JPH11178575A - Apparatus for analyzing dna base sequence, analysis and recording medium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To determine the DNA sequence, analyze the sequence and determine a plan for adopting or not adopting the results of the analysis in an early stage during the midst of electrophoresis. SOLUTION: This apparatus for analyzing a DNA sequence is composed of a conventional device for eletrophoresis for determining the DNA sequence, a signal memory device capable of obtaining a signal from a signal detecting part thereof at a prescribed time interval and accumulating the obtained signal, a computer capable of accessing the signal memory device at a prescribed time interval, determining the sequence and similarly searching for data groups of the known DNA sequences or the known amino acid sequences using the sequence as a query and a display device. The results of determination of the sequence are displayed together with the time to the completion of the determination of the whole sequence or the number of bases and the results of the search are then displayed as a top score list 302, a progress of the number of sequences having the prescribed minimum value or more or similarity with the passage of time 301 or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an apparatus, a method and a recording medium for DNA sequencing using electrophoresis used in biological research, health and medical care.

[0002]

2. Description of the Related Art Conventionally, Sanger's nucleotide sequencing method (F. San
ger, S. Nicklen, and AR Coulson. "DNA sequencin
g with chain-terminating inhibitors. "Proc. Natl.
Acad. Sci. USA, 74: 5463-5467 (1977)).
When determining the A base sequence, after the electrophoresis of the DNA fragment was completed, the accumulated migration pattern was analyzed to determine the sequence. However, as long as this method is adopted, information on the sequence of the sample cannot be obtained by a method other than reading the concentration pattern of the electrophoretic components by inspection until the electrophoresis is completed. Sequencing by inspection of the electrophoresis pattern requires user skill and takes time,
For the average user, sequencing during electrophoresis may be said to be impossible in most cases with conventional procedures.

[0003] Unnecessary electrophoresis patterns have been collected in some cases because sequencing during electrophoresis is practically impossible. Also, usually, a D-base having a length of about 500 bases
The sequence of NA is determined by a single electrophoresis. The time required for this electrophoresis is as long as several hours. It is important in the conversion and rapid use of analysis results.

[0004]

SUMMARY OF THE INVENTION DN during electrophoresis
A To provide a DNA sequence analysis apparatus, method and recording medium that facilitates sequencing and sequence analysis,
It is an object of the present invention to enable early decision-making to adopt or reject the analysis result during electrophoresis.

[0005]

SUMMARY OF THE INVENTION The present invention provides the above-mentioned object,
In particular, in order to facilitate DNA sequencing during electrophoresis, the invention according to claim 1 employs an electrophoresis path (101) for electrophoretically separating DNA fragments and each component of the DNA fragments separated by electrophoresis. A detection device (102) for detecting, a storage device (103) for storing a detection signal from the detection device, and a sequence determination device for determining the sequence of a DNA fragment to be analyzed based on the signal stored in the storage device (104) and a DNA base sequence analysis device comprising a display device (105) for displaying a sequence determined by the sequence determination device, wherein the sequence determination device is stored in the storage device by that time during electrophoresis. A part of the sequence of the DNA fragment is determined based on the signal.

According to a second aspect of the present invention, the display device (10
5) indicates the length of a base sequence already determined and the length of a base sequence not yet determined during electrophoresis. The invention according to claim 3 is characterized in that, during the migration, the DNA
A sequence search device (106) for accessing the sequence data group (107) and searching the DNA sequence data group for a DNA sequence that matches or is similar to the base sequence already determined by the sequence determination device. is there.

The invention according to claim 4 is that, during the electrophoresis, the amino acid sequence data group (107) is accessed to match the amino acid sequence after the translation of the base sequence already determined by the sequencing device, or Sequence search apparatus for searching for a similar amino acid sequence from the amino acid sequence data group
(106). The invention according to claim 5, wherein the display device (105), the information on the sequence searched by the sequence search device (106), by a predetermined number of sequences in order from the highest similarity in a predetermined similarity scale, Alternatively, up to an array having a similarity greater than or equal to a predetermined first minimum value is displayed.

[0008] The invention according to claim 6 is the display device (10)
5) determines the number of sequences having a similarity greater than or equal to a predetermined second minimum value on a predetermined similarity scale among the sequences searched by the sequence search device (106), as a function of the signal acquisition time. Or as a function of the number of bases in the determined sequence. With such a display, it is possible to make an early decision on the adoption or non-employment of the search result of the sequence search apparatus.

[0009] The invention according to claim 7 is characterized in that the electrophoretically separated D
In a DNA base sequence analysis method for determining the sequence of a DNA fragment to be analyzed based on a signal obtained by detecting each component of an NA fragment, during the electrophoresis, the sequence of the DNA fragment is determined based on the signal obtained up to that time. Is a method for analyzing a DNA base sequence, wherein a part of the DNA base sequence is determined.
The invention according to claim 8 is a computer-readable recording medium that records a program for causing a computer to execute the DNA base sequence analysis method according to claim 7.

[0010]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring to FIG. 1, the overall configuration of a DNA base sequence analyzer of the present invention is shown. 101 is an electrophoresis path through which the solution after the extension reaction in Sanger's base sequence determination method passes, 102 is a detection device that detects each component separated by electrophoresis, 103 is a signal storage device that stores the signal detected in 102 , 104 is a sequence determination device that determines a part of the sequence of the DNA fragment to be analyzed based on the change over time of the signal stored in 103, 105 is a display device, 106 is a sequence search device, 107 is a known DNA sequence or It is a sequence storage device that stores amino acid sequence data.

Examples of the electrophoresis path 101 include a plate gel electrophoresis path in which a polymer gel is sandwiched between two flat plates, and a capillary gel electrophoresis path in which a polymer gel is filled in a tube. An example of the detection device 102 includes a camera that detects light emission from an electrophoretic component. Examples of the signal storage device 103 include a random access memory, a hard disk, and a magneto-optical disk. Sequence determination device 104
One example is a digital computer loaded with sequencing software. As an example of the display device 105,
Monitors used in personal computers, workstations, and the like are included. An example of a sequence search device 106 is a digital computer that loads sequence match or similarity search software. Sequence storage device 107
Examples include a random access memory, a hard disk, and a magneto-optical disk.

In FIG. 1, the sequence determination device 104 and the sequence search device 106 are shown separately. However, in some cases, these devices may be replaced with a single digital computer having a sequence determination function and a sequence search function. . Alternatively, these digital computers may be parallel computers using a plurality of central processing units. Using FIG.
The operation of the DNA base sequence analyzer of the present invention will be described.

The detection device 102 detects a signal from the electrophoresis component at a predetermined time interval during the electrophoresis, and transfers the signal detected at the time to the signal storage device 103. The signal storage device 103 stores the received signal. Therefore, the signal storage device 103 accumulates the signals at each time. The sequencing device 104 accesses the signal storage device 103 at predetermined time intervals, and retrieves signals added since the previous access.
The data is copied to the storage device in 104, and the access time is also recorded. After the copy is completed, the lanes are determined, and the sequence is determined based on the time-dependent change in the signal intensity on each lane. The arrangement determined in 104 is displayed on the display device 105. By displaying the determined sequence directly, it is easier to analyze the DNA sample currently being electrophoresed. Another display method related to the determined sequence will be described later.

Further, the determined sequence is stored in a sequence search device.
The sequence search device 106 searches the known DNA sequence group stored in the sequence storage device 107 for a DNA sequence that matches or is similar to the passed DNA sequence, or The amino acid sequence that matches or is similar to the amino acid sequence obtained by translating is searched from the group of known amino acid sequences stored in the sequence storage device 107. The found similar sequences are annotated with each sequence, and in a predetermined similarity scale (refer to the explanation of terms), in descending order of similarity (refer to explanation of terms),
A predetermined number or an array having a predetermined minimum similarity is displayed on the display device 105. By displaying such an annotated similar sequence, the user can know information for knowing the position of the DNA sequence of the sample during electrophoresis on the genome, the function of the gene, and the like more quickly. further,
Another display method related to the similar sequence will be described later.

Another display method related to the determined sequence will be described with reference to FIG. The arrangement determined by the arrangement determining device 104 is displayed by, for example, arranging the symbols of A, C, G, and T horizontally at equal intervals, as shown in FIG. The part where the symbols are arranged is the part where the sequencing has been completed. The length of the undetermined region is the length of a DNA sample (base unit; this length can be predicted from the retention time in separation of the sample by electrophoresis or the like) which has been input to the sequencing device 104 in advance. Is obtained by subtracting the length of the determined area from, and this is displayed as a blank of the corresponding length adjacent to the determined area, for example, as shown in FIG. With such a display, it is easy to see what percentage of the analysis target has been sequenced. In addition, the time required for completing the sequencing may be directly indicated by a numerical value after converting the number of remaining bases as shown in the lower part of FIG.

Such information on the ratio of the determined regions is a material for judging to what extent the sequence information can be refined by extending the electrophoresis. That is, by waiting for electrophoresis, a criterion for determining how much information can be expected to be added is given. Such information is particularly useful in cases where time for decision making is limited, such as diagnosis based on the DNA sequence of a sample, personal identification, and the like.

As another method of displaying the time until the entire region of the sample sequence is determined, a time scale based on the acquisition time of the signal data of the electrophoresis component is displayed as shown in FIG. cheap. In addition, time
If a scale is displayed in units of the number of bases in addition to the scale, the number of remaining bases becomes easier to read. Another display method related to a similar sequence will be described with reference to FIG.

At 301, the sequence retrieving device 106 uses the sequence determined based on the signal data received at a certain time by the sequence determining device 104 as a search key (query) (see the explanation of terms). 5 is an example of display of information on similar sequences obtained as a result of a search for a group of known sequences in the list.
302, the searched similar sequences, in order from a sequence having a high degree of similarity in a predetermined similarity scale, a predetermined number of sequences,
Alternatively, a list displayed using the similarity of each sequence and an annotation of the sequence up to a sequence having a similarity equal to or greater than a predetermined minimum value is referred to as a top score list (refer to the explanation of terms). Here, as an example of the similarity measure,
A similarity statistic calculated based on the distribution of scores between the "score" (see the explanation of terms) calculated based on the sum of similarities between the contiguous array elements and the known sequence group of Queries Importance.

FIG. 4 shows an example of the top score list. In this example, a DNA base sequence having a length of 15 bases was used as a querily and about 60,000 amino acid sequence groups SWISS • PRO
This is the result of searching for T (about 20 million amino acids). The score column in FIG. 4 shows a score in consideration of the length of the amino acid sequence. In this example, information of 17 sequences is displayed in order of the score from the amino acid sequence having the largest score. P (z> x)
And E (z> x) are columns indicating statistical significance. P (z>
x) is the probability that the query and the random amino acid sequence have a similarity greater than or equal to the score shown on the left. E (z>
x) is the expected value of the number of the amino acid sequence. Therefore, the smaller the value of both P (z> x) and E (z> x), the higher the statistical significance. The target column is a column in which annotations regarding the amino acid sequence are displayed.
Here, only the beginning of each annotation (keywords related to the function of species and proteins are mainly written)
Is displayed.

In the portion other than 302 in 301, the number of sequences in the sequence storage device having a similarity with the query or higher than a predetermined limit value among the searched sequences is stored in the signal storage device 104 by the sequence determination device 104. It is displayed as a function of the number of bases of the sequence determined by the sequence determination device 104 based on the signal data acquisition time from the device 103 or the data updated by the signal data acquisition. Here, the threshold value of similarity with Queries is a value equal to the similarity (score, statistical significance, etc.) of the sequence that matches Queries, or
A value multiplied by a certain factor (positive value) is preferable.

In FIG. 3, the number of similar sequences is displayed on a log scale, but a normal scale may be employed. The time dependence of the number of similar sequences or the dependence on the number of determined bases is represented by a line graph in FIG. 3, but another expression method may be adopted. The solid line portion of the polygonal line indicates the result of searching for the determined sequence as a query. The broken line portion of the broken line is a prediction of a change in the number of similar sequences in the future.

According to the display contents of 301 and 302, the sequence determination device 104 acquires new data, determines a new sequence,
Using the determined sequence as a query, the sequence search device 106
And is updated each time a new result is obtained. By estimating future changes in the number of similar sequences, sample D
The time at which NA can be identified can be predicted. That is,
When the number of sequences having a similarity equal to or more than a certain limit becomes about several, the user can start the identification work while looking at the annotations of the top score list 302. The time at which the number of sequences becomes about several may be read using a predicted straight line or curve of the number of similar sequences. As shown in the example of FIG. 3, when the identifiable expected time is automatically displayed, it becomes easier to read.

As an example of the extrapolation method of the similar curve, there is a method using a model function shown below. Sequence storage device 107
A very long random array is adopted as a model of the array group stored in the. The number f of subarrays in the random array of length N (a sufficiently large value) that matches the Quarry array of length n is f = N * (1 / k) ** n. Here, k is the number of types of array elements (DN
K = 4 for the A sequence and k = 20 for the amino acid sequence). Therefore, log (f) = log (N) -n * log (k), that is, the number f of subsequences that match the Quarry array
Is linearly dependent on the length n of the Quarry array. It can be expected that the number of arrays having a similarity equal to or greater than a predetermined limit value to the Quarry array is also linearly dependent on the length n of the Quarry array. Therefore, as shown in FIG. 3, a change in the number of similar sequences can be predicted by linear extrapolation using a log scale of the number of similar sequences.

Thus, during the electrophoresis, the sample DN
The display of the time at which A can be identified is very useful information when the time limit for decision-making is severe in diagnosis, personal recognition, and the like. That is, it is possible to determine whether or not this DNA sequence analysis information can be used as a material for decision making.

[0025]

According to the present invention, DNA by electrophoresis
In the sequence analyzer, it is possible to determine the sequence during the electrophoresis, and it is possible to directly display the determined sequence. Therefore, it is easier to analyze the DNA sample during the electrophoresis.

Further, since it is possible to compare the DNA sequence determined during electrophoresis with the known DNA sequence or known amino acid sequence, it is possible to display annotated similar sequences during electrophoresis. The information for knowing the position of the DNA sequence of the sample on the genome, gene function, and the like can be obtained more quickly. In addition, it is possible to provide information on the ratio of the sequenced region during electrophoresis, so that waiting for electrophoresis gives a criterion of how much information can be expected to be added, diagnosis, and personal identification. For example, it is useful for decision making in a situation where time is limited.

In addition, since the expected time at which the sample DNA can be identified can be displayed during the electrophoresis, this DNA sequence analysis information can be used as a material for decision making in diagnosis, personal recognition and the like. It is possible to judge whether or not it is possible, and to provide useful information for decision making under a situation where time is severe.

[Explanation of Terms] Queries: When a sequence matching or similar to a certain sequence is to be found (searched) from a plurality of sequences,
The former "certain arrangement" is called a query. Library; refers to a sequence group to be searched. Similarity: refers to the degree of similarity when comparing two sequences. However, when the two sequences are of different types (for example, amino acid and DNA base sequences), one sequence may be translated and compared with the other as the same type. For example, when a DNA base sequence is translated into an amino acid sequence and compared with another amino acid sequence. Score; refers to a numerical value expressing the similarity between two sequences.
Usually, the score between two sequences is calculated from the sum of similarities between opposing sequence elements (characters representing DNA bases, amino acids, etc.) when the two sequences are arranged in a certain arrangement. However, depending on the arrangement, there may be a gap (gap) where the partner array element is missing in the middle of the arrangement of the opposing array elements. In this case, the sum of the similarities between the above elements is penalized (negative numerical value). ). In addition, there is a score calculation method that takes into account the length of the library sequence in the search. Similarity measure: A method of calculating a numerical value expressing the degree of similarity between two sequences. For example, there are various score calculation methods and various statistical importance calculation methods. Top score list; coined word in this specification. List showing the sequences of the searched libraries. In order from the sequence with the highest similarity of the predetermined similarity measure, only the predetermined number of sequences,
Alternatively, for sequences having a similarity greater than or equal to a predetermined minimum value, the similarity and annotation of each sequence are displayed.

[Brief description of the drawings]

FIG. 1 is a diagram illustrating an overall configuration of the present invention.

FIG. 2 is a view for explaining an example of display in the present invention (part 1).

FIG. 3 is a view for explaining an example of display in the present invention (part 2).

FIG. 4 is a diagram illustrating a top score list display according to the present invention.

[Explanation of symbols]

 Reference Signs List 101 electrophoresis path 102 detection device 103 signal storage device 104 sequence determination device 105 display device 106 sequence search device 107 sequence storage device

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI G06F 17/30 G06F 15/40 370F (72) Inventor Satoshi Takahashi 882 Ma, Hitachinaka-shi, Ibaraki Measuring Instruments Business Hitachi, Ltd. (72) Keiichi Nagai, Inventor 1-280 Higashi Koigabo, Kokubunji-shi, Tokyo Inside Central Research Laboratory, Hitachi, Ltd.

Claims (8)

[Claims] An electrophoresis path for electrophoretically separating DNA fragments, a detection device for detecting each component of the DNA fragments separated by the electrophoresis, a storage device for storing a detection signal from the detection device, and the storage device A sequence determination device that determines the sequence of the DNA fragment to be analyzed based on the signal stored in the storage device; and a display device that displays the sequence determined by the sequence determination device. During the electrophoresis, based on the signal stored in the storage device up to that time, the DN
DNA for determining a part of the sequence of fragment A
Base sequence analyzer. 2. The DN according to claim 1, wherein the display device displays the determined base sequence and the length of the base sequence not yet determined during the electrophoresis.
A base sequence analyzer. 3. During the electrophoresis, a DNA sequence data group is accessed, and a DNA sequence that matches or is similar to the base sequence already determined by the
2. The DNA base sequence analyzer according to claim 1, further comprising a sequence search device for searching from the NA sequence data group. 4. Accessing the amino acid sequence data group during the electrophoresis to find an amino acid sequence that matches or is similar to the amino acid sequence after translating the base sequence already determined by the sequencer. 2. The DNA base sequence analyzer according to claim 1, further comprising a sequence search device for searching from a group. 5. The display device according to claim 1, wherein the information on the sequences searched by the sequence search device is a predetermined number of sequences in order from a sequence having a high degree of similarity on a predetermined similarity scale, or a predetermined first minimum value or more. The DNA base sequence analyzer according to claim 3 or 4, wherein up to the sequence having the similarity of (1) is displayed. 6. The display device obtains the number of sequences having a similarity greater than or equal to a predetermined second minimum value on a predetermined similarity scale among the sequences searched by the sequence search device. The DNA base sequence analyzer according to any one of claims 3 to 5, wherein the information is displayed as a function of time or as a function of the number of bases of the determined sequence. 7. A DNA base sequence analysis method for determining the sequence of a DNA fragment to be analyzed based on a signal obtained by detecting each component of a DNA fragment subjected to electrophoresis separation, wherein the DNA base sequence analysis method comprises the steps of: A DNA base sequence analysis method, comprising determining a part of the sequence of the DNA fragment based on a signal. 8. A computer-readable recording medium on which a program for causing a computer to execute the DNA base sequence analysis method according to claim 7 is recorded.