JPH08173197A - Method for determining wave form peak for determining dna base sequence and device for determining dna base sequence - Google Patents

Abstract

PURPOSE: To provide a method for determining the peak of the wave form for the determination of a base sequence, capable of extracting a substantially true wave form, even when the wave form signal of fluorescent strength per time (occurrence frequency) contains connected waves. CONSTITUTION: The left and right sides of a wave form peak are grasped as a Gaussian distribution and a Cauchy distribution, respectively, and the wave form peak is thus grasped as the peal having different wave form characteristics on the left and right sides, respectively. The half-value widths of the left and right sides are computed, respectively, and used as barometers. A base having wave form data little in the fluctuation of the position of the peak is first extracted as a normal wave form from data comprising the computed half-value widths and the strength of the peak of each wave form length. The pitch ΔTn of the wave form is determined on the basis of the data of the wave signals of the normal wave form for each interval. The strength I and position of the peak of a true base signal peak in continuous connected wave form signals not selected as the base of the true base signal are determined from the above-described equation and the above-described value: ΔTn.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a waveform peak determining method and a DNA nucleotide sequencer for determining a DNA nucleotide sequence. In a DNA base sequencer device (hereinafter referred to as a DNA sequencer) that detects fluorescence as a waveform characteristic of time-reception intensity signal by receiving fluorescence with a line sensor for the migration state of each DNA base fragment using a dye marker, Even if the waveform signals overlap due to the appearance, the correct peak value and its position can be obtained, and for example, the DNA base determination rate for a long DNA base having a length of 400 or more base sequences is improved. Method and apparatus that can be performed.

[0002]

2. Description of the Related Art A conventional method for determining a base sequence is D
As NA bases, A (adenine), C (cytosine), G
For (guanine) and T (thymine), each fragment is electrophoresed in the electrophoresis plate, and the reaching state of these fragments is detected by receiving the fluorescence generated by laser light irradiation at a predetermined position of each detection lane. However, the peak position of the waveform signal detected by referring to the table of the base pitch ΔT stored in advance and various determination conditions such as the intensity thereof are combined.

However, in the case of a long DNA base sequence, the migration speed becomes slower depending on the length, and the emission width becomes wider,
Overlapping of waveforms due to continuous bases. In this case, the detected waveform signal does not become a signal of a normal waveform by a single base, but the detected waveform overlaps with an adjacent waveform signal and becomes a concatenated waveform signal. In addition, the smiley of each base and the pitch fluctuation that occur during the migration also cause overlap of the waveform signals, which causes a concatenated waveform signal. further,
Since the DNA base whose reaction has stopped in the deoxy state is detected simultaneously with the original DNA base sequence, it appears as a ghost signal with respect to the detection signal of the DNA base of each complete bond. Such a signal waveform also causes a waveform concatenation.

[0004]

The base pitch ΔT fluctuates according to the electrophoretic conditions, and the occurrence of a concatenated waveform is a cause of a missed reading or a reading error when there is a weak base before and after a strong one. Therefore, the probability of determining the base sequence is reduced. Especially, 400 or 50 long DNA base sequences
For 0 or even longer DNA bases, the half-width ΔT _1/2 becomes larger than the base pitch ΔT, and the overlapping of the waveforms becomes significant, so that the decision probability is, for example, 9
It decreases to about 0%.

In order to improve the decision probability, it is necessary to rescue the signal waveform embedded in the concatenated waveform, but even if each peak is used as the peak position, the peak position itself changes. Therefore, the true peak position cannot be set, and it is not possible to determine the base sequence by taking them as an accurate pitch. Further, when the peaks disappear due to the overlapping of the waveform signals, the waveform signal selected according to the intensity and the calculated base pitch ΔT itself become inaccurate. The present invention solves such a problem of the conventional technique, and it is possible to extract a substantially true waveform even if there is a concatenated waveform in the waveform signal of time versus fluorescence intensity (frequency of appearance). It is to provide a method for determining a waveform peak for determining a base sequence. Another object of the present invention is to provide a DNA sequencer capable of improving the probability of determining a base of a long DNA base sequence.

[0006]

[Means for Solving the Problems]
Waveform peak determination for base sequencing of the present invention for
The fixed method and sequencer are characterized by A (adenine), C
(Cytosine), G (guanine), T (thymine) DNA
Calculate the sum of the waveform signals of bases as a function of migration time
Average value ΔTm of the appearance pitch of the peaks obtained
And peak value above a certain level
Target waveform extractor for extracting base waveform signal data
And the waveform data of the extracted base waveform signal.
The first threshold width ΔTR1 / 2 of the waveform after the peak position
And the second threshold width Δ of the waveform temporally before the peak position
TL1 / 2 and threshold value calculation as a function of migration time
Outputting means and first and second threshold widths ΔTR1 / 2, ΔTL1 / 2
And the peak intensity is less
Extract the basic waveform of the waveform data as a normal waveform base.
Base wave extraction means for normal waveform and base wave extraction for this normal waveform
The waveform data of the bases extracted by
A smile correction method for ring correction and this smile
Waveform signal data of the base corrected by the ring correction means
For each interval divided into predetermined time intervals
Appearance pitch of peaks obtained from waveform signals of DNA bases
Base pitch to obtain base pitch ΔTn according to the average value of
Calculation means, and of each section obtained by this base pitch calculation means
The value ΔTn and the first and
The salt of a normal waveform is formed by the second threshold widths ΔTR1 / 2 and ΔTL1 / 2.
For the concatenated waveform signal not selected by the base extraction means,
And the intensity * I of the peak of the true base signal is_n = {I_n + 2β₁ β₂× I_n-3-(Β₁-2α x
β₂ ) I_n-1-ΑI_{n + 1}} / (1-2α × β₁ ) However, I_n Is the nth observed intensity in the interval, I
_{n + 1} , I_n-1, I_n-3Is I_n From the position of ΔTn, -Δ
Tn, observation intensity at -3ΔTn apart, α = exp (-ln2 · P²), Β₁ = 1 / (1 + Q²), Β₂ = 1 / (1 + 4
Q²) P = 2ΔT_n / ΔT_{L1 / 2}, Q = 2ΔT_n / ΔT_{R1 / 2} And extract as a normal waveform based on the value ΔTn
The peak position based on the waveform position of the adjacent base
And a true waveform specifying means for specifying the position.

[0007]

[Function] Regarding the waveform of the base waveform signal, the left side of the peak is regarded as a Gaussian distribution, and the right side of the peak is regarded as a Cauchy distribution. Then, the base of the waveform data with a small fluctuation in the peak position is extracted as a normal waveform from the data of the calculated left and right threshold widths as an index and the peak intensity of each waveform signal, and first extracted,
The pitch ΔTn of the waveform is obtained for each section based on the data of the waveform signal of this normal waveform, and the base pitch of the normal waveform is selected by this and the left and right threshold widths ΔTR1 / 2 and ΔTL1 / 2. The peak intensity I of the true base signal and the position of the continuous continuous waveform signal which is not present are determined by the above equation and the value ΔTn. As a result, many base waveform signals including the base signal having the concatenated waveform can be obtained as substantially correct waveform data.

By the way, the DNA base whose reaction is stopped in the deoxy state is generated by the generation of incomplete bond due to the influence of the reaction inhibiting factor. This is the original fully coupled DN
Usually, about several percent occurs with respect to A base. Therefore,
The frequency of appearance of DNA bases in which the reaction has stopped in the deoxy state is several% of the frequency of appearance of completely bonded DNA bases. However, this becomes a ghost signal component. The ghost signal is
Since it has a similar shape, it has almost no effect on its own waveform signal, but since other DNA bases have different waveform shapes, its effect becomes a problem. On the other hand, if the ghost component of the own base is removed, it has a great influence on the waveform signal component having a weak intensity, and this has an influence on obtaining a high base decision probability of 98% to 99%. Therefore, in addition to the above, the signal of the remaining three DNA bases is used to effectively remove the ghost signal without reducing the original signal intensity without removing the ghost component of one DNA base. The fluorescence intensity (appearance frequency) of the ghost signal component from the component is calculated by multiplying the ghost occurrence rate ε. By subtracting this from the original waveform signal, it is possible to detect a larger number of substantially true waveform signals including the signal waveform with weak intensity. This can further improve the decision probability.

[0009]

1 is a block diagram of an embodiment of a DNA sequencer to which the waveform peak determining method for base sequence determination of the present invention is applied. FIG. 2 is a flowchart of the ghost signal removing process. 4 is an explanatory diagram of the waveform data generation method, FIG. 4 is a flowchart for extracting true peak waveform data, FIG. 5 is an explanatory diagram of pitch frequency, waveform distribution, and half width, and FIG. 6 is a concatenated waveform and true waveform. It is explanatory drawing of the relationship with the peak of. In FIG. 1, reference numeral 1 denotes a DNA sequencer, which uses a lens (not shown) to fluoresce a base fragment group having a predetermined width from the electrophoretic plate 2 irradiated with the laser beam L.
The light is received by the one-dimensional line image sensor (CCD) 3 via. As a result, fluorescence intensity in each lane in which A (adenine), C (cytosine), G (guanine), and T (thymine) were migrated as each DNA base using a single-color fluorescent dye marker was detected as an analog signal. Then, the CCD drive / control circuit 4 receives it and continuously outputs the analog signal to the arithmetic processing unit 5.

The CCD drive / control circuit 4 has a built-in timing controller, timing generation circuit, multiplexer, etc., and outputs the signal read from the line image sensor 3 to the arithmetic processing unit 5. The line image sensor 3 generates, for example, a signal of 125 μm per pixel, integrates 4 pixels for each lane, and outputs about 60 pixels in the lane direction.
A signal of 0 μm width is output as one unit. The arithmetic processing unit 5 includes a microprocessor (MPU) 50, an amplifier 51 that receives a light-receiving signal from the CCD drive / control circuit 4, and amplifies it. A low-pass filter (LPF) 52 and an A / D.
Conversion circuit (A / D) 53, memory 54, waveform memory 5
5, CRT display (CRT) 56, keyboard 5
7, printer (not shown), and timer (not shown)
Etc., and these circuits are connected to the MPU 50 via the bus 58.
Are interconnected with. Then, the received fluorescence intensity is output to the CRT display 56 or a printer as waveform data of time-dependent fluorescence intensity (frequency of appearance of DNA bases).

The MPU 50 sends a sampling pulse to the A / D 53 at a predetermined cycle to convert the noise-removed light-receiving signal through the LPF 52 into a digital value, and the bus 5
The digital value is received via 8 and stored in the waveform memory 55 as measurement data in sequence. As a result, the waveform memory 55 stores the data of the fluorescence intensity versus time corresponding to the sampling time as a digital value at each measurement time point, for example, as a waveform signal as shown in FIG. Become. On the other hand, in the memory 54, a base waveform data extraction program 54a, a ghost data calculation program 54b, a ghost data removal program 54c,
Waveform display / output program 54d, calculation target waveform extraction program 54e, open range calculation program 54f, regular waveform base extraction program 54g, smileing correction program 54h, base pitch calculation program 54i, and true waveform identification program 54j, and various other types. A processing program is stored.

The base waveform data extraction program 54a is
Each DN from the measured waveform data stored in the waveform memory 55
Waveform data corresponding to A bases A, C, G, and T are extracted, and a predetermined amount of data value corresponding to background noise is subtracted from the read data corresponding to each measurement time point of the waveform data memory. . Furthermore, this is the memory 54
DNA bases A, C, and
The data extracted at the timing corresponding to each lane of G and T is separated so as to correspond to each lane, divided into each storage area, and sequentially stored. The background noise is, for example, a process of obtaining a local minimum value in the waveform data measured corresponding to each predetermined measurement section and sequentially subtracting the local minimum value.

The ghost data calculation program 54b is
According to the following formula (1) gi = ε (d1 + d2 + d3 + d4) -εdi ... (1), each of the DNA bases A, C, G, and T is sequentially assigned to i and the ghost signal is assigned. Ingredient g
The calculation of i is calculated for a certain measurement time point, the measurement time point is sequentially updated, and the level of the ghost signal component gi is calculated as data in the time relation corresponding to each original data over the entire measurement period. It is a program for sequentially storing in a predetermined area of the memory 55 in association with each other in terms of time. However, d1, d2, d3 and d4 are waveform data values for the respective DNA bases A, C, G and T, and n is 4 in this embodiment. di is a DNA base A, C,
It is a waveform data value of a certain DNA base i of G and T, and ε is a ghost occurrence rate.
Is. For each of the DNA bases A, C, G, and T, the occurrence rate at which the reaction stops in the deoxy state is D, which is the original complete bond.
This is because it is about 3% with respect to the base of NA. In addition,
The ghost occurrence rate ε may be such that a weak fluorescence intensity signal of a base sequence having a long sequence to be detected is not excluded from detection, and usually about 0.02 to 0.1 is appropriate.

The equation (1) is for calculating the signal level of the ghost signal component at the time of measurement by multiplying the total value of the waveform data of each DNA obtained at each time of measurement by the ghost occurrence rate. Yes, and the second term of the equation, “εd
By subtracting "i", only the ghost signal component of other DNA is calculated. As a result, the self weak signal waveform can be relieved. The ghost data removal program 54c is the ghost data calculation program 5 described above.
The measurement data corresponding to each measurement time point calculated by 4b is removed from the original waveform data at that measurement time point, and the original data is generated for each of the bases A, C, G, and T of each DNA. It is a program stored in the memory 54 corresponding to the measurement time point.

In this case, the ghost data removing program 54c uses the bases A, C, G, T of each DNA described above.
However, instead of subtracting the ghost signal component of each other DNA, the ghost signal of each other DNA is sequentially applied to the total waveform data of the bases A, C, G, T of each DNA at each measurement time point. The waveform data may be generated by subtracting the components and adding them in the total state.

Next, the removal of the ghost signal component will be described with reference to the flow of processing shown in FIG. 2 and FIG. First,
The MPU 50 executes the base waveform data extraction program 54a by interrupt processing at the time of measurement when a predetermined amount of measurement data is stored in the waveform memory 55. Then, the background noise is removed (step 10).
1), from the waveform data at a certain measurement time point shown in FIG. 3A (for convenience of explanation, the digital value is shown in an analog state in FIG. 3), from each of the DNA bases A, C, G, T The waveform data is extracted and stored in the memory 54 (step 1
02). When this storage state is continuously illustrated for a large number of measurement points, it becomes as shown in FIG.

Next, the MPU 50 executes the ghost data calculation program 54b, calculates each ghost signal component according to the equation (1), and stores it in the memory 54 (step 103). This is the state shown as a representative waveform of the mesh portion shown as Gst for the DNA base A in FIG. 3 (c). Then, the ghost data removal program 54
By executing c, the MPU 50 removes the measurement data of the ghost signal corresponding to each measurement time point calculated by the ghost data calculation program 54b from the original data at the measurement time point and removes the original data from the base A of each DNA. , C, G,
It is generated in T and stored in the memory 54 corresponding to each measurement time point (step 104). In this case, the ghost signal component is included in its own waveform data.

This data is then processed to find the peak of the true waveform signal for the concatenated waveform signal (step 10).
In the process of 5), the peak position of each waveform data and its value are calculated as the respective base intensities through the calculation of the full width at half maximum of the waveform data and the extraction of the bases of the normal waveform in the waveform data. The base sequence is determined by performing the analysis process described in 1. Note that this step 10
Details of the process of 5 are shown in FIG. Also, in the step 10
Waveform display / output program 54d after the MPU50
May be executed to output the measurement result data excluding the ghost signal component to the CRT display 56 or the printer (step 106).

Before describing the processing for obtaining the peak of the true waveform signal in FIG. 4, a program related to this processing will be described first. Calculation target waveform extraction program 54e
Is the sum of the waveform signals of the DNA bases of A (adenine), C (cytosine), G (guanine) and T (thymine).
Data is calculated as a function of migration time, and data of this sum is subjected to discretized differential processing, for example, second-order five-term smoothing differential processing to detect peaks for the data. Then, frequency distribution data about the pitch of the detected peak is generated. The frequency distribution data is shown in FIG. In addition, the arithmetic expression of the sum of the waveform signals of DNA bases is given by equation (2)
become. Isum = IA + IC + IG + IT (2) From the peak position of the frequency distribution data, ΔT, Δ2T,
The value of Δ3T is obtained, and the base pitch ΔTm as the average value of ΔT is calculated from these values. Further, the average value ΔTm of the appearance pitch of the peak and a threshold value of a predetermined level are set, and the data of the base waveform signal to be calculated is extracted based on the peak value higher than that.

In this case, further, only the single waveform base may be extracted as the calculation target. This means that at each peak position of the waveform from the waveform data of the calculation target base, b ×
The waveform data in which another peak exists in the range of ΔTm (for example, a certain value in the range of b = 1.2 to 1.8) is shaken off. Then, the following conditional expression is applied to the remaining waveform data, and data of only a single waveform base is extracted by arithmetic processing. In this case, the full width at half maximum calculation program 54f for each waveform will be described in detail. However, the threshold value width ΔT _{R1 / 2} of the waveform that is after the peak position (on the right side) and before the peak position in time. Assuming that the threshold width of the waveform on the left side of is ΔTL1 / 2, | (ΔTL1 _{/ 2−} ΔTR1 _{/ 2)} / (ΔTL1 _{/ 2} + ΔTR1 _{/ 2} ) |
<Δ However, only the peak waveform satisfying the condition of δ ≈ 0.1 to 0.3 is extracted. here,
It is also possible to select only the base of the single waveform by selecting the value of δ in an appropriate range and to calculate the basic data such as the full width at half maximum thereafter based on this. As shown in the graph of FIG. 5 (b) for the waveform data of the calculation target base waveform signal extracted by the above-described processing, the threshold value calculation program 54f determines the threshold value width ΔTR1 / 2 of the waveform on the right side of the peak. And the threshold width ΔT _{L1 / 2} of the waveform on the left side of the peak are averaged as a function of the migration time to create a table of the threshold widths.

The normal waveform base extraction program 54g is
The bases of the waveform data in which the fluctuation of the peak position is small are extracted as a normal base waveform with reference to the threshold widths ΔT _{R1 / 2} and ΔT _{L1 / 2} and the obtained peak value (intensity) of each waveform.
Specifically, the extraction reference widths of the left and right half-value widths are set as the range of data variation in the above-mentioned price range data table as shown in Fig. (B), and the range of 3σ is set as shown in Fig. (C). , Dotted lines 3σLA and 3σLB of the selection range of the left price range, respectively
And the right-hand side of the selection range of the price range, the dashed-dotted line 3σRA,
Let 3σ RB. Then, the discrimination width is obtained corresponding to the migration time of each base waveform, and the waveform data whose left and right threshold widths are within this range is, for example, about 80% (70% to 90%).
A value selected from the range of%) is set as a threshold value equal to or higher than a predetermined peak intensity to be extracted, and the target waveform data is sequentially selected as a function of the migration time. The smiley correction program 54h corrects the waveform position of a base that is temporally shifted by smileing to the waveform signal data of each base extracted by the normal waveform base extraction program 54g.

This is the base pitch ΔT with respect to the sum S of the data of each base waveform for the base of the selected normal waveform.
The sequence of each base waveform data is obtained so that the variance of is minimized. For example, the positions of the waveform data of other bases are shifted with reference to the sequence of the base waveform data of A so that Σ | ΣΔT / S-ΔTi | is minimized (ΣΔT / S is an average value). ), And then the base sequence to be the reference is changed from the base of A to another base, and the same processing is sequentially repeated to obtain the same. The base pitch calculation program 54i divides the corrected base waveform signal data after the smileing correction into predetermined time intervals and outputs the sum of the DNA base waveform signals in each section.
Average value ΔTn of the appearance pitch of peaks obtained from
And the process of asking for. The section width is preferably such that several tens to hundreds of waveform data are included in one section. In this case, connect the values of ΔTn obtained in each section with a straight line, and
Similar to the table of the judgment widths ΔT _{R1 / 2} and ΔT _{L1 / 2,} a continuous value table can be obtained as a function of the migration time.
In this way, ΔT _{R1 / 2} and ΔT _{L1 / 2} are obtained from the half widths that are sequentially calculated on the straight line at the time positions of the respective waveforms corresponding to the respective migration times, and the following formula (3) is obtained. The true peak intensity can be obtained by the calculation of, and the peak position can be calculated more accurately by ΔTn sequentially calculated on the straight line.

The true waveform identification program 54j
Value calculated by the switch calculation program 54i
The section within each section calculated by the output program 54f.
Threshold width ΔT of each waveform_{R1 / 2}, ΔT_{L1 / 2}And each ward
A normal waveform is extracted by the base extraction means of the normal waveform between
True for continuous concatenated waveform signals that are not selected
The intensity I of the peak of the base signal of is calculated by the following equation (2),
The peak position is specified. * I_n = {I_n + 2β₁ β₂× I_n-3-(Β₁-2α × β₂ ) I_n-1-ΑI_{n + 1}}  / (1-2α × β₁ ) ……………… (3) However, I_n Is the nth observed intensity in the interval, I
_{n + 1} , I_n-1, I_n-3Is I_n From the position of ΔTn, -Δ
Tn, observation intensity at -3ΔTn apart, α = exp (-ln2 · P²), Β₁ = 1 / (1 + Q²), Β₂ = 1 / (1 + 4
Q²) P = 2ΔT_n / ΔT_{L1 / 2}, Q = 2ΔT_n / ΔT_{R1 / 2} As shown in Fig. 5 (b), this equation (3) is for the base waveform signal.
Regarding the waveform, the Gaussian distribution is on the left side of the peak.
And the right side of the peak is Cauchy distribution
Consider the formula to obtain the value for each peak value h
Thus, in the Gaussian distribution, some distribution value y is y = hexp
{-ln2 (2T / ΔT_1/2)²} Becomes. And in the Cauchy distribution,
One distribution value y is y = h / {1+ (2T / ΔT_1/2)²} become.
In accordance with this, the strength of the concatenated waveform shown in Fig. (C) is calculated.
Then, equation (3) is obtained.

Further, together with this intensity, the waveform of adjacent bases extracted as a normal waveform based on the pitch value ΔTn, for example, the waveform data K at the position of intensity In-2 in FIG. The peak position of the n-th waveform data can be obtained by ts + {n- (n-2)} * [Delta] Tn with respect to the time position ts of K. Similarly, the n + 1-th and n + 2-th peak positions and intensities are obtained in the same manner. FIG. 6 shows that n = 5 calculated in this way.
The states of the 48th, n + 1 = 549th, and n + 2 = 550th are shown. It should be noted that the n number is not the rank of the section, but the number counted from the beginning. The ranking in the section is, for example, a value obtained by subtracting 500 from the above numerical value.

Next, the processing for obtaining the peak of the true waveform signal together with the normal waveform signal will be described with reference to the flow of processing shown in FIG. 4 and FIG. First, the MPU 50 executes the calculation target waveform extraction program 54e, and A (adenine) is applied to the waveform data of each base processed to remove the background noise and the ghost signal obtained in step 104 of FIG. ), C (cytosine), G (guanine), T (thymine) DNA base waveform signal sum data
Is calculated as a function of migration time according to equation (2) (step 105A). Then, second-order five-term smoothing differential processing (as an example of discretization processing) is performed on the data of this sum (step 105B), and a peak is detected for the data and the detected peak is detected. The frequency distribution data for the pitch is generated (step 105C). Further, the base pitch ΔTm as an average value is calculated from this frequency distribution data (step 105D).

The MPU 50 includes a price range calculation program 54.
As shown in FIG. 5 (b) for the waveform data of the base waveform signal of the calculation target extracted by the above process by executing f, as shown in FIG. 5 (b), the threshold value width ΔTR1 / 2 of the waveform on the right side of the peak and the peak A process of calculating the threshold width ΔTL1 / 2 of the waveform on the left side as a function of the migration time is performed. Then, as shown in FIG. 6C, a data table of the average value of the price range calculated as a function of the migration time is created (step 105E). next,
The MPU 50 executes the normal waveform base extraction program 54g, refers to the above-described threshold value width data table, and determines the peak position based on the threshold value widths ΔT _{R1 / 2} and ΔT _{L1 / 2} and the peak intensity. The base of the waveform data with little fluctuation is extracted as the base of the normal waveform (step 105F). As described above, the extraction of the base waveform at this time is, for example, a selection range of the left and right threshold widths (a range in which 70% to 90% of the entire waveform signal is extracted). For example, the above 3σ) and the threshold value of the peak intensity are set.

Next, the MPU 50 executes the smileing correction program 54h to perform smileing correction on the base data that can be regarded as the selected normal waveform (step 105G). Next, the MPU 50 executes the base pitch calculation program 54i and obtains the sum of the waveform signals of the DNA bases in each section with respect to the corrected base waveform signal data after the smileing correction. Then, an average value ΔTn of the appearance pitch of the peaks is obtained (step 105H). Next, the MPU 50 executes the true waveform specifying program 54j and obtains the peak position and the intensity of the concatenated waveform data that has not been extracted by the base extraction processing of the normal waveform (step 105I). Using the peak positions and intensities of all the waveform data of the normal waveform and the concatenated waveform thus obtained as data, the MPU 50 analyzes the base sequence as in the conventional case (step 105).
J).

As described above, the waveform data of the embodiment obtains the half width ΔT _1/2 and the waveform pitch ΔT with respect to the raw waveform data. However, including the waveform data, the half width ΔT
More accurate data can be obtained by extracting _1/2 or the waveform pitch ΔT as a function of the second derivative and extracting a normal base waveform or the like. When the waveform data having a certain mountain-shaped peak is differentiated (first derivative), the position of the maximum value becomes the zero cross point, and waveform data having positive and negative peaks is generated. When this is further differentiated (second derivative), it becomes waveform data in which each peak is a zero cross point,
The waveform on the negative side becomes waveform data having a sharp peak with a width narrower than that of the first waveform data. The waveform on the positive side becomes a waveform with a wide low peak. Therefore, by inverting the waveform on the negative side and setting it on the positive side, it becomes possible to handle it in the same manner as the original waveform data. That is, the calculation target waveform extraction program 54e secondarily differentiates the value ΔTm with respect to the data of the sum of the waveform signals of the DNA bases, inverts the positive and negative values, and obtains it based on the positive side waveform data. The threshold width calculation program 54f inverts the positive / negative of the data obtained by second-order differentiating the waveform data of the extracted base waveform signal, and converts the positive and negative from the positive side waveform data. 2 threshold width ΔT
_{R1 / 2} and ΔT _{L1 / 2} are obtained, and the base extraction program 54g uses the second differential of the second differential of the waveform data of each of the bases. Based on ΔT _{R1 / 2} , ΔT _{L1 / 2 and} the intensity _of the peak, the base waveform of the waveform data in which the fluctuation of the peak position is small is extracted as the base of the normal waveform.

Regarding the elimination of the ghost signal in this embodiment, as described above, the total sum of the respective waveform data is calculated, and the other three ghost signal components are sequentially updated while sequentially updating the bases to be calculated. By calculating and subtracting,
It is possible to efficiently obtain the total waveform data from which the ghost signal component has been removed. However, it goes without saying that the ghost signal component and noise may be removed by other methods.
The full width at half maximum in the example is calculated for each of the left and right sides of the base signal waveform. This is calculated by the average value of the left and right sides, that is, ΔT _1/2 = (ΔT _{L1 / 2} + ΔT _{R1 / 2} ) / 2. May be. In this case, ΔT _{L1 / 2} = ΔT _1/2 , ΔT _{R1 / 2}
= ΔT _1/2 .

[0030]

According to the present invention, the left side of a peak is regarded as a Gaussian distribution, and the right side of a peak is regarded as a Cauchy distribution. Then, the bases of the waveform data with less fluctuation of the peak position are extracted as the normal waveform from the data of the calculated left and right threshold widths as an index and the peak intensity of each waveform signal. Then, the pitch ΔTn of the waveform is obtained for each section based on the data of the waveform signal of the regular waveform, and the base of the regular waveform is calculated from this and the left and right judgment widths ΔT _{R1 / 2} and ΔT _{L1 / 2.} For the continuous concatenated waveform signal that is not selected, the peak intensity I of the true base signal and its position are obtained for each section by the above equation and the value ΔTn. You can get a lot of base waveform signal as a nearly correct waveform data including a group signal.
As a result, 400 or 50 long DNA base sequences
The probability of decision about 0 DNA bases can be improved.

[Brief description of drawings]

FIG. 1 is a block diagram of an embodiment of a DNA sequencer to which a waveform peak determining method for determining a base sequence of the present invention is applied.

FIG. 2 is a flowchart of the ghost signal removal processing.

FIG. 3 is an explanatory diagram of the waveform data generation method.

FIG. 4 is a flowchart for extracting true peak waveform data.

FIG. 5 is an explanatory diagram of pitch frequency, waveform distribution, and half width.

FIG. 6 is an explanatory diagram of a relationship between a concatenated waveform and a true peak.

[Explanation of symbols]

1 ... DNA sequencer, 2 ... electrophoresis plate, 3 ... line image sensor, 4 ... CCD drive / control circuit, 5 ... arithmetic processing unit, 50 ... MPU, 51 ... amplifier, 52 ... low pass filter (LPF), 53 ... A / D conversion circuit (A / D), 5
4 ... Memory, 55 ... Waveform memory, 56 ... CRT display, 57 ... Keyboard, 58 ... Printer, 54a ... Base waveform data extraction program, 54b ... Ghost data calculation program, 54c ... Ghost data removal program, 54d ... Waveform display / output Program, 54e ... Calculation target waveform extraction program 54, 54f ... Opening width calculation program, 54g ... Normal waveform base extraction means, 54h ... Smileing correction program, 54i ... Base pitch calculation program, 54j ... True waveform identification program.

Claims (7)

[Claims] 1. A DNA base fragmentation using a single color dye marker.
Electrophoresis one group, and at the position where it is run for a certain distance
The reaching state is detected by receiving light emitted from the dye,
The received light intensity is used as a waveform signal as a function of time, and this waveform signal
Sequence determination to determine the sequence of DNA bases based on the number
In the device, A (adenine), C (cytosine), G (guanine), T
Electrophoresis of sum data of waveform signals of DNA base of (thymine)
Appearance pitch of peaks calculated as a function of time
Based on the average value ΔTm and the peak value above a certain level.
Then, the data of the base waveform signal to be calculated is extracted.
A calculation target waveform extracting means, and a peak from the waveform data of the extracted base waveform signal.
First threshold width ΔTR1 / 2 of waveform after time
And the second threshold width Δ of the waveform temporally before the peak position
TL1 / 2 and threshold value calculation as a function of migration time
Output means and the first and second threshold widths ΔTR1 / 2 and ΔTL1 / 2
Waveform with less fluctuation in peak position based on
Normal to extract the base waveform of data as a base of normal waveform
Waveform base extraction means and the base wave extracted by this normal waveform base extraction means
Smiley that corrects smiley for shape data
Correction means and the waveform of the base corrected by this smiley correction means
Each section is divided into predetermined time intervals for signal data.
Of the peak obtained from the waveform signal of the DNA base between
Calculate base pitch ΔTn according to the average value of appearance pitch
Base pitch calculating means, and the value ΔTn of each section obtained by this base pitch calculating means
The first and second values obtained by the notation price range calculation means
Of the normal waveform by the judgment widths ΔTR1 / 2 and ΔTL1 / 2 of
For the concatenated waveform signal not selected by the base extraction means,
And the intensity * I of the peak of the true base signal is_n = {I_n + 2β₁ β₂× I_n-3-(Β₁-2α x
β₂ ) I_n-1-ΑI_{n + 1}} / (1-2α × β₁ ) However, I_n Is the nth observed intensity in the interval, I
_{n + 1} , I_n-1, I_n-3Is I_n From the position of ΔTn, -Δ
Tn, observation intensity at -3ΔTn apart, α = exp (-ln2 · P²), Β₁ = 1 / (1 + Q²), Β₂ = 1 / (1 + 4
Q²) P = 2ΔT_n / ΔT_{L1 / 2}, Q = 2ΔT_n / ΔT_{R1 / 2} And a normal waveform based on the value ΔTn
Based on the position of the waveform of the extracted adjacent base, its peak
A DNA salt having a true waveform specifying means for specifying a position
A waveform peak determination method for determining a base sequence. 2. The half-value width is calculated as ΔT _1/2 as an average value of the first price width ΔT _{R1 / 2} and the second price width ΔT _{L1 / 2,} and the first price width ΔT _{R1 / 2 and} ΔT _1/2 are used in place of the second threshold value width ΔTL _1/2.
A method for determining a waveform peak for determining the DNA base sequence as described. 3. The light emission is fluorescence and the waveform signal is
It is stored in the memory as a digital value and the ghost occurrence rate ε
Is 0.02 to 0.1, and A (adenine), C
The data gi for the ith ghost signal gi (where i is a subscript 1 to 4) of the four DNA bases of (cytosine), G (guanine), and T (thymine) is given by gi = ε (d1 + d2 + d3 + d4 )-. Epsilon.di and subtracting the obtained gi from the data of the waveform signal, wherein d1, d2, d3 and d4 are data values of the waveform signal for each DNA base. Waveform peak determination method for determining the DNA base sequence of the above. 4. The calculation target waveform extracting means is configured to output the value ΔT.
m is secondarily differentiated with respect to the data of the sum of the waveform signals of the DNA bases, positive and negative are inverted, and is obtained on the basis of the waveform data on the positive side thereof. The positive and negative of the second-order differentiated data of the waveform data of the base waveform signal thus obtained are inverted, and the first and second threshold widths ΔTR1 / 2 and ΔTL1 / 2 are obtained from the waveform data on the positive side. Therefore, the base extracting means sets the first and second threshold widths ΔTR1 / 2 and ΔTL1 / 2 of the second derivative and the peak intensities thereof to the data obtained by second-derivating the waveform data of each base. The waveform peak determination method for determining a DNA base sequence according to claim 1, wherein the base waveform of the waveform data with a small fluctuation in the peak position is extracted as the base of the normal waveform with reference to the above. 5. A DNA base breakage using a single color dye marker.
Electrophoresis one group, and at the position where it is run for a certain distance
The reaching state is detected by receiving light emitted from the dye,
The received light intensity is used as a waveform signal as a function of time, and this waveform signal
Sequence determination to determine the sequence of DNA bases based on the number
In the device, the migration plate is irradiated with a laser and the DN is detected by a line sensor.
Depending on the arrival state of the base fragment group of A or RNA,
Of an electric signal obtained from the line sensor having an optical detection system for detecting emitted light and a memory.
Detect levels as a function of time and convert them to digital values
And a processing unit that stores the data as data in the memory.
The arithmetic processing unit includes A (adenine) and C (sitoshi).
Waves of DNA bases of G), G (guanine) and T (thymine)
Obtained by calculating the sum of the shape signals as a function of migration time.
The average value ΔTm of the appearance pitches of the generated peaks and the predetermined value
Base wave to be calculated based on peak value of bell or higher
Waveform extraction means for extracting the data of the shape signal, and a peak from the waveform data of the extracted base waveform signal.
First threshold width ΔTR1 / 2 of waveform after time
And the second threshold width Δ of the waveform temporally before the peak position
TL1 / 2 and threshold value calculation as a function of migration time
Output means and the first and second threshold widths ΔTR1 / 2 and ΔTL1 / 2
Waveform with less fluctuation in peak position based on
Normal to extract the base waveform of data as a base of normal waveform
Waveform base extraction means and the base wave extracted by this normal waveform base extraction means
Smiley that corrects smiley for shape data
Correction means and the waveform of the base corrected by this smiley correction means
Each section is divided into predetermined time intervals for signal data.
Of the peak obtained from the waveform signal of the DNA base between
Calculate base pitch ΔTn according to the average value of appearance pitch
Base pitch calculating means, and the value ΔTn of each section obtained by this base pitch calculating means
The first and second values obtained by the notation price range calculation means
Of the normal waveform by the judgment widths ΔTR1 / 2 and ΔTL1 / 2 of
For the concatenated waveform signal not selected by the base extraction means,
And the intensity * I of the peak of the true base signal is_n = {I_n + 2β₁ β₂× I_n-3-(Β₁-2α x
β₂ ) I_n-1-ΑI_{n + 1}} / (1-2α × β₁ ) However, I_n Is the nth observed intensity in the interval, I
_{n + 1} , I_n-1, I_n-3Is I_n From the position of ΔTn, -Δ
Tn, observation intensity at -3ΔTn apart, α = exp (-ln2 · P²), Β₁ = 1 / (1 + Q²), Β₂ = 1 / (1 + 4
Q²) P = 2ΔT_n / ΔT_{L1 / 2}, Q = 2ΔT_n / ΔT_{R1 / 2} And a normal waveform based on the value ΔTn
Based on the position of the waveform of the extracted adjacent base, its peak
DNA salt having a true waveform specifying means for specifying the position
Base sequencing device. 6. The light emission is fluorescence and the waveform signal is
It is stored in the memory as a digital value and the ghost occurrence rate ε
Is 0.02 to 0.1, and A (adenine), C
The data gi for the ith ghost signal gi (where i is a subscript 1 to 4) of the four DNA bases of (cytosine), G (guanine), and T (thymine) is given by gi = ε (d1 + d2 + d3 + d4 )-[Epsilon] di and subtracting the obtained gi from the waveform signal data, wherein d1, d2, d3 and d4 are the data values of the waveform signal for each DNA base and the half width Is calculated as an average value of the first price range ΔTR1 / 2 and the second price range ΔTL1 / 2, and the first price range ΔTR1 / 2 and the second price range Δ
The DNA nucleotide sequencer according to claim 3, wherein the ΔT1 / 2 is used in place of TL1 / 2. 7. The calculation target waveform extracting means is configured to output the value ΔT.
m is secondarily differentiated with respect to the data of the sum of the waveform signals of the DNA bases, positive and negative are inverted, and is obtained on the basis of the waveform data on the positive side thereof. The positive and negative of the second-order differentiated data of the waveform data of the base waveform signal thus obtained are inverted, and the first and second threshold widths ΔTR1 / 2 and ΔTL1 / 2 are obtained from the waveform data on the positive side. Therefore, the base extracting means sets the first and second threshold widths ΔTR1 / 2 and ΔTL1 / 2 of the second derivative and the peak intensities thereof to the data obtained by second-derivating the waveform data of each base. The DNA base sequence determination device according to claim 3, wherein the base waveform of the waveform data with a small fluctuation in the peak position is extracted as a base of the normal waveform with reference to the above.