by a simpler, smaller, and lower cost device configuration. For example, by N = 3 color detection using an RGB color sensor whose performance and cost are rapidly increasing, M = 4 or more types of components labeled with M = 4 or more types of phosphors are identified. The analysis to be detected becomes possible. As a result of the above, analysis by multicolor detection with high accuracy and low cost becomes possible. For example, M = by detecting N = 3 colors using a low-cost RGB color sensor while electrophoretically separating M = 4 types of DNA fragments labeled with M = 4 types of phosphors by the Sanger reaction. Even if DNA fragments of different lengths labeled with 4 types of phosphors are measured in a mixed state, time-series data of the concentration of M = 4 types of DNA fragments can be obtained, and the DNA sequence can be obtained. Can be performed well.

The present invention is not limited to the above-mentioned examples, and includes various modifications. The above-mentioned embodiment has been described in detail in order to explain the present invention in an easy-to-understand manner, and is not necessarily limited to the one including all the configurations described. It is also possible to replace a part of the configuration of one embodiment with the configuration of another embodiment. It is also possible to add the configuration of another embodiment to the configuration of one embodiment. In addition, other configurations can be added / deleted / replaced with respect to a part of the configurations of each embodiment.

Further, each configuration, function, processing unit, processing means and the like of the above-mentioned computer 520 may be realized by hardware by designing a part or all of them by, for example, an integrated circuit. Further, each of the above configurations, functions, and the like may be realized by software by the processor interpreting and executing a program that realizes each function. Information such as programs, tables, and files that realize each function can be stored in various types of non-transitory computer readable medium. As the non-temporary computer-readable medium, for example, a flexible disk, a CD-ROM, a DVD-ROM, a hard disk, an optical disk, a magneto-optical disk, a CD-R, a magnetic tape, a non-volatile memory card, a ROM, or the like is used.

In the above embodiment, the control lines and information lines are shown as necessary for explanation, and not all control lines and information lines are necessarily shown in the product. All configurations may be interconnected.

c Fluorescent signal detected in wavelength band c g Fluorescent signal detected in wavelength band g y Fluorescent signal detected in wavelength band y Fluorescent signal r Fluorescent signal detected in wavelength band r Fluorescent signal C Fluorescent substance C or base type C
A Fluorescent substance A or base species A
G Fluorescent substance G or base species G
T Fluorescent substance T or base type T
1 Capillary 2 Sample injection end 3 Sample elution end 4 Cathode side electrolyte solution 5 Anode side electrolyte solution 6 Negative electrode 7 Positive electrode 8 High pressure power supply 9 Sample solution 10 Electrophoresis direction 11 Laser light source 12 Laser beam 13 Fluorescence 14 Multicolor detector 15 Laser beam irradiation position 16 Lens 17 Two-dimensional color sensor 510 Analyzer 520 Computer 530 Display device 540, 550 Database

Claims (11)

Samples containing 4 or more types of phosphors and
Capillaries for performing electrophoretic analysis of the sample,
A light source that irradiates the capillary with a laser beam,
An optical system that collects fluorescence emitted from the emission point of the capillary by irradiation with the laser beam, and an optical system.
In a capillary electrophoresis apparatus including a sensor for measuring an image of the emission point generated by the optical system.
A capillary electrophoresis apparatus, wherein the sensor is an RGB color sensor. In the capillary electrophoresis apparatus according to claim 1,
A capillary electrophoresis apparatus characterized in that the DNA sequence of the sample is performed by the electrophoresis analysis. In the capillary electrophoresis apparatus according to claim 1,
A capillary electrophoresis apparatus characterized in that DNA fragment analysis of the sample is performed by the electrophoresis analysis. In the capillary electrophoresis apparatus according to any one of claims 1 to 3,
Time-series data of the signal of the RGB color sensor obtained during the electrophoretic analysis of the sample, and
A capillary electrophoresis apparatus, characterized in that a step of comparing time-series data of signals of the RGB color sensor obtained by electrophoretic analysis of each of the plurality of types of phosphors is executed. In the capillary electrophoresis apparatus according to any one of claims 1 to 3,
The time-series data of the signal of the RGB color sensor obtained at the time of the electrophoretic analysis of the sample is obtained.
A capillary electrophoresis apparatus characterized in that a step of expressing a combination of time-series data of signals of the RGB color sensor obtained by electrophoretic analysis of each of the plurality of types of phosphors is executed. Multiple types of samples, each containing four or more types of phosphors, and
Multiple capillaries for performing electrophoretic analysis of the multiple types of samples,
A capillary array in which the measured parts of the plurality of capillaries are arranged on the same plane, and
A light source that irradiates the capillary array with a laser beam,
An optical system that collects fluorescence emitted from the emission points of the plurality of capillaries by irradiation with the laser beam, and an optical system.
In a capillary electrophoresis apparatus including an area sensor for measuring an image of fluorescence from the plurality of emission points generated by the optical system.
A capillary electrophoresis apparatus, wherein the area sensor is an RGB color sensor. In the capillary electrophoresis apparatus according to claim 6,
A capillary electrophoresis apparatus characterized in that DNA sequences of the plurality of types of samples are performed by the electrophoresis analysis. In the capillary electrophoresis apparatus according to claim 6,
A capillary electrophoresis apparatus characterized in that DNA fragment analysis of the plurality of types of samples is performed by the electrophoresis analysis. In the capillary electrophoresis apparatus according to any one of claims 6 to 8, in the capillary electrophoresis apparatus.
Time-series data of each signal of the RGB color sensor obtained during the electrophoretic analysis of the plurality of types of samples, and
A capillary electrophoresis apparatus, characterized in that a step of comparing time-series data of signals of the RGB color sensor obtained by electrophoretic analysis of each of the plurality of types of phosphors is executed. In the capillary electrophoresis apparatus according to any one of claims 6 to 8, in the capillary electrophoresis apparatus.
The time-series data of each signal of the RGB color sensor obtained during the electrophoretic analysis of the plurality of types of samples can be obtained.
A capillary electrophoresis apparatus characterized in that a step of expressing a combination of time-series data of signals of the RGB color sensor obtained by electrophoretic analysis of each of the plurality of types of phosphors is executed. In the capillary electrophoresis apparatus according to claim 6,
The optical system includes a plurality of lenses that individually collect the fluorescence emitted from the emission points of the plurality of capillaries.
A capillary electrophoresis apparatus, characterized in that a plurality of individually condensed fluorescence is directly incident on the RGB color sensor.

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