JP2003216616A - Method of selecting base sequence waveform data with base inserted or deleted part by use of reference sequence extracting filter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of determining, using a reference sequence extracting filter, whether or not there is insertion or deletion of bases in a DNA sequence to be tested or determining the number of bases inserted or deleted. <P>SOLUTION: A reference sequence and a DNA sequence to be tested are converted respectively into a reference sequence extracting filter and a sample matrix. The filter and the starting and ending terminals of the matrix are arranged to overlap one another, and as bases are moved one by one, the sum of the products of matrixes is calculated for the distances traveled and for overlapping bases. These bases are used to enable evaluation as to whether or not there is insertion or deletion of bases in the DNA sequence to be tested or determine the number of bases inserted or deleted. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for determining the presence or absence of insertion or deletion of bases in a test DNA sequence and the number of inserted or deleted bases using a reference sequence extraction filter.

[0002]

2. Description of the Related Art Diploid organisms such as humans have two copies of each chromosome, and when a heterozygous base insertion deletion occurs, the sequence becomes a state where two waveforms are overlapped and noise occurs. Is difficult to determine. The base sequence waveform data output from the sequencer is obtained through a measurement system including a base modification reagent, electrophoresis, and a detection scanning system. Therefore, deterioration factors such as noise are added to the waveform data. The waveform data thus detected by deterioration is indistinguishable from the waveform having base insertions and deletions, and it is difficult to automatically select these. Usually, SSCP (Si
DNA such as ngle‐Strand Conformational Polymorphism)
There is a method to detect these from the difference in the higher order structure of
It has been a problem that the experimental method is difficult to achieve high throughput because some experimental condition settings have to be distributed. Sequencing (base sequence determination) has achieved remarkable high throughput in recent years, and the spread of the device is progressing. If it is possible to determine the sample with insertion or deletion from the sequencing result including noise,
Although it could be the standard and fast means of finding insertional deletion samples, there were no reports of the development of such methods.

[0003]

The present invention has been made in view of such a situation, and its object is to test DN.
It is an object of the present invention to provide a new method capable of quickly and automatically determining the insertion or deletion of a base in an A sequence. More specifically, the presence or absence of insertion or deletion of bases in the test DNA sequence, or the insertion or deletion from the test DNA sequence indicated by the waveform data to which deterioration elements such as noise are added, is determined using a reference sequence extraction filter. It is an object of the present invention to provide a method for determining the number of generated bases.

[0004]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to solve the above problems. First, the test DNA sequence represented by the reference sequence and the waveform data is
A set of vectors decomposed into base components was converted into a two-dimensional matrix and named as a reference sequence extraction filter and a sample matrix, respectively. Then, overlap the reference sequence extraction filter and the start and end ends of the sample matrix, and move the reference sequence extraction filter and the sample matrix one base at a time, moving distance from the first overlapping point, and overlapping bases. The sum of matrix products was calculated. Moreover, the homologous region at each position was identified. Furthermore, the maximum homologous nucleotide sequence detection point, the standard reference sequence extraction filter homology region, and the standard sample matrix homology region were specified, and the reference sequence extraction filter predicted homology region was calculated. The predicted homology region and the measured homology region were compared to confirm the data consistency. Next, a detection positional relationship diagram was created with the moving distance on the vertical axis and the reference array matrix position on the horizontal axis. From the above homologous base number, reference sequence extraction filter homology region, sample matrix homology region, maximum homology base sequence detection point, standard reference sequence extraction filter homology region, and standard sample matrix homology region, the test DNA sequence In, the presence or absence of base insertions or deletions was evaluated, and it was shown that the number of bases inserted or deleted can be identified. Furthermore, it was shown that by performing the above steps on a computer, it is possible to realize high throughput evaluation of the presence or absence of insertion or deletion of bases in the test DNA sequence, or identification of the number of inserted or deleted bases.

From the above, the insertion or deletion of a base in a test DNA sequence can be determined rapidly and automatically. In the above method, the data deterioration element such as noise can be removed by converting the test DNA sequence represented by the waveform data into a matrix. Therefore, it is highly expected that the method of the present invention facilitates the determination of insertion or deletion of a base in a test DNA sequence, particularly from waveform data in which a deterioration element such as noise is added.

That is, the present invention relates to a new method capable of rapidly and automatically determining the presence or absence of insertion or deletion of bases in a test DNA sequence, or the number of inserted or deleted bases, and more specifically, [1 ] A method for determining the presence or absence of base insertions or deletions in a test DNA sequence, or the number of bases inserted or deleted, comprising the steps of (a) decomposing a vector into four base components for each base: Creating a reference sequence extraction filter consisting of a set, and a sample matrix consisting of a set of vectors obtained by decomposing the test DNA sequence into four base components for each base, (b) starting the reference sequence extraction filter and the sample matrix A step of superimposing the end and the terminating end, (c) while moving the reference sequence extraction filter and the sample matrix one by one, the reference sequence extraction Calculating a sum of products of matrices for a moving distance from a point where a filter and a start end and a stop end of the sample matrix are overlapped, and overlapping bases; (d) overlapping bases from the sum of products of the matrix In the step of calculating the number of continuous homologous bases, migration distance, reference sequence extraction filter homology region, and sample matrix homology region, (e) maximum homologous base sequence detection point which is the migration distance in the case of showing the most number of homologous bases, Standard reference sequence extraction filter homology region that is the reference sequence extraction filter homology region at the detection point,
And a step of calculating a standard sample matrix homology region which is a sample matrix homology region at the detection point,
(F) a step of calculating an inter-matrix position correction value from the standard reference sequence extraction filter homology region and the standard sample matrix homology region,
From the reference sequence extraction filter homology region, sample matrix homology region, maximum homologous base sequence detection point, standard reference sequence extraction filter homology region, and standard sample matrix homology region, the presence or absence of base insertion or deletion in the test DNA sequence is evaluated. , A method for determining the number of inserted or deleted nucleotides, [2] computer (1) (a) ~
A program for executing the step (f) to determine the presence or absence of base insertions or deletions in the test DNA sequence, or the number of bases inserted or deleted, [3] [2]
A computer-readable recording medium recording the program according to [4], including the means of (1) to (3) below, and the presence or absence of insertion or deletion of bases in the test DNA sequence, or insertion or deletion. A device for determining the number of bases, (1) the reference sequence and the input means for inputting the test DNA sequence information (2) by the steps (a) to (f) of [1], the number of homologous bases and the reference sequence Provided are an extraction filter homology region, a sample matrix homology region, a maximum homologous base sequence detection point, a standard reference sequence extraction filter homology region, and a calculation unit (3) a display unit for displaying the calculation result. It is a thing.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention provides a method for determining the presence or absence of insertion or deletion of bases in a test DNA sequence, and the number of inserted or deleted bases.

In the present invention, first, a reference sequence extraction filter consisting of a set of vectors obtained by decomposing the reference sequence into 4 base components for each base, and a vector obtained by decomposing the test DNA sequence into 4 base components for each base. A sample matrix composed of a set of is created (step (a)).

As the test DNA sequence for determining the presence or absence of a base insertion or deletion, or the number of inserted or deleted bases by the method of the present invention, for example, DNA sequencing generally performed by those skilled in the art can be used. The desired DNA sequence data obtained may be mentioned. The presence or absence of base insertions or deletions, or the number of bases inserted or deleted, is usually determined by comparing the test DNA sequence with a DNA sequence whose DNA sequence has already been determined (for example, D of the wild-type gene).
NA sequences, etc.) are compared. For example, in the case of determining the presence or absence of insertion or deletion of bases, or the number of inserted or deleted bases by the method of the present invention for a test DNA sequence obtained by sequencing Gene A of a certain test sample , The test DNA sequence,
The determination of the present invention can be made by comparing with the base sequence of the wild-type gene A.

The origin of the test DNA of the present invention is not particularly limited, and examples thereof include chromosomal DNA or cDNA prepared from a haploid or diploid organism. When using a chromosomal DNA prepared from a diploid organism as a test DNA, specifically, as insertion or deletion of bases,
(1) A type in which a chromosomal DNA has a base insertion or deletion (hetero insertion or deletion),
(2) A type in which a base insertion or deletion occurs in both chromosomes (homo insertion or homo deletion) is included, and the method of the present invention includes the presence or absence of base insertion or deletion in such a type. , Or the number of inserted or deleted bases can be determined. Further, the present invention is 2
Since it is effective against homozygous deletions and insertions in polyploid organisms, it is also applicable to chromosomal DNA sequences of haploid organisms. In addition, the present invention is capable of determining insertions and deletions at multiple positions in a test DNA sequence.

In the present invention, the DNA sequence to be compared with the test DNA sequence is called the reference sequence. In the present invention, the reference sequence refers to a general sequence of these sequences when the nucleotide sequences encoding the same region of the same species differ slightly from one individual to another. The general-type sequence can be identified by, for example, determining the base sequences of the same region in a plurality of samples by a sequencer and connecting them by an assembler. If the area is registered in the existing database, it may be used. The reference sequence has the same base sequence as the test DNA sequence and should not be a complementary strand.

In the present invention, the above-mentioned reference sequence extraction filter is a set of vectors obtained by decomposing the reference sequence (base sequence represented by A, T, G, C) into 4 base components for each base, and is a two-dimensional array. In the present invention, it is also referred to as a reference sequence matrix. Further, the sample matrix in the present invention is a set of vectors obtained by decomposing the test DNA sequence into four base components, and the sequence is translated (developed) into a two-dimensional matrix.
Means what you have done.

Specifically, when the type of the base at a certain position in the test DNA sequence is A, T, G, or C, the above vectors are, for example, (1,0,0,0), respectively. , (0,0,0,
1), (0,1,0,0), or (0,0,1,0), and the test DNA sequence is expressed as a matrix which is a set of these vectors. That is, the test DNA sequence is represented as a matrix in which the vectors represented as described above are assembled according to the base sequence of the sequence. More specifically, when the test DNA sequence is, for example, waveform data detected as a result of performing a sequence, the matrix of the present invention can be obtained, for example, in Table 1 or Table 2 of Example 1 described later. It can be expressed as shown. Specifically, 1 is detected as a waveform, 0 is not detected, and each base is, for example, A (1,0,0,0), G (0,1,0,
0), C (0,0,1,0) and T (0,0,0,1). However, as long as the test DNA sequence can be expressed as digital information (binary system) as described above, it is not limited to the above expression form. It is also possible that the waveform signal corresponding to one base in the waveform data is not specified as one and has two or more waveform signals. In this case, the vector is, for example, 2 of A and T.
If there are two waveform signals, it can be represented as (1,0,0,1), and if there are two waveform signals of AG, they can be represented as (1,0,1,0). In the present invention, the waveform data to which a deterioration element such as noise is added is 1 or 0 including the deterioration element.
To digitize. Since the appearance of deterioration elements has no regularity, continuous homology cannot be obtained in comparison with the reference sequence extraction filter. Therefore, the deterioration factor can be removed. Furthermore, even if two waveform signals are output, these two signals can be added as information for determining the insertion or deletion, so that it is possible to determine whether the insertion or deletion is hetero or homo. It was

In the present invention, the reference sequence extraction filter and the starting end and the ending end of the sample matrix are then superposed (step (b)).

In a preferred mode in this step, first, two matrices, which are a set of vectors, are made to correspond to the first vector (start end) of one matrix and the last vector (stop end) of the other matrix. Then, overlay. In the present invention, this state is called a "measurement start point". As the measurement start point, specifically, a state as shown in FIG. 5 of Example 1 described later can be exemplified. In the present invention, the "measurement start point" usually refers to a place where the first vector and the last vector of the other matrix are superposed so as to correspond to each other, and usually the sequence reaction start point in the reference sequence extraction filter can be presented. Only when the measurement start point can be moved. In addition, the measurement start point is not limited to the ends of the above-mentioned start end and end end, and when the sequence reaction start point in the reference sequence extraction filter is known, overlapping other than the above is performed to reduce the calculation amount. It is also possible to use the state of as the measurement starting point.

In the present invention, next, the reference sequence extraction filter and the sample matrix are moved one base at a time, while the start and end ends of the reference sequence extraction filter and the sample matrix are overlapped (measurement start point).
And the sum of the products of the matrices for the overlapping bases is calculated (step (c)).

In the above step, "moving the reference sequence extraction filter and the sample matrix by one base at a time" means that the overlapping region between the reference sequence extraction filter and the sample matrix is one base (1 This is an operation that shifts the vector so that it changes.
This operation is finished as soon as the matrices do not overlap each other, or when the sample matrix start point overlaps with the sequence reaction end point in the reference sequence extraction filter.

The moving distance means the number of vectors moved from the measurement starting point, that is, the number of moving bases, when the reference sequence extraction filter and the sample matrix are moved one base at a time.

To calculate the sum of matrix products for overlapping bases means to calculate the sum of products of overlapping vectors for all the vectors included in the overlapping matrix region in the region. .

The sum of the vector products can be calculated, for example, by two vectors
Toru is (a₁, b₁, c₁, d₁) And (a₂, b₂, c ₂, d₂),
{(a₁× a₂) + (B₁× b₂) + (C₁× c₂) + (D₁× d₂)}
It Specifically, two vectors are (1,0,0,0) and
The sum of the products of these vectors when (1,0,0,1)
Is (1 × 1) + (0 × 0) + (0 × 0) + (0 × 1) = 1.
Also, the two vectors are (1,0,0,0) and (0,0,1,0)
The sum of the products of these vectors is (1 × 0) +
(0x0) + (0x1) + (0x0) = 0.

In the present invention, the number of consecutive homologous bases in the overlapping bases, the migration distance, the reference sequence extraction filter homology region, and the sample matrix homology region are then calculated from the sum of the matrix products (step (d)). .

In the present invention, whether or not the bases are homologous is judged by, for example, that the overlapping bases are homologous when the sum of the products of the matrix calculated in the step (c) is 1 or more. If the sum of products of the matrix is 0, it is determined that they are not homologous (non-homologous).

The term "consecutive homologous base sequences in overlapping bases" means a matrix area in which "one or more numbers" of the sum of products of matrices showing the homologous bases in the above judgment appear consecutively. is there. "Consecutive appearance"
Means that there is a homology of a certain number or more. The criterion for the fixed number of bases is not particularly limited. For example, when multiple waveforms appear in one vector (a state where there is a lot of noise), a large number of regions with homology of 20 bases or less are detected. In the invention, it is usually recorded when there is a homology of 25 bases or more.

The "number of homologous bases" refers to the number of times the "numerical value of 1 or more" of the sum of products of the matrix appears consecutively. "Reference sequence extraction filter homologous region" is on the reference sequence extraction filter where "1 or more numbers" appear at the beginning and end of the region where "1 or more numbers" of the sum of matrix products appear consecutively. It is the position information in. The same applies to the “sample matrix homologous region”. The migration distance, the number of homologous bases, the positions of both ends of the region having homology of the reference sequence extraction filter, and the positions of both ends of the region having homology of the sample matrix are determined by the matrix in the above step (c).
Each base is moved, and a record is made each time a "consecutive homologous base sequence in overlapping bases" is detected. In addition, in the present invention, the above-mentioned “number of 1 or more”
Is usually "1".

Further, in the data obtained in the step (d), there are a lot of false positive data derived from noises and repetitive sequences, which usually must be removed. In the present invention, then, the maximum homologous base sequence detection point, which is the migration distance when showing the highest number of homologous bases, the standard reference sequence extraction filter homology region which is the reference sequence extraction filter homology region at the detection point, and the detection A standard sample matrix homology region that is a sample matrix homology region at a point is calculated (step (e)), and then an inter-matrix position correction value is calculated from the standard reference sequence extraction filter homology region and the standard sample matrix homology region. (Step (f)).

The above "maximum homologous nucleotide sequence detection point" is considered to be where all the regions showing homology are usually present on the detection point if there is no insertion / deletion site in the sequence sequence. On the other hand, if there is a homologous region at a migration distance away from the “maximum homologous nucleotide sequence detection point”, it is considered that insertion and / or deletion has occurred.

The "inter-matrix position correction value" is the difference in the standard homology region between the matrices, and if either "reference sequence extraction filter homology region" or "sample matrix homology region" is known, "inter-matrix position correction value" is obtained. It is possible to calculate the original position on the matrix of each other using "value". The calculation of the “inter-matrix position correction value” is performed specifically when the “standard reference sequence extraction filter homology region” is (c1, c2) and the “standard sample matrix homology region” is (s1, s2). The correction value is expressed as c1-s1 or c2-s2.

Next, in a preferred embodiment of the present invention, the difference in each of the above moving distances is added to the standard sample matrix homology region, and further inter-matrix position correction is performed to calculate the reference sequence extraction filter predicted homology region. To be done.

The "difference in moving distance" is calculated by calculating the difference in moving distance from the "detection point of maximum homologous base sequence".
When this is added to the sample matrix homology region, the sample matrix homology region when there is no insertion or deletion can be calculated.

"Reference sequence extraction filter predicted homology region"
The calculation of, for example, "inter-matrix position correction value" is a,
Moving distance when "maximum homologous nucleotide sequence detection point" is b
b ', "Sample matrix homology region" (s1, s2), "reference sequence extraction filter predicted homology region" (c1,
c2) is {c1 = s1 '+ a- (b'-b)}, {c2 = s2' + a-
(b'-b)}. Specifically, when the "standard reference sequence extraction filter homology region" is (100,500) and the "standard sample matrix homology region" is (150,550), the inter-matrix position correction value is 100-150 = -50. is there. When the "maximum homologous nucleotide sequence detection point" is 1200, the moving distance is 12
10, "Sample sequence homology region" (300, 550) "reference sequence extraction filter predicted homology region" (c1, c
2) is c1 = 300 + (-50)-(1210-1200) = 240, c2 = 550
It is represented by + (-50)-(1210-1200) = 490.

Detected at a migration distance that is 10 bases more than the "maximum homologous base sequence detection point" means that 10 bases have been deleted. The homology region of the sample matrix is 300-550 due to the deletion of 10 bases. Therefore, when the inter-matrix position correction is performed, the expected homology region of the reference sequence extraction filter is 240-490.
Is. If the predicted homologous region thus obtained and the actually detected reference sequence extraction filter homologous region are the same, it is confirmed that the information is homologous region information based on insertion and deletion. In this way, the consistency of the data can be confirmed by calculating the predicted homology region of the reference sequence extraction filter and comparing it with the actual homology region of the reference sequence extraction filter.

In the present invention, the number of homologous bases, the reference sequence extraction filter homology region, the sample matrix homology region, the maximum homologous base sequence detection point, the standard reference sequence extraction filter homology region, and the standard sample matrix homology region are used for the test. The presence or absence of base insertions or deletions in the DNA sequence is evaluated to determine the number of bases inserted or deleted.

As a preferable mode in the above judgment,
A detection positional relationship diagram that describes all "reference sequence extraction filters" calculated in the above steps is created, and the above determination is performed based on this detection positional relationship diagram. 7 to 16,
A specific example of the detected positional relationship diagram created in this way is shown, but the type of graph in the present invention is not particularly limited.

In the detection positional relationship diagram prepared, in the case of a normal sample having neither insertion nor deletion, all the reference sequence extraction filters and sequence extraction homology regions are described on the maximum homologous nucleotide sequence detection point. On the other hand, in the sample in which insertion or deletion occurs, a plurality of homologous regions appear according to the number, and the forms of insertion, deletion, homo, hetero, etc. can be predicted from the detected form.

In the drawn detection pattern, insertion and deletion are determined by evaluating one homologous region downstream with respect to the homologous region upstream of the reference sequence matrix position. By evaluating insertion and deletion in order from the upstream, it becomes easy to make sense of mutations when multiple mutations exist. For example, when the presence of a homologous region at another migration distance overlapping almost the entire region with respect to the upstream homologous region is recognized, it indicates that the insertion or deletion is a hetero. In addition, when the homologous region is observed at a migration distance different from that of the upstream homologous region with almost no overlap, it is found that the homologous region is a homologous insertion or deletion. The determination of insertion or deletion is insertion if the migration distance of the target homologous region with respect to the upstream homologous region is long, and deletion is short. Due to the characteristics of the detection method, the position of insertion or deletion cannot be accurately determined when it is on a single-nucleotide repeat sequence, but it can also be determined if there is little noise otherwise.

Furthermore, the method of the present invention is also effective for measuring the number of repetitions of a tandem repeat array such as microsatellite. When a tandem repeat sequence having a different number of repeats is sequenced, the sample matrix is detected by overlapping a plurality of waveforms as in the case of insertion deletion. This is to detect the sequences after the tandem repeat sequence is overlaid due to the difference in the number of repetitions. The only difference in the detection method is the creation of the reference sequence extraction filter matrix. In other words, create a reference sequence extraction filter matrix with a base sequence excluding the tandem repeat sequence, and perform this test on a sample matrix containing the tandem repeat sequence to detect the base number of the tandem repeat sequence as the number of inserted bases. Is possible. If there is a difference in the number of repetitions, different numbers of inserted bases will be detected in the same region.

The method of the present invention is effective for measuring the number of bases in a tandem repeat sequence such as microsatellite. When tandem repeat sequences with different numbers of repeats are sequenced, the sample matrix is detected by overlapping a plurality of waveforms as in the case of insertion deletion. This is to detect the sequences after the tandem repeat sequence is overlaid due to the difference in the number of repetitions.

The difference in the detection method is only the creation of the reference sequence extraction filter matrix. Create a reference sequence extraction filter matrix with the base sequence excluding the tandem repeat sequence. By performing this test on a sample matrix containing a tandem repeat sequence, the number of tandem repeat sequence bases is detected as the number of inserted bases. If there is a difference in the number of repetitions, different numbers of inserted bases will be detected in the same region (Fig. 17).

The present invention also causes a computer to execute the above steps (a) to (f) to determine the presence or absence of insertion or deletion of bases in the test DNA sequence, or the number of inserted or deleted bases. Provide a program for judgment.

The computer program of the present invention includes a part or all of the following programs (1) to (7). (1) A computer program for causing a computer to create a reference sequence extraction filter and a sample matrix in the method of determining the presence or absence of insertion or deletion of the present invention, or the number of inserted or deleted bases (“matrix creating program”). Is also described). (2) In the method of the present invention for determining the presence or absence of insertions or deletions, or the number of inserted or deleted bases, the reference sequence extraction filter and the start end and end end of the sample matrix are superposed on each other. A computer program for calculating the moving distance (also referred to as a "moving distance calculating program"). (3) In the method of the present invention for determining the presence or absence of insertion or deletion, or the number of inserted or deleted bases, a computer is caused to calculate the sum of matrix products for overlapping bases of a reference sequence extraction filter and a sample matrix. A computer program (also referred to as "a program for calculating the sum of matrix products"). (4) In the method of the present invention for determining the presence or absence of insertion or deletion, or the number of inserted or deleted bases, a computer for causing a computer to calculate continuous homologous regions in overlapping bases from the sum of products of matrices Program (also referred to as "homologous region calculation program"). (5) Standard reference sequence extraction filter homologous region and standard sample having a maximum homologous nucleotide sequence detection point among homologous regions in the method of the present invention for determining the presence or absence of insertion or deletion, or the number of inserted or deleted nucleotides A computer program for calculating a matrix homology region (also referred to as "maximum homologous nucleotide sequence detection point calculation program"). (6) In the method for determining the presence or absence of insertion or deletion, or the number of inserted or deleted bases of the present invention, a reference sequence extraction filter predicted homology region is calculated in each homology region, and an actual reference sequence extraction filter homology region is calculated. A computer program to be compared with (also referred to as a “predicted homology region calculation program”). (7) A computer program (also referred to as a “detection position relationship diagram drawing program”) for drawing a detection position relationship diagram including a movement distance and a reference sequence extraction filter homology region on a computer.

The present invention further provides a computer-readable recording medium in which the computer program of the present invention is recorded. The recording medium of the present invention can be read by a general-purpose computer, and the computer program of the present invention is recorded therein. The recording medium of the present invention includes both portable and fixed media, and examples thereof include a CD-ROM, a flexible disk (FD), a DVD, a hard disk, and a semiconductor memory.

Further, the program of the present invention is stored in the portable recording medium for sale and sale, or stored in a recording device of a computer connected through a network and transferred to another computer through the network. You can also do it. The providing medium that provides the user with the computer program that executes the processing steps of the present invention can be distributed as various forms of computer-readable media, and the present invention can be realized by using the specific type of computer program used for actual distribution. Applies regardless of medium.

The present invention also provides an apparatus for determining the presence or absence of insertion or deletion of bases in a test DNA sequence, or the number of inserted or deleted bases. The apparatus comprises an input means for inputting reference sequence and test DNA sequence information, and the number of homologous bases, reference sequence extraction filter homology region, sample matrix homology region, maximum homologous base sequence detection by the above steps (a) to (f). Calculating means for calculating points, standard reference sequence extraction filter homologous areas, and standard sample matrix homologous areas, and display means for displaying calculation results.

A preferred embodiment of the above apparatus of the present invention is a computer in which the program of the present invention is stored in an auxiliary storage device such as a hard disk device. The apparatus includes a matrix creating program, a moving distance calculating program, a matrix product sum calculating program, a homologous region calculating program, a maximum homologous base sequence detection point calculating program, an expected homologous region calculating program, a detection position relationship diagram drawing program,
Further, it includes some or all of the programs for controlling these programs.

FIG. 1 shows an example of a system configuration diagram of the apparatus of the present invention. The input means 1 and the display means 2 are connected to the bus line 3. The temporary storage means 4 stores the input data,
The calculated numerical data and the like are temporarily stored. The central processing unit (CPU) 5 performs various calculations in response to the instructions of the program of the present invention. The main memory 6 stores various programs for executing the processing of the present invention. This program includes a matrix creation program 61, a moving distance calculation program 62, a matrix product sum calculation program 63, a homologous region calculation program 64, a maximum homologous base sequence detection point calculation program 65, an expected homologous region calculation program 66, and a detection position relationship. Drawing drawing program 67 and program 68 for controlling these programs
Can be roughly divided into 1 of these programs 61-68
It is also possible to combine them into one program.

FIG. 2 shows an example of the flow of processing executed by the apparatus. First, a reference sequence and test DNA sequence information (hereinafter, the two are collectively referred to as “sequence information”, and the sequence information includes waveform data) are input from the input means. This sequence information is directly input to the device of the present invention from an input unit such as a keyboard, and also a portable recording medium on which the sequence information is recorded, a fixed medium such as a hard disk,
Alternatively, it can be supplied to the computer from a communication network such as a data bank of the Internet using a receiving means such as a modem. Usually, as the sequence information of the present invention, information obtained from a nucleotide sequence database in Japan or overseas or output by a DNA autosequencer can be used. It can also be created using a commercially available text editor. Further, it is also possible to store appropriate array information in advance in a fixed medium such as a hard disk and appropriately load from the fixed medium for use. The input sequence information can be stored in the main memory or temporary storage means of the computer. The sequence information input by the input means is
Converted to matrix. Normally, this processing step is carried out by an arithmetic means such as a central processing unit (CPU) receiving an instruction of a matrix creating program in the main memory based on the array information stored in the main memory or the temporary storage means.
In particular, the process of converting waveform data into a matrix is generally called base call. In the present invention, it is also possible to manually perform this base call operation while observing the waveform data.

Next, from the inputted sequence information, the steps (a) to (d) of the present invention are carried out so that the sum of the products of the matrix, the number of consecutive homologous bases in the overlapping bases, the moving distance, the homology of the reference sequence extraction filter Calculate the region and the sample matrix homology region. Normally, in this processing step, arithmetic means such as a central processing unit (CPU) receives instructions from a matrix product sum calculation program in the main memory, a movement distance calculation program, a homologous region calculation program, etc., and reads array information. , The sum of matrix products, the number of consecutive homologous bases in overlapping bases, the migration distance, the reference sequence extraction filter homology region, and the sample matrix homology region are calculated. The calculated sum of products of matrix, movement distance, reference sequence extraction filter homology region, and calculation result of sample matrix homology region are stored in the storage means or the temporary storage means of the computer and used in the calculation in the following steps. To be done.

Then, according to the above steps (e) and (f), the maximum homologous base sequence detection point, which is the migration distance when the maximum number of homologous bases is shown, and the standard sequence extraction filter homology region at the detection point. The reference sequence extraction filter homology region and the standard sample matrix homology region that is the sample matrix homology region at the detection point are calculated. Furthermore, a reference sequence extraction filter predicted homology region is calculated in each homology region and compared with the actual reference sequence extraction filter homology region. These processing steps include a maximum homologous region calculation program in the main memory and a maximum homologous base sequence detection based on the arithmetic result calculated by the above step stored in the storage means by the arithmetic means such as the central processing unit (CPU). Calculation is performed in response to commands such as the point calculation program and the predicted homology region calculation program.

Next, the calculated calculation result is displayed by the display means. The calculation result can be displayed as text, for example, or as a graph with the moving distance and the reference array matrix position as an axis as described above, but the display form is not particularly limited. Normally, this processing step is executed in response to a command from the detected positional relationship diagram drawing program in the main memory based on the calculation result calculated in the above step. This display means includes a display monitor, a printer, and the like.

[0050]

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Example 1] (1) First, a reference sequence extraction filter for recognition of a common base pattern was prepared. The reference sequence extraction filter was created by expanding the reference sequence (normal waveform without insertion deletion (Fig. 3)). In this embodiment, in the case of A (1,0,0,0) in the case of T (0,0,0,1) in the case of G (0,1,0,
0) In the case of C, it was set to (0,0,1,0). A matrix for each base of the reference sequence was prepared and used as a reference sequence extraction filter (Table 1).

[0051]

[Table 1]

(2) Next, the sample waveform was developed into a matrix. The sample waveform matrix was created by expanding the sample waveform (FIG. 4). For example, when the AT waveform is output (1,0,0,1), it is output when the AG waveform is output (1,0,1,0). A matrix for each base of the sample waveform was created and used as a sample matrix. In this example, a sample in which one base was deleted was used (Table 2).

[0053]

[Table 2]

(3) Next, the starting end of the reference sequence extraction filter and the ending end of the sample matrix were overlapped and used as the measurement starting point (FIG. 5). (4) The product of each base and the sum of the products at that position were calculated. When the reference array (1,0,0,0) sample (1,0,0,1) is (1 × 1) + (0 × 0) + (0 × 0) + (0 × 1) = 1 . On the other hand, when the reference array (1,0,0,0) sample (0,0,1,0) is (1 × 0) + (0 × 0) + (0 × 1) + (0 × 0) = 0. When 1 appears consecutively, it means that each nucleotide sequence at this site has homology. The site with the longest homology was recorded. (5) The overlap between the reference sequence extraction filter and the sample matrix was shifted by one base, and the homologous region at each position was identified. (6) The calculation ends when the matrices do not overlap each other. (7) The maximum homologous nucleotide sequence detection point, the standard reference sequence extraction filter homology region, and the standard sample matrix homology region were identified. (8) Reference sequence extraction filter The predicted homology region was calculated. (9) The predicted homology region and the measured homology region were compared, and only the data in which the insertion or deletion was reflected were selected. FIG. 6 shows a calculation method for obtaining the predicted homologous region from the homologous region and the movement distance by the reference sequence extraction filter. After the slide of the matrix was completed, the migration distance with the maximum homologous region was 29, so the maximum homologous nucleotide sequence detection point was set to 29 and each standard homologous region was defined. After calculating the inter-matrix position correction value,
The reference sequence extraction filter homology region was calculated from the sample matrix homology region at each movement distance, and this was compared with the actual value of the reference sequence extraction filter homology region to confirm the data consistency. (10) A detection positional relationship diagram was created with the moving distance on the vertical axis and the reference array matrix position on the horizontal axis (FIG. 7). Homology domain
From the relationship of homologous region B (moving distance 30 homologous region 1 to 29) to A (moving distance 29 homologous region 1 to 29), the two homologous regions overlap, and since the moving distance of B is long, it is the 15th base. It can be seen that there is a single base hetero insertion.

[Example 2] A normal sample without insertion deletion was analyzed (Fig. 8). As a result, the "maximum homologous nucleotide sequence detection point" was at the migration distance of 300, and the standard reference sequence extraction filter homology region was 150 to 550. In this way, it was revealed that the homologous region exists at a single migration distance in the absence of insertion deletion.

[Example 3] Tero insertion at one site (at the 400th base)
As a result of analysis of a sample of 150 base hetero insertion),
Homologous region A (moving distance 300, homologous region 150 to 550) and homologous region
B (moving distance 450 homology region 400 to 550) was detected (Fig. 9).
(Left figure). In this case, the relationship between the homologous region B and the homologous region A indicates that the two homologous regions overlap and the B migration distance is long, so that there is a 150-base hetero insertion at the 400th base.

In addition, as a result of analyzing a sample with one site heterozygous deletion (hetero deletion of 100 bases from 300 to 400 bases), homology region A (migration distance 300 homology region 150 to 550) and homology region B ( A movement distance of 200 homologous regions 400 to 550) was detected (Fig. 9, right). In this case, homologous region B to homologous region A
From the relationship, it can be seen that the two homologous regions overlap and the B migration distance is short, so that there is a heterodeletion of 100 bases at the 400th base.

[Example 4] 1-site homo insertion (10 at the 350th base)
As a result of analyzing the sample, homologous region A (movement distance 300, homologous region 150 to 350) and homologous region B were analyzed.
(Moving distance 400 homology region 350-500) was detected (Fig. 1
0 left figure). In this case, the relationship between the homologous region B and the homologous region A indicates that the two homologous regions do not overlap with each other and that B has a long migration distance, so that there is a 100-base homozygous insertion at the 350th base.

Also, one-site homodeletion (1 to 350-450 bases
As a result of analysis of a sample (homo-deletion of 00 bases), homology region A (migration distance 300, homology region 150 to 350) and homology region B
(Movement distance 200 homology region 450-550) was detected (Fig. 1
0 right figure). In this case, the relationship between the homologous region B and the homologous region A indicates that the two homologous regions do not overlap with each other and the B migration distance is short, so that there is a 100 base homozygous deletion at the 450th base.

[Example 5] As a result of analysis of a sample after homo insertion and hetero insertion (100 bases homo insertion at 250th base, 50 bases hetero insertion at 400th base), homology region A (movement distance 300 homology) Area 150-250), homology area B (travel distance 400
The homologous regions 250 to 550) and the homologous region C (moving distance 450, homologous regions 400 to 550) were detected (FIG. 11, left diagram). In this case, because of the relationship between the homologous region A and the homologous region B, the two homologous regions do not overlap, and the moving distance of B is long.
It can be seen that there is a 100 base homo insertion at the 50th base. Further, from the relationship of the homologous region C to the homologous region B, the two homologous regions overlap and the movement distance of C is long, so 400
It can be seen that there is a hetero insertion of 50 bases at the base position.

Further, as a result of carrying out a sample after homo insertion and hetero deletion (a homo insertion of 100 bases at the 250th base, a hetero deletion of 50 bases from 350 to 400 bases), a homologous region A (movement distance 3
00 homology region 150-250), homology region B (travel distance 400 homology region 250-550) and homology region C (travel distance 350 homology region 40)
0-550) was detected (the right figure in FIG. 11). In this case, the relationship between the homologous region B and the homologous region A indicates that the two homologous regions do not overlap with each other, and the migration distance of B is long, so that there is a 100-base homozygous insertion at the 250th base. Further, from the relationship of the homologous region C to the homologous region B, the two homologous regions are overlapping, and because the migration distance of C is short, 50 at the 400th base.
It can be seen that there is a heterodeletion of bases.

[Example 6] Homo-insertion after hetero insertion (150 base hetero insertion at 200 bases, 50 base homo insertion at 400 bases) As a result of analysis, a homology region A (movement distance 300 homology) Region 150-400), homology region B (travel distance 450)
Homologous region 200-400), homologous region C (moving distance 350 homologous region)
400 to 550), and homology region D (moving distance 500 homology region 40
0-550) was detected (the left figure of FIG. 12). In this case, from the relationship of the homologous region B to the homologous region A, the two homologous regions overlap, and the migration distance of B is long, so at the 200th base.
It can be seen that there is a heterogeneous insertion of 150 bases. Further, at the reference array matrix position 400, the homologous region C does not overlap with the homologous region A with respect to the homologous region A, and the homologous region D does not overlap with the homologous region B.
It can be seen that there is a homozygous insertion of 50 bases at the base position.

In addition, as a result of analysis of a homozygous deletion sample after hetero insertion (a hetero insertion of 150 bases at the 200th base, a homo deletion of 50 bases from 400 to 450 bases), a homologous region A (300 homologous migration distances) Area 150-400), homology area B (moving distance 45
0 homology region 200-400), homology region C (travel distance 250 homology region 450-550), and homology region D (travel distance 400 homology region)
450-550) was detected (the right figure in FIG. 12). In this case, the relationship between the homologous region B and the homologous region A indicates that the two homologous regions overlap and the B migration distance is long, so that there is a hetero insertion of 150 bases at the 200th base. Further, at the reference array matrix position 450, the homologous region C does not overlap the homologous region A with respect to the homologous region A, and the homologous region D does not overlap with the homologous region B.
It can be seen that there is a homozygous deletion of 50 bases at the 0th base.

[Example 7] Homo-insertion after hetero-deletion (200-2
A heterozygous deletion of 50 bases up to 50 bases, a 100 base homo insertion at the 400th base) As a result of analysis of the sample, homology region A
(Movement distance 300 homology region 150 to 400), homology region B (movement distance 250 homology region 250 to 400), homology region C (migration distance 400 homology region 400 to 550), and homology region D (movement distance 350 homology region 400 ~ 550) was detected (Fig. 13, left). In this case, because of the relationship of the homologous region B to the homologous region A, the two homologous regions overlap, and because the moving distance of B is short, 250
It can be seen that there is a 50-base heterodeletion at the base position. Further, at the reference sequence matrix position 400, the homologous region C does not overlap with the homologous region A with respect to the homologous region A, and the homologous region D does not overlap with the homologous region B. It can be seen that there is a 100 base homo insertion.

Further, as a result of analysis of the homo-deleted sample after hetero-deletion (50-base hetero-deletion from 200 to 250 bases, 50-base homo-deletion from 400 to 450 bases), homologous region A
(Movement distance 300 homology region 150 to 400), Homology region B (Movement distance 250 homology region 250 to 400), Homology region C (Movement distance 250 homology region 450 to 550), and Homology region D (Movement distance 200 homology region 450) (-550) was detected (the right figure of FIG. 13). In this case, because of the relationship of the homologous region B to the homologous region A, the two homologous regions overlap, and because the moving distance of B is short, 250
It can be seen that there is a 50-base heterodeletion at the base position. In the reference sequence matrix position 450, the homologous region C does not overlap with the homologous region A with respect to the homologous region A and the homologous region D does not overlap with the homologous region B. It can be seen that there is a homozygous deletion of 50 bases.

[Example 8] After homo-insertion, homo-insertion (100-base homo-insertion at 250th base, 50-base homo-insertion at 400th base) was analyzed. As a result, homology region A (movement distance 300 homology) Regions 150 to 250), homologous region B (moving distance 400 homologous region 250 to 400), and homologous region C (moving distance 450 homologous region 400 to 550) were detected (FIG. 14, left diagram). In this case, because of the relationship between the homologous region A and the homologous region B, the two homologous regions do not overlap, and the moving distance of B is long.
It can be seen that there is a 100 base homo insertion at the 50th base. Also, due to the relationship of homologous region C to homologous region B, the two homologous regions do not overlap and C has a long moving distance.
It can be seen that there is a homozygous insertion of 50 bases at the 00th base.

In addition, after the homo insertion, the homo deletion (1 at the 200th base)
As a result of analysis of a sample of 50 base homo insertion, 50 base homo deletion from 400 to 450 bases, homology region A (migration distance 300 homology region 150 to 200), homology region B (migration distance 450 homology region 200) .About.400), and homology region C (moving distance 400, homology region 450 to 550) were detected (right diagram in FIG. 14). In this case, because of the relationship between the homologous region A and the homologous region B, the two homologous regions do not overlap, and the moving distance of B is long.
It can be seen that there is a homozygous insertion of 150 bases at the 00th base. Also, due to the relationship of homologous region C to homologous region B, the two homologous regions do not overlap and the migration distance of C is
It can be seen that there is a 50 base homozygous deletion at the 50th base.

[Example 9] Insertion at two sites on one side of the chromosome (insert of 100 bases at 250th base, insertion of 50 bases at 400th base) As a result of analysis of the sample, homologous region A (movement distance 300 homology region 150-550), homology region B (migration distance 400 homology region 250-400), and homology region C (migration distance 450 homology region 400-550) were detected (Fig. 15, left figure). In this case, from the relationship of the homologous region B to the homologous region A, the two homologous regions overlap, and since the moving distance of B is long, 250
It can be seen that there is a 100 base hetero insertion at the base position. Also, because of the relationship of homologous region C to homologous region B, the two homologous regions do not overlap and B has a long moving distance, so
It can be seen that there is a hetero insertion of 50 bases at the 00th base. When there is no duplication of homologous regions, it is usually a homozygous mutation, but it is considered that mutations occur continuously on the same chromosome because a heterozygous mutation has already occurred in the upstream.

In addition, two deletions (100
~ 100 bases deleted up to 200 bases, 100 bases deleted up to 350-450 bases) As a result of sample analysis, homologous region A
(Moving distance 300 homology region 50 to 550), homology region B (moving distance 200 homology region 200 to 350), and homology region C (moving distance
100 homologous regions 450 to 500) were detected (Fig. 15, right panel).
In this case, the relationship between the homologous region A and the homologous region A indicates that the two homologous regions do not overlap with each other, and the migration distance of B is short, so that there is a 100-base heterodeletion at the 200th base. Also, from the relationship of the homologous region C to the homologous region B,
The two homologous regions do not overlap and the C migration distance is short, indicating that there is a 100-base heterodeletion at the 450th base. When there is no duplication of homologous regions, it is usually a homozygous mutation, but it is considered that mutations occur continuously on the same chromosome because a heterozygous mutation has already occurred in the upstream.

[Example 10] Deletion and insertion on one chromosome (deletion of 100 bases from 200 to 300 bases, insertion of 50 bases at the 400th base) As a result of analysis of the sample, homologous region A ( Movement distance 300 homology region 150 to 550), homology region B (movement distance 20
0 homology region 300 to 400), and homology region C (travel distance 250)
The homologous region 400-500) was detected (the left figure in FIG. 16). In this case, the relationship between the homologous region A and the homologous region A indicates that the two homologous regions do not overlap with each other, and the migration distance of B is short, and thus there is a 100-base heterodeletion at the 300th base. Further, from the relationship between the homologous region C and the homologous region B, it is clear that the two homologous regions do not overlap and the B migration distance is long, so that there is a hetero insertion of 50 bases at the 400th base. When there is no duplication of homologous regions, it is usually a homozygous mutation, but it is considered that mutations occur continuously on the same chromosome because a heterozygous mutation has already occurred in the upstream.

In addition, insertion and deletion on one chromosome (350
Insertion of 200 bases at the base position, deletion of 50 bases from 450 to 500 bases was analyzed. As a result, homology region A (movement distance 300 homology region 150 to 550), homology region B (movement distance 500 homology region) 350-450) and homology region C (moving distance 450 homology region 500-550) were detected (FIG. 16, right panel). In this case, because of the relationship of the homologous region B to the homologous region A, the two homologous regions overlap, and since the moving distance of B is long, 350
It can be seen that there is a 200-base hetero insertion at the base position. Further, from the relationship of the homologous region C to the homologous region B, the two homologous regions do not overlap, and the moving distance of C is 5
It can be seen that there is a hetero deletion of 50 bases at the 00th base. When there is no duplication of homologous regions, it is usually a homozygous mutation, but it is considered that mutations occur continuously on the same chromosome because a heterozygous mutation has already occurred in the upstream.

Example 11 The number of bases in the tandem repeat sequence was measured (FIG. 17). Two alleles of microsatellite tandem repeat sequences were used as samples. In allele 1, the GA motif repeats 4 times (small),
Repeat 7 times for Allele 2 (large). First, the tandem repeat sequence sites were sequenced, and the tandem repeats were typed by performing insertion and deletion analysis. The difference from the usual insertion / deletion analysis is that the tandem repeat sequence is removed from the reference sequence filter and the analysis is performed.
By removing the tandem repeat sequence, this could be measured as the number of inserted bases. Four repeats of the GA motif were detected as 8 base insertions, and 7 repeats were detected as 14 base insertions.

[0073]

INDUSTRIAL APPLICABILITY The present invention provides a method for determining the presence or absence of insertion or deletion of bases in a test DNA sequence, and the number of inserted or deleted bases. The method can determine a sample in which insertion or deletion of bases is occurring regardless of whether it is homo or hetero, and the number of insertion-deletion bases can be specified with high sensitivity. In addition, the speeding up of the nucleotide insertion / deletion site test can be realized along with the speeding up of the sequencing. Furthermore, since the method of the present invention can remove the data deterioration element such as noise, it is highly expected that the determination of the insertion or deletion of the base will be easy especially in the waveform data in which the deterioration element such as noise is added. To be done.

[Brief description of drawings]

FIG. 1 is a system configuration diagram of an apparatus of the present invention.

FIG. 2 is a diagram showing an example of a flow of processing executed by the device of the present invention.

FIG. 3 shows the waveform of a normal sample without insertion deletion.

FIG. 4 is a diagram showing a waveform of a sample in which one base is deleted.

FIG. 5 is a conceptual diagram of a measurement start point where a reference sequence extraction filter and a sample matrix are superposed.

FIG. 6 is a diagram showing a calculation method for obtaining a predicted homology region from a homology region and a movement distance by a reference sequence extraction filter.

FIG. 7 is a detection positional relationship diagram showing an example of analysis result of 1-site hetero insertion.

FIG. 8 is a detection positional relationship diagram showing an example of analysis results of a normal sample having no insertion deletion.

FIG. 9 is a detection positional relationship diagram showing an example of analysis results of 1-site hetero insertion and 1-site hetero deletion samples.

FIG. 10 is a detection positional relationship diagram showing an example of the analysis result of a 1-site homo-insertion and 1-site homo deletion sample.

FIG. 11 is a detection positional relationship diagram showing an example of analysis results of a hetero-deleted sample after homo-insertion and a hetero-inserted sample after homo-insertion.

FIG. 12 is a detection positional relationship diagram showing an example of analysis results of a homo insertion after hetero insertion and a homo deletion sample after hetero insertion.

FIG. 13 is a detection positional relationship diagram showing an example of analysis results of homo-insertion after hetero-deletion and homo-deletion sample after hetero-deletion.

FIG. 14 is a detection positional relationship diagram showing an example of analysis results of homo-insertion after homo-insertion and homo-deletion sample after homo-insertion.

[Fig. 15] Fig. 15 is a detection positional relationship diagram showing an example of analysis results of two insertions on one chromosome and two deletions on one chromosome.

FIG. 16 is a detection positional relationship diagram showing an example of analysis results of deletion and insertion in one chromosome and insertion and deletion samples in one chromosome.

FIG. 17 is a diagram showing a method for measuring the number of bases in a tandem repeat sequence.

[Explanation of symbols] 1 Input means 2 display means 3 bus lines 4 Temporary storage means 5 Central processing unit (CPU) 6 main memory

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Misao Oki             5-1-1 Tsukiji, Chuo-ku, Tokyo National Cancer Center             Center Institute (72) Inventor Tsuyoshi Maeno             5-1-1 Tsukiji, Chuo-ku, Tokyo National Cancer Center             Center Institute (72) Inventor Tokuoki Sakiyama             5-1-1 Tsukiji, Chuo-ku, Tokyo National Cancer Center             Center Institute F-term (reference) 5B056 BB22 BB37 BB42 BB71                 5B075 ND03 UU19

Claims (4)

[Claims] 1. A method for determining the presence or absence of insertion or deletion of bases in a test DNA sequence, or the number of inserted or deleted bases, wherein (a) the reference sequence is divided into 4 base components for each base. A reference sequence extraction filter consisting of a set of decomposed vectors, and a step of creating a sample matrix consisting of a set of vectors into which a test DNA sequence is decomposed into 4 base components for each base, (b) the reference sequence extraction filter and the Superimposing the starting end and the ending end of the sample matrix, (c) while moving the reference sequence extraction filter and the sample matrix one by one,
Calculating the sum of the products of the matrices for the moving distance from the point where the reference end extraction filter and the start end and the end end of the sample matrix are overlapped, and the overlapping bases, (d) from the sum of the products of the matrix , A step of calculating the number of consecutive homologous bases in overlapping bases, migration distance, reference sequence extraction filter homology region, and sample matrix homology region, (e) maximum homologous base sequence that is the migration distance when showing the largest number of homologous bases Calculating a detection point, a standard reference sequence extraction filter homology region that is a reference sequence extraction filter homology region at the detection point, and a standard sample matrix homology region that is a sample matrix homology region at the detection point, (f) the standard reference Sequence extraction filter homology region and the standard sample matrix homology region And the step of calculating the inter-matrix position correction value, including the number of homologous bases, the reference sequence extraction filter homology region, the sample matrix homology region, the maximum homology base sequence detection point, the standard reference sequence extraction filter homology region, and the standard. A method for determining the number of inserted or deleted bases by evaluating the presence or absence of bases in the test DNA sequence from the sample matrix homology region. 2. A computer according to claim 1, comprising:
A program for executing the step (f) to determine the presence or absence of insertion or deletion of bases in the test DNA sequence, or the number of inserted or deleted bases. 3. A computer-readable recording medium in which the program according to claim 2 is recorded. 4. An apparatus for determining the presence or absence of insertion or deletion of bases in a test DNA sequence, or the number of inserted or deleted bases, which comprises the following means (1) to (3). (1) Input means for inputting the reference sequence and test DNA sequence information (2) By the steps (a) to (f) of claim 1, the number of homologous bases, the reference sequence extraction filter homology region, the sample matrix homology region, Calculation means for calculating the maximum homologous nucleotide sequence detection point, the standard reference sequence extraction filter homology area, and the standard sample matrix homology area (3) Display means for displaying the calculation result